A SIOP Renal Tumours Study Group Prospective Clinical Study

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UMBRELLA PROTOCOL SIOP-RTSG 2016

Integrated research and guidelines for standardized diagnostics and therapy

Therapeutic Recommendations WT Non-WT St I-III St IV St V CCSK MRTK CMN RCC Relapse Adults

EudraCT number: 2016-004180-39

VERSION 1.2 October 2016 UMBRELLA Protocol SIOP 2016

Table of contents

PART A – Evaluation of tissue, imaging and biomarkers to optimize risk stratification….16

PART B - Treatment Guidelines …………………………………………………………………………………...56

1 General introduction ...... 10 2 Introduction UMBRELLA protocol SIOP 2016 ...... 17 3 Overall aim of the UMBRELLA protocol SIOP 2016 ...... 17 3.1 Primary aims ...... 18 3.2 Secondary aims ...... 19 3.3 Expected impact ...... 19 4 Background and rationale of the UMBRELLA protocol SIOP 2016 ...... 20 4.1 Molecular biology ...... 20 4.1.1 1q gain as a prognostic biomarker in WT ...... 20 4.1.2 Other genomic biomarkers ...... 20 4.2 Pathology and Blastemal volume ...... 21 4.3 Radiology ...... 22 5 Samples collection and storage ...... 24 5.1 Further usage of biomaterial in upcoming research questions ...... 24 6 Patient enrolment ...... 24 6.1 Inclusion criteria ...... 27 6.2 Exclusion criteria ...... 27 7 Centralized review (CR) ...... 27 7.1 Central Radiology Review (CRR) ...... 27 7.1.1 Time points for CRR ...... 27 7.1.2 Central radiology review ...... 28 7.2 Central Pathology Review (CPR) ...... 28 7.3 Central Biology Logistics ...... 30 7.3.1 Biological sample collection, processing and storage ...... 30 7.3.2 Fresh Tissue Sampling for cell culture (limited centres only) ...... 32 7.3.3 Blood Sample collection in MRTK ...... 32 7.3.4 Renal tumour biology study specimen collection protocol ...... 33 7.3.5 Analysis and quality control of DNA ...... 34 7.3.6 Urine Sample Requirements (for full details, participating centres should refer to the relevant Laboratory Manual) ...... 34 7.3.7 Blood Sample Guidelines (for full details, participating centres should refer to the relevant Laboratory Manual) ...... 34 7.4 Central surgical review ...... 35 7.5 Central radiotherapy review ...... 35 8 Statistical Considerations with specific focus on 1q gain including sample size calculations ...... 36 9 IT infrastructure and data management ...... 36

UMBRELLA Protocol SIOP 2016

10 Patient Empowerment ...... 37 10.1 Impact on the well-being of patients ...... 37 11 Diagnostic work-up ...... 38 11.1 Clinical work-up ...... 38 11.2 Laboratory work-up ...... 38 11.3 Radiological work-up ...... 39 11.4 Genetic counselling ...... 39 11.5 Flow diagram of initial diagnostic work-up ...... 40 11.6 Timeframe of diagnostic work-up ...... 41 11.6.1 Standard investigations during the pre-operative phase in clinically localized disease ...... 41 11.6.2 Standard diagnostics during the pre-operative phase in stage IV ...... 42 11.6.3 Postoperative standard investigations after renal surgery in patients that received preoperative chemotherapy ...... 43 11.6.4 Postoperative investigations in patients after primary surgery (i.e. without pre-operative chemotherapy) ...... 44 11.6.5 Investigations in patients during follow-up after end of treatment ...... 45 11.7 Details on radiology ...... 46 11.7.1 Imaging diagnostics ...... 46 11.7.2 Optional and functional diagnostics imaging ...... 46 11.7.3 Classification of lung lesions ...... 47 11.7.4 Cutting Needle Biopsy ...... 47 11.8 Pathology issues ...... 47 11.8.1 Nephrectomy handling ...... 47 11.8.2 Tissue for research (see also section 7.3 and Appendix 7) ...... 50 12 Plan of integrated research...... 51 12.1 List of some research proposals ...... 51 13 References ...... 53 14 General Treatment Guidelines ...... 57 14.1 General remarks ...... 57 14.2 General chemotherapy guidelines ...... 57 14.2.1 Drugs and dosage ...... 57 14.3 Toxicity ...... 61 14.3.1 Hematological toxicity...... 61 14.3.2 Neutropenic fever ...... 61 14.3.3 Isolated gastrointestinal complications ...... 61 14.3.4 Hepatic complications ...... 62 14.3.5 Exposure to infection with varicella or herpes ...... 62 14.3.6 Cardiac toxicity ...... 62 14.3.7 Neurological toxicity ...... 62 14.3.8 Bladder and renal toxicity ...... 63 14.3.9 Gonadotoxicity ...... 63 14.3.10 Major intolerance during pre-operative therapy ...... 63 14.4 Supportive care ...... 64 14.5 Modifications for small children or children below 7 months of age ...... 64 14.5.1 General remarks ...... 64

UMBRELLA Protocol SIOP 2016

14.5.2 Treatment recommendations for patients with renal tumours below 7 months of age ...... 66 14.5.3 Chemotherapy dose adjustment for infants and/or < 12 kg body weight ...... 67 14.5.4 Radiotherapy ...... 67 14.6 References...... 67 15 Treatment guidelines for Wilms Tumours ...... 68 15.1 Introduction / background ...... 68 15.2 Treatment guidelines for localized Wilms Tumours (stage I – III)...... 69 15.2.1 Treatment recommendations localized WT...... 69 15.2.2 Surgical recommendations ...... 74 15.2.3 Radiotherapeutic recommendations ...... 74 15.2.4 Chairs and members of the Stage I-III WT Panel ...... 74 15.3 Treatment guidelines for metastatic Wilms Tumours (stage IV) ...... 76 15.3.1 General remarks ...... 76 15.3.2 Pre-operative chemotherapy ...... 76 15.3.3 Surgery ...... 77 15.3.4 Post-operative chemotherapy ...... 77 15.3.5 Treatment schedules for Stage IV ...... 81 15.3.6 Recommended Treatment adjustments – Steering during treatment ...... 84 15.3.7 Radiotherapy ...... 85 15.3.8 Follow-up ...... 85 15.3.9 Treatment Recommendation Overview ...... 85 15.3.10 Chairs and members of the Stage IV WT Panel ...... 86 15.4 Management of bilateral disease (stage V) and bilaterally-predisposed unilateral Wilms Tumour 88 15.4.1 BACKGROUND ...... 88 15.4.2 GOALS AND OBJECTIVES (scientific aims of the study) ...... 91 15.4.3 TREATMENT PLAN ...... 93 15.4.4 Treatment plans ...... 94 15.4.5 Chairs and members of the Bilateral WT Panel ...... 98 15.4.6 References...... 99 15.5 Treatment guidelines after primary surgery ...... 101 15.5.1 Staging ...... 101 15.5.2 Histological classification ...... 101 15.5.3 Post-operative chemotherapy regimens for tumours having primary excision ...... 101 15.5.4 Chairs and members of the primary surgery panel ...... 104 15.5.5 References...... 105 15.6 Treatment guidelines for relapsed Wilms tumours ...... 106 15.6.1 Introduction and background ...... 106 15.6.2 Prognostic factors and risk stratification at relapse ...... 106 15.6.3 Rationale for treatment of relapsed WT with standard-risk features from previous studies 109 15.6.4 Rationale for treatment of relapsed WT with high-risk features from previous studies ...... 109 15.6.5 Comparison between high-dose therapy and standard-dose therapy ...... 112 15.6.6 Topoisomerase inhibitors ...... 112 15.6.7 Relapsed WT with very high risk features ...... 113 15.6.8 “Local” therapies: role for surgery and radiation therapy ...... 114 15.6.9 Biological studies at relapse ...... 115

UMBRELLA Protocol SIOP 2016

15.6.10 Combined rationale for the proposed therapy recommendations: summary ...... 116 15.6.11 Specific objectives ...... 118 15.6.12 Diagnostic and staging investigations at relapse ...... 118 15.6.13 Eligibility and risk stratification ...... 119 15.6.14 Flow diagram with treatment recommendations ...... 120 15.6.15 Therapeutic recommendations ...... 121 15.6.16 Surgical guidelines for relapse ...... 126 15.6.17 Radiotherapy guidelines for relapse ...... 126 15.6.18 Biological studies ...... 126 15.6.19 Chairs and members of the Relapsed WT Panel ...... 127 15.6.20 References ...... 128 15.7 Treatment guidelines for adults with Wilms tumours ...... 133 15.7.1 Introduction ...... 133 15.7.2 Treatment Regimens ...... 133 15.7.3 Summary of radiotherapy recommendations...... 137 15.7.4 Diagnostic procedures during and after treatment ...... 137 15.7.5 Chairs and members of the Adult WT Panel ...... 137 15.7.6 References...... 138 16 Surgical Guidelines ...... 139 16.1 General surgical guidelines ...... 139 16.1.1 Nephrectomy ...... 139 16.1.2 Nephron sparing surgery (NSS) in unilateral cases [20-29] ...... 140 16.1.3 Laparoscopic nephroureterectomy [30-31] ...... 142 16.1.4 Comments regarding pathology specimens ...... 143 16.1.5 The SIOP form ...... 143 16.2 Surgical guidelines Wilms Tumour ...... 143 16.2.1 Wilms Tumour Stage I-III ...... 143 16.2.2 Wilms Tumour Stage IV [13–15] ...... 143 16.2.3 Bilateral Wilms Tumours (Stage V) [16 – 19] ...... 144 16.2.4 Relapsed Wilms Tumour ...... 145 16.3 Surgical guidelines for non-Wilms Tumours ...... 146 16.3.1 Clear cell Sarcoma of the Kidney (CCSK) ...... 146 16.3.2 Renal Cell Carcinoma (RCC) [32-41] ...... 146 16.3.3 Malignant rhabdoid tumour of the kidney (MRTK) ...... 147 16.3.4 Congenital mesoblastic nephroma (CMN) ...... 148 16.4 Chair and Members of the Surgical Panel ...... 148 16.5 References...... 149 17 Radiotherapeutic guidelines ...... 153 17.1 RADIATION THERAPY TREATMENT OF LOCAL ABDOMINAL DISEASE ...... 153 17.1.1 Indications for post-operative local or flank RT: ...... 153 17.1.2 Indications for post-operative whole abdominal RT: ...... 153 17.1.3 Start of RT...... 153 17.2 LOCAL FLANK RADIOTHERAPY - dose and fractionation - ...... 154 17.2.1 Intermediate risk histology – local flank RT ...... 154 17.2.2 High risk histology – local flank RT ...... 154 5

UMBRELLA Protocol SIOP 2016

17.3 WHOLE ABDOMEN RADIOTHERAPY – dose and fractionation - ...... 155 17.4 Summary recommendation of radiation therapy treatment of abdominal disease ...... 155 17.5 Dose reduction of chemotherapy ...... 155 17.6 RADIATION THERAPY TREATMENT OF STAGE V (BILATERAL WILMS TUMOUR) ...... 155 17.6.1 Indications for postoperative local radiotherapy ...... 156 17.6.2 Radiation therapy treatment after nephron sparing surgery - dose and fractionation - ...... 156 17.7 RADIATION THERAPY TREATMENT OF METASTATIC SITES ...... 156 17.7.1 Indications for pulmonary radiotherapy (primary treatment) ...... 156 17.7.2 Timing of pulmonary radiotherapy ...... 157 17.7.3 Indications for hepatic radiotherapy ...... 157 17.7.4 Indications for radiotherapy to other metastatic sites ...... 157 17.8 Summary recommendations of radiation therapy treatment of metastatic sites ...... 158 17.9 RADIATION THERAPY TREATMENT OF RECURRENT DISEASE ...... 158 17.9.1 RADIATION TREATMENT OF LOCAL RELAPSES IN THE ABDOMEN ...... 158 17.9.2 RADIATION TREATMENT OF RELAPSES IN THE LUNG ...... 159 17.10 INTERRUPTIONS and BREAKS ...... 159 17.11 EQUIPMENT AND TREATMENT TECHNIQUE (SIMULATION/TREATMENT PERFORMANCE) ...... 160 17.12 TARGET VOLUME DEFINITION ...... 160 17.12.1 Dose uniformity and Reference Points (ICRU) ...... 160 17.13 TARGET VOLUMES...... 160 17.13.1 Localisation of primary tumour and kidney for flank/abdominal RT ...... 160 17.14 CLINICAL TARGET VOLUME (CTV) AND PLANNING TARGET VOLUME (PTV) ...... 161 17.14.1 Flank-Radiotherapy ...... 161 17.14.2 Whole abdominal RT ...... 161 17.14.3 Pulmonary RT ...... 161 17.14.4 Liver RT ...... 161 17.14.5 RT for brain metastases ...... 161 17.14.6 RT for haematogenous metastases to bone ...... 162 17.15 NORMAL TISSUE SPARING ...... 162 17.15.1 Critical organ dose ...... 162 17.15.2 Shielding: ...... 162 17.16 EXAMPLES FOR TYPICAL TARGET VOLUMES AND RADIATION PORTALS ...... 163 17.16.1 Stage II high risk, stage III (Fig. 1a-c) ...... 163 17.16.2 Stage III intermediate and high risk (Fig. 2a, b) ...... 165 17.16.3 Stage III (all histologies): major intra-/retroperitoneal rupture ...... 166 17.16.4 PULMONARY RADIOTHERAPY (Stage IV) ...... 168 17.17 GENERAL GUIDELINES FOR RADIATION THERAPY FOR PATIENTS WITH RHABDOID TUMOURS OF THE KIDNEY (RTK) ...... 170 17.18 Clear Cell Sarcoma of the Kidney (CCSK) ...... 171 17.19 ORGANS AT RISK ...... 173 17.19.1 Bone and soft tissue ...... 173 17.19.2 Liver ...... 173 17.19.3 Gastrointestinal tract ...... 173 17.19.4 Kidney ...... 174 17.19.5 Ovary ...... 174

UMBRELLA Protocol SIOP 2016

17.19.6 Testes ...... 174 17.19.7 Mammary gland/areola ...... 174 17.19.8 Cardiac toxicity ...... 174 17.20 QUALITY ASSURANCE DOCUMENTATION – Case Report Form (CRF) ...... 175 17.21 Chair and Members of the Radiotherapy Panel ...... 175 18 Non-Wilms tumours of the kidney ...... 176 18.1 Clear Cell Sarcoma ...... 176 18.1.1 Introduction ...... 176 18.1.2 Aims and endpoints ...... 176 18.1.3 Background information ...... 177 18.1.4 Treatment recommendations ...... 181 18.1.5 Follow-up guideline for surveillance ...... 182 18.1.6 Recommendations treatment relapsed CCSK ...... 182 18.1.7 References...... 182 18.1.8 Chairs and members of the CCSK Panel ...... 185 18.2 Renal Cell Carcinoma (RCC) ...... 187 18.2.1 Introduction ...... 187 18.2.2 Aims and endpoints ...... 188 18.2.3 Background information ...... 188 18.2.4 Genetics and Biology ...... 192 18.2.5 Inclusion criteria prospective RCC registry and guideline ...... 193 18.2.6 Treatment ...... 194 18.2.7 Treatment recommendations ...... 195 18.2.8 Medical treatment: ...... 197 18.2.9 Radiotherapy ...... 200 18.2.10 Follow-up guideline for surveillance ...... 200 18.2.11 Second line treatment after relapse of RCC ...... 200 18.2.12 Side effects of medical treatment ...... 200 18.2.13 References ...... 208 18.2.14 Chairs and members of the RCC Panel ...... 213 18.3 Malignant Rhabdoid Tumour of the Kidney (MRTK) ...... 215 18.3.1 Introduction ...... 215 18.3.2 Aims ...... 215 18.3.3 Endpoints ...... 216 18.3.4 Inclusion criteria ...... 216 18.3.5 Exclusion criteria ...... 216 18.3.6 Background Information ...... 216 18.3.7 Recommendations ...... 222 18.3.8 References...... 223 18.3.9 Chairs and members of the MRTK Panel ...... 226 18.4 Congenital Mesoblastic Nephroma (CMN) ...... 228 18.4.1 Introduction / background ...... 228 18.4.2 3.4.2 Aims and endpoints ...... 229 18.4.3 Treatment recommendations ...... 230 18.4.4 Follow-up guideline for surveillance ...... 232 7

UMBRELLA Protocol SIOP 2016

18.4.5 Treatment recommendations of relapsed CMN ...... 232 18.4.6 References...... 232 18.4.7 Chairs and members of the CMN Panel ...... 234 18.5 Other Kidney Tumours ...... 235 18.6 Follow-up and late effects...... 236 18.6.1 References...... 237 18.6.2 Chairs and members of the Late Effects Group ...... 238 19 Appendices ...... 239 19.1 Appendix 1: Contract form for participation in the SIOP 2016 UMBRELLA protocol ...... 239 19.2 Appendix 2: Information and Consent forms ...... 241 19.2.1 Parent / Guardian Information Sheet ...... 242 19.2.2 Information sheet for young adults ...... 247 19.2.3 Information Sheet for patients over 14 years and below 18 years ...... 252 19.2.4 Information Sheet for children aged 8 – 14 years ...... 257 19.2.5 Information Sheet to be read to children less than 8 years old ...... 260 19.2.6 Consent for enrolment in the UMBRELLA SIOP 2016 registry ...... 261 19.2.7 Consent for data storage and exchange ...... 262 19.2.8 Consent for storing and usage of biomaterial ...... 263 19.3 Appendix 2a: Information and Consent forms (Mother language version) ...... 264 19.4 Appendix 3: CRFs of SIOP 2016 UMBRELLA protocol ...... 265 19.5 Appendix 4: Technical details and guidelines regarding radiology ...... 267 19.5.1 Abdominal ultrasound ...... 267 19.5.2 Abdominal MRI ...... 267 19.5.3 Abdominal CT (only if MRI is not available) ...... 269 19.5.4 Chest CT...... 270 19.5.5 Chest X-Ray ...... 270 19.5.6 Optional diagnostic imaging...... 270 19.5.7 Imaging definitions ...... 272 19.5.8 Cutting Needle Biopsy ...... 273 19.6 Appendix 5: Details of pathology ...... 275 19.6.1 Histological classification ...... 275 19.6.2 Differential diagnosis of renal tumours of childhood ...... 284 19.6.3 Other tumours included in the study: ...... 285 19.6.4 Histological staging ...... 285 19.6.5 References...... 288 19.7 Appendix 6: Possible treatment regimen for Stage IV group D in exceptional cases ...... 290 19.7.1 High risk and high dose regimen (HR & HD) for group D patients ...... 290 19.7.2 References...... 292 19.8 Appendix 7: Integrated Research Projects and list of laboratories ...... 293 19.8.1 Wilms Tumour ...... 293 19.8.2 Non-Wilms Tumour ...... 300 19.8.3 Novel models ...... 301 19.8.4 Radiological Projects ...... 302 19.8.5 Radiotherapeutic Projects ...... 303 19.8.6 Surgical Projects ...... 303

UMBRELLA Protocol SIOP 2016

19.8.7 References...... 304 19.8.8 Table of laboratories doing molecular genetic analyses ...... 306 19.9 Appendix 8: Rules and regulations of SIOP-RTSG ...... 309 19.9.1 Structure of SIOP-RTSG ...... 309 19.9.2 Membership of SIOP-RTSG...... 315 19.9.3 International Data Centre ...... 315 19.9.4 Publication Policy and Authorship ...... 316 19.9.5 Biobanking and tissue sharing...... 318 19.10 Appendix 9: IT infrastructure ...... 321 19.10.1 Legal framework ...... 322 19.10.2 Virtual samples collection and storage ...... 322 19.10.3 Imaging tool for reference radiology ...... 322 19.10.4 Pharmacovigilance (SAE and SUSAR reporting) ...... 323 19.10.5 Statistics ...... 323 19.11 Appendix 10: Flowcharts and treatment schedules ...... 324 19.11.1 Localized Wilms Tumours (stage I – III) ...... 325 19.11.2 Metastatic Wilms Tumours (stage IV) ...... 326 19.11.3 Bilateral WT ...... 327 19.11.4 WT after primary surgery ...... 327 19.11.5 Relapsed WT ...... 328 19.11.6 Adults with WT ...... 330 19.12 Appendix 11: List of members of the different Subgroups ...... 331 19.12.1 Biology ...... 331 19.12.2 Radiology ...... 331 19.12.3 Surgery ...... 332 19.12.4 Pathology ...... 332 19.12.5 Radiotherapy ...... 333 19.12.6 Late effects ...... 333 19.12.7 Data management, Statistics and IT ...... 334 19.13 Appendix 12: Abbreviations ...... 335

UMBRELLA Protocol SIOP 2016

1 General introduction The main mission of the International Society of Paediatric Oncology (SIOP) Renal Tumour Study Group (RTSG) is to increase survival and to reduce acute treatment toxicity and late effects in all children, adolescents and young adults diagnosed with any renal tumour. In this context, SIOP-RTSG is aiming to offer all these patients the same standardized high quality diagnostics and treatment, independent of the tumour type, the socio-economic status or the geographic region where the patient is living. To achieve these goals, the UMBRELLA protocol was developed. In this respect kidney cancer in childhood will serve as a paradigm for other diseases, which is in line with the goals of SIOP Europe, ENCCA and ExPO-r-Net. Around 90% of paediatric renal tumour cases are nephroblastomas or Wilms tumours (WT). The other tumours comprise rare entities such as Clear Cell Sarcoma of the Kidney (CCSK), Renal Cell Carcinoma (RCC), Malignant Rhabdoid Tumors of the Kidney (MRTK), Congenital Mesoblastic Nephroma (CMN), and a few other, even rarer tumour types (Fig. 1).

Fig. 1: Distribution of renal tumours in childhood. (CMN: congenital mesoblastic nephroma, CCSK: Clear cell sarcoma of the kidney, MRTK: Malignant Rhabdoid tumour of the kidney)

Given the relative rarity of paediatric renal tumours and in particular rare subgroups, our previous studies demonstrated that it is necessary to recruit as many patients as are available at a population level. Over the last decades more than 10,000 children have been prospectively enrolled in SIOP Wilms Tumour studies and trials (Fig. 2). Since SIOP 93-01 SIOP-RTSG registered nearly 8,000 patients with a renal tumour from 261 centres across 28 countries. All of them have been treated according to consensus European trials and protocols. This has resulted in more standardised diagnostic procedures, improved risk stratification, and adjusted treatment recommendations for most renal tumours. The hallmark of the SIOP RTSG approach is the preoperative chemotherapy (Vincristine and Actinomycin-D in localized and with the addition of Doxorubicin in metastatic disease) without

UMBRELLA Protocol SIOP 2016 preceding mandatory histological assessment. This has the clear evidence-based benefit of down staging tumours, thereby sparing survivors the late effects of doxorubicin or radiotherapy by around 20% compared to patients treated with immediate surgery [1]. Nevertheless, this approach carries the risk of misdiagnosis (< 5%), as currently the so-called non-Wilms tumours cannot be identified by standard radiology or existing discriminating biomarker assessment.

Fig. 2: Number of patients enrolled in prospective SIOP Wilms Tumour trials.

The current SIOP 2016 integrated research and diagnostic UMBRELLA protocol (part A) serves as an entry for including all children with a renal tumour in Europe and other participating centres in the SIOP-RTSG. Subsequently, treatment of each participant’s renal tumour is recommended according to the SIOP 2016 treatment guidelines (part B), which provides treatment strategies for all WT patients and all children with other renal tumours. These recommendations are mainly based on the results from the previous SIOP and COG trials. According to the results of the recently closed SIOP 2001 trial all children with localized stage II and III intermediate risk tumours will receive no doxorubicin in the postoperative chemotherapy anymore as the new standard of care. The detailed clinical treatment guidelines and follow up protocols for all renal tumours in children and young adults are available to all participating partners. For nephroblastoma treatment guidelines are according to SIOP 2001 to allow a prospective validation of prognostic biomarkers like 1q gain and others.

UMBRELLA Protocol SIOP 2016

Steering Committee SIOP-RTSG and collaborating countries SIOP 2016 This proposal is prepared by the SIOP-RTSG Steering Committee (Table 1A) and by the chairs of each Sub-Committee (Table 1B).

Steering committee:

Name Country Email Speciality

Norbert Graf Germany [email protected] Paediatric (Chair) Oncologist Marry van den The Netherlands m.m.vandenheuvel-eibrink@ Paediatric Heuvel-Eibrink (Co- prinsesmaximacentrum.nl Oncologist chair) Arnauld Verschuur France [email protected] Paediatric Oncologists Christophe Bergeron France [email protected] Paediatric Oncologists Beatriz de Camargo Brazil [email protected] Paediatric Oncologist Kathy Pritchard-Jones UK [email protected] Paediatric Oncologist Filippo Spreafico Italy [email protected] Paediatric Oncologist Tomás Acha Spain [email protected] Paediatric Oncologist Harm van Tinteren The Netherlands [email protected] Statistician Anne Smets The Netherlands [email protected] Radiologist Jan Godzinski Poland [email protected] Surgeon Gordan Vujanic UK [email protected] Pathologist Ivo Leuschner Germany [email protected] Pathologist Christian Rübe Germany [email protected] Radiotherapist Manfred Gessler Germany [email protected] Biologist Richard Williams UK [email protected] Bioinformatician

Table 1A: SIOP-RTSG Steering Committee

UMBRELLA Protocol SIOP 2016

Chairs and Co-Chairs of various discipline groups (alphabetical order):

Name Country Email Speciality

Christophe France [email protected] Chair bilateral disease Bergeron Rhoikos Germany [email protected] Co-Chair stage IV WT Furtwängler Jan Godzinski Poland [email protected] Chair Surgical group Norbert Graf Germany [email protected] Chair RTSG Chair localised Wilms Marry van den The m.m.vandenheuvel-eibrink@ Co-Chair RTSG Heuvel-Eibrink Netherlands prinsesmaximacentrum.nl Chair non-WT Ivo Leuschner Germany [email protected] Chair Pathology group (research) Kathy Pritchard- UK [email protected] Chair Biology committee Jones Christian Rübe Germany [email protected] Chair Radiotherapy group Anne Smets The [email protected] Chair Radiology group Netherlands Filippo Italy [email protected] Chair relapsed Wilms Spreafico Harm van The [email protected] Chair Statistician group Tinteren Netherlands Arnauld France [email protected] Chair stage IV WT Verschuur Gordan Vujanic UK [email protected] Chair Pathology group (review process) Manfred Germany [email protected] Co-chair Biology Gessler committee

Table 1B: SIOP-RTSG chairs and co-chairs of Sub-Committees

UMBRELLA Protocol SIOP 2016

National coordinators:

Name Country Email Speciality Confirmed

Europe Leo Kager Austria [email protected] Oncologist yes Heidi Segers Belgium [email protected] Oncologist yes Jelica Predojevic Bosnia- [email protected] Herzegovina Srdjana Culic Croatia [email protected] yes Visnja Armanda [email protected]. Viera Bajciova Czech [email protected] Oncologist yes Republic Jon Helgestad Denmark [email protected] pending Catherine Rechnitzer Kadrie Saks Estonia [email protected] Marika Gronroos Finland [email protected] Oncologist yes Christophe Bergeron France [email protected] Oncologist yes Arnauld Verschuur [email protected] Norbert Graf Germany [email protected] Oncologist yes Apostolos Pourtsidis Greece [email protected] Oncologist pending Dimitrios Doganis [email protected] Helen Kosmidis [email protected] Gábor Ottóffy Hungary [email protected] Oncologist yes Sólveig Hafsteinsdóttir . Iceland [email protected] Filippo Spreafico Italy [email protected] yes Marika Grutupa Latvia [email protected] yes Elizabete Cebura [email protected] Rolanda Nemanienė Lithuania [email protected] Oncologist yes Eva Widing Norway [email protected] Oncologist yes Wojciech Pietras Poland [email protected] Oncologist yes Jan Godzinski [email protected] Surgeon Nuno Farinha Portugal [email protected] Oncologist yes Alexander Karachunskiy Russia [email protected] Oncologist pending Denis Kachanov [email protected] Margarita Belogurova [email protected] Dragana Janic Serbia [email protected] Ladislav Deak Slovakia [email protected] yes Simona Avcin Slovenia [email protected] Oncologist yes Tomas Acha Spain [email protected] Oncologist yes Niklas Pal Sweden [email protected] Oncologist yes Felix Niggli Switzerland [email protected] Oncologist yes Marry van den Heuvel- The m.m.vandenheuvel-eibrink@ Oncologist yes Eibrink Netherlands prinsesmaximacentrum.nl Rejin Kebudi Turkey [email protected] pending Kathy Pritchard-Jones UK [email protected] Oncologist yes

Africa Wael Zekri Egypt [email protected] Oncologist yes Ibraheem Abosoudah Saudi Arabia [email protected] Oncologist pending

Asia Matthew Shing HongKong [email protected] Oncologist yes Sajid S Qreshi India [email protected] yes Tsugumichi Koshinaga Japan [email protected] pending Mei Yoke Chan Singapore [email protected] yes Amos Loh [email protected]

UMBRELLA Protocol SIOP 2016

North America Ronald Grant Canada [email protected] Oncologist pending (Toronto)

Australasia Ross Pinkerton Australia [email protected] Oncologist pending Rob Corbett New Zealand [email protected] Oncologist pending Jane Skeen

South America Pedro Zubizaretta Argentina [email protected] Oncologist yes Beatriz de Camargo Brazil [email protected] Oncologist yes Milena Villarroel Chile [email protected] yes Luis Castillo Uruguay [email protected] Oncologist yes Table 2: National coordinators. (pending: decision will be done after a Meeting of the National Renal Tumour Group

A list of contact persons per country and a list of responsible pathologists, radiologists, surgeons, radiotherapists and biologists, are provided by the different Sub-Committees and is provided as Appendix 10. This information is also regularly updated at our website in the Intranet (http://siop- rtsg.eu).

The EudraCT number of the UMBRELLA protocol is: 2016-004180-39

UMBRELLA PROTOCOL SIOP 2016 Part A

Part A: Evaluation of tissue, imaging and biomarkers to optimize risk stratification

UMBRELLA Protocol SIOP 2016

2 Introduction UMBRELLA protocol SIOP 2016 The UMBRELLA protocol is based on the UK IMPORT (Improving Population Outcomes for Renal Tumours of Childhood) 2013 protocol, version 4.0 September 2013 and the experiences from SIOP 2001 [2]. The study will identify and refine new (bio-)markers (molecular, histopathological, and imaging defined) for the management of all children, adolescents and young adults with WT and other ‘non- Wilms’ renal tumours of childhood. Hence, this protocol will lay the foundation for better stratification parameters for upcoming SIOP-RTSG clinical trials. This will be important for subgroups of patients with a poor but also for those with an excellent prognosis to adapt treatment accordingly, which requires a patient centered assessment of clinical, molecular data, histopathological and imaging. Such a strategy needs increasing parental and patient involvement. In addition based on recommendations, there will be ‘real time’ central reviews of imaging studies (DICOM files) and pathology to standardise assessment of diagnosis and response to treatment and to minimize the risk of suboptimal stratified patients. Furthermore, the feasibility of integrating all of these complex datasets within a newly developed e- health tool project known as “p-medicine” [3], which is designed to improve clinical decision making in future clinical trials, will be explored in the German speaking countries (Germany, Switzerland and Austria) against the backbone of the ALEA data management system that will be applied in all other countries as well. It is advised that patients registered in this study should be treated according to recommendations given in part B of the UMBRELLA protocol SIOP 2016.

3 Overall aim of the UMBRELLA protocol SIOP 2016 The overall aim of the SIOP 2016 UMBRELLA protocol is to harmonize the clinical relevant standard diagnostic procedures for all paediatric renal tumours within SIOP and to provide imaging studies and biomaterial from all of these patients, to find new and better risk factors for treatment stratification and molecular targets for novel therapeutic approaches. This will help to improve short and long term outcomes for all children with renal tumours through the introduction of a more ‘personalised’ approach.

UMBRELLA Protocol SIOP 2016

3.1 Primary aims 1. To show the feasibility of storing serial blood, urine samples, tumour and germline material at diagnosis and at specific time points during treatment for international collaborative studies. These will be used to validate and quantify in multivariate analysis, the relative adverse prognostic significance of specified somatic molecular biomarkers (listed in aim 2) in relation to blastemal volume (aim 3). They will also be used for exploratory analyses of potential novel biomarkers, including circulating nucleic acids detectable in blood and urine, for diagnosis and prognosis.

2. 1q gain and genomic copy number biomarkers;

 To assess genomic 1q gain as a prognostic biomarker in WT.  To analyse other genomic copy number biomarkers that may have potential prognostic relevance in WT: o Simultaneous allele loss of 1p and 16q o MYCN gain o 17p loss encompassing the TP53 locus.  To test the feasibility of returning biomarker results to treatment centres within a clinically relevant time frame.

3. Volume of blastema: To optimise the definition of high risk WT, ‘blastemal type’ through:

 Accurate measurement of the residual blastemal cells volume including centralised ‘real time’ pathology and radiology review.  Correlation of blastemal cell volume and corresponding biomarkers with event free and overall survival.  Assessment of the molecular characterisations of those that meet the current definition (crude proportion) of ‘blastemal type’ or that of relapse.

4. Radiology review: To optimise radiological diagnostics by (real time) central review to:

 Monitor and give appropriate feedback on diagnostic imaging quality.  Harmonise diagnostic procedures and standardise reporting of radiology findings.  To stratify treatment based on central radiological review.

5. Pathology review: To optimise pathological diagnostics by (real time) central review to:

 Monitor and give appropriate feedback on local pathological diagnosis.  To stratify treatment based on central pathological review.  To store biological material according to the guidelines given in chapter 7.3

UMBRELLA Protocol SIOP 2016

3.2 Secondary aims 1. To explore whether aberrations in any or a combination of the following genes have a significant impact on event-free or overall survival WT: WT1, CTNNB1, AMER1, TP53, MYCN, FBXW7, GPC3, MLLT1, DIS3L2, DICER1, DROSHA, DGCR8, SIX1 and SIX2. 2. To explore the role of miRNAs in blood and tumour as biomarkers for kidney tumours. 3. To establish a surgical review process for guiding local centres in performing nephron sparing surgery (NSS) in uni - and bilateral WT or minimal invasive surgery in unilateral renal tumours. 4. To perform explorative epidemiological analyses of the collected data. 5. To validate new tools developed within the e-Health project such as p-medicine [4] and similar projects, to improve clinical treatment decision-making based on integrated risk assessment and response modelling in silico, in a selected well documented large subset of patients. The validation will be performed against the background of the ALEA registered complete dataset. 6. To characterise at a molecular level all subtypes of WT and non-WT and their associated nephrogenic rests, using whole genome, epigenomic and proteomic approaches. 7. To assess the feasibility of developing a targeted ‘next generation’ sequencing panel for WT and other childhood renal tumours. 8. To contribute to the development of ex vivo models for functional validation of newly discovered molecular aberrations and biomarkers.

3.3 Expected impact 1. To conclude whether 1q or other biomarkers is a clinically relevant independent prognostic factor for WT. The results will be considered in the design of future clinical trials, where 1q gain may be tested as a biomarker for use in treatment planning. 2. To obtain a better definition of high risk ‘blastemal type’ and the prognostic value of residual blastemal volume after pre-operative chemotherapy. 3. To conclude whether gain of 1q, residual blastemal volume or both factors should be included in risk stratification in the design of a future clinical trial, taking account of current prognostic factors (tumour stage and histological risk group) and the age of the patient. 4. Improved infrastructure for data and samples collection, in due time resulting in better and complete data and more biomaterial for translational research. 5. Improved central radiology, pathologic and surgical review resulting in standard diagnostic procedures and treatment for all paediatric renal tumours 6. Strengthened translational research in paediatric renal tumours by fostering networking between basic and clinical scientists to enhance the portfolio of novel therapeutic approaches for children and adolescents with renal tumours. 7. Provide a better risk stratification for upcoming prospective and randomized the SIOP-RTSG trial for renal tumours. 8. Provide the biological rationale for at least one phase I/II trial of a targeted therapy approach for relapsed patients.

UMBRELLA Protocol SIOP 2016

4 Background and rationale of the UMBRELLA protocol SIOP 2016 The background and rationale of the SIOP 2016 UMBRELLA protocol is based on the results of former SIOP, UKW, AIEOP and NWTSG/COG trials. The overall outcome of patients with nephroblastoma is excellent, but there are still subgroups of patients with unacceptable numbers of relapses and deaths. Without the addition of biological markers and imaging features as stratification parameters, further improvement of outcome cannot be gained. Thus, further knowledge needs to be generated from imaging, pathological and biological studies. The seamless sharing and joining of this data is mandatory as well as refining logistics for the availability of high quality data and information from reference centres of radiology, pathology, surgery and radiotherapy in due time. New ways of data analysis need to be established to select those biomarkers and imaging features that will have the highest impact as new stratification parameters for subsequent randomized clinical trials. The provision of an integrated IT-infrastructure as well as the inclusion of bio-informaticians in this process is mandatory.

4.1 Molecular biology 4.1.1 1q gain as a prognostic biomarker in WT Approximately 28% of WTs have 1q gain. Several relatively small retrospective studies have suggested that 1q gain is associated with poor outcome in WT [4 – 9]. More recently, the SIOP-RTSG has analysed 586 WT samples [10] from the SIOP WT 2001 clinical study, and also concluded that 1q gain is associated with significantly poorer event-free survival (hazard ratio >2.3) in patients treated with pre-operative chemotherapy. A comparable study conducted by the Childhood Oncology Group [8], using samples from the North American NWTS-5 study, has reached similar conclusions for patients treated by immediate nephrectomy. There is therefore a compelling argument to assess this biomarker prospectively in an independent sample series. Therefore, UMBRELLA will include 1q copy number analysis of all available WT samples. A multiplex ligation-dependent probe amplification (MLPA) assay was developed by the SIOP-RTSG in association with MRC-Holland b.v. (Amsterdam) for the previous copy number study, and is now available commercially (P380 Wilms Tumour probe mix). This assay has the advantages of low cost, rapid turnaround, direct comparability with existing data, and compatibility with both frozen and paraffin- fixed material. It is able to provide biomarker data in a clinically relevant timeframe, and can potentially be implemented using equipment and expertise available in a routine diagnostic laboratory. The probe mix also detects copy number aberrations at other loci of interest in WT. It is currently intended that MLPA will be the primary 1q gain assay for UMBRELLA, both to generate study-wide data, and to test the feasibility of returning results to treatment centres quickly enough to inform treatment decisions. However, higher resolution genomic technologies are evolving rapidly, provide much more genome- wide data, and are becoming increasingly affordable. Thus, new methods may be adopted during the lifetime of the protocol.

4.1.2 Other genomic biomarkers In addition to 1q gain, several other genomic copy number biomarkers have potential prognostic relevance in WT. Simultaneous allele loss of 1p and 16q has been shown to be associated with poor outcome in COG cases treated by immediate nephrectomy [11], though the relative rarity of this aberration limits its usefulness. MYCN gain was recently found to be associated with adverse outcome in

UMBRELLA Protocol SIOP 2016

a retrospective SIOP-RTSG study [12]. 17p loss encompassing the TP53 locus, usually concomitant with point mutation of the other allele, appears to be associated with poor outcome in anaplastic tumours [13]. With the exception of TP53, mutations in individual known WT genes have not generally been associated with adverse outcome. However, initial studies suggest that cases in which both the SIX1/SIX2 and the microRNA processing pathways are targeted by mutation have particularly poor outcome at least in one of the two studies [14, 15]. A large-scale comprehensive survey of single nucleotide variants and small indels in all genes known to be frequently mutated in WT, including WT1, CTNNB1, AMER1, TP53, MYCN, FBXW7, GPC3, MLLT1, DIS3L2, DICER1, DROSHA, DGCR8, SIX1 and SIX2, will allow us to detect whether aberrations in any of these genes, alone or in combination, have a significant impact on event-free or overall survival. A targeted ‘next generation’ sequencing panel for WT is currently in development by the UK SIOP-RTSG in association with the Crick Institute. It is intended that this panel will be suitable for genomic DNA extracted from both frozen and paraffin-fixed material, and should therefore be widely applicable across the UMBRELLA study.

4.2 Pathology and Blastemal volume All renal tumour slides will be reviewed on a national/regional level and peer reviewed by the international SIOP RTSG pathology panel.

Fig. 3: Comparison of local and central pathology (GPOH data of SIOP 93-01 and 2001)

UMBRELLA Protocol SIOP 2016

The introduction of central pathology review (CPR) has played an important role in correctly diagnosing and staging specific subtypes of WT (fig. 3). Also, less frequently occurring renal tumours will be diagnosed in a real-time setting allowing timely counselling and intensifying (CCSK, MRTK), or alternatively de-intensifying (CMN) treatment. The improvement in the quality of material submitted for CPR, mainly due to the introduction of simple standard operating procedures (please see Pathology protocol, chapters 7.2, 11.8, 19.5) and the introduction of rapid (‘real-time’) CPR have contributed to decreased discrepancies between CPR and local pathologists. “Real-time” CPR allowed clinicians to modify treatment (if required) within a clinically acceptable timeframe. The overall and event-free survival of the majority of renal tumours is excellent. However, nearly 40% of all relapses occur in children whose tumours were not classified as high risk at diagnosis. Blastemal type nephroblastoma represents a subtype which has already been shown to benefit from intensive treatment in SIOP 2001 [16, 17]. This highlights the need to identify ‘high risk’, chemo resistant blastema in order to avoid unnecessary failure of first line therapy. Hence, the aim of UMBRELLA is to optimise the definition of high risk, ‘blastemal type’ Wilms tumour, which is currently defined according to the crude proportion of resistant blastemal cells that survived pre-operative chemotherapy. The UMBRELLA protocol will consider ‘absolute blastemal volume’ and standardize the accurate measurement of the volume of residual blastemal cells in all WT in patients receiving pre-operative chemotherapy. A new retrospective analysis of SIOP 2001 trial data has shown that a threshold of between 20-50 ml residual blastemal volumes in localised WT could be used a potential new stratifying biomarker. In addition, a detailed quantification of residual blastema needs more objective criteria to minimize inter-individual variation and to allow local pathologists to make a correct diagnosis [16]. Furthermore, the blastemal volume threshold at which the risk of relapse rises sharply enough needs to be confirmed and refined drivers and markers. In addition, UMBRELLA will oversee the molecular characterisations of this group of chemo-resistant blastemal tumours to discover their biological drivers and markers. Ultimately, this will lead to more objective criteria for identifying high-risk blastema.

4.3 Radiology Over recent years radiological developments in tumour imaging did improve anatomical depiction and assessment of tumour composition. However, standardisation and quality improvement of imaging studies are still needed in multicentre trials and studies. Up to now imaging hass not been able to differentiate benign from malignant small lung nodules nor round nephrogenic rests from nephroblastoma. The North-American Children’s Oncology Group (COG) and Gesellschaft für Pädiatrische Onkologie und Hämatologie (GPOH) group reported a discrepancy rate between local image interpretation and central radiology review of around 15% with the down staging of lung involvement and upstaging of local disease in renal tumours in a significant proportion of imaging studies [18]. Therefore, the UMBRELLA protocol introduces a centralised radiology review of imaging studies on a nationwide basis. The IT infrastructure will facilitate secure uploading of DICOM files on a centralized DICOM server for radiological review in due time for decision support, mainly to judge if the imaging is compatible with a renal tumour and if the tumour is localized, metastasized or bilateral to start with the correct preoperative chemotherapy or to go to core needle biopsy or primary surgery in case of doubt of diagnosis. Secondly, response to preoperative chemotherapy needs a uniform assessment for all patients done by the radiology reviewers. After reviewing for clinical purposes the imaging files will be pseudonymized/anonymized and uploaded into the data warehouse, where they will be available for further research. In addition specific tools like DoctorEye [19] a novel, open access

UMBRELLA Protocol SIOP 2016

interactive platform for 3D medical image analysis, simulation and visualization, focusing on oncology images will be made available While chest X-ray and US scans were the standard of care for SIOP 2001 trial, a variety of improved imaging studies have been implemented into clinical practice in paediatric cancer patients today. CT scan of the chest and MRI of the abdomen have gradually become standard in most but not all countries. CT is the most sensitive imaging for the detection of very small pulmonary lesions but carries a much higher burden of ionising radiation exposure compared to chest X-ray [20, 21]. Recent literature suggests that the detection of small pulmonary lesions, not detectable with chest X-ray (CT-only lesions), might be of benefit for refinement of treatment for those particular patients [22, 23]. It could potentially lead to high evidence supporting a rationale for avoiding whole lung radiation. On the other hand, there is the potential of over-diagnosing patients with metastatic disease, as not all lung lesions are metastases [24].

Techniques such as Diffusion Weighted Imaging (DWI) with apparent diffusion coefficient (ADC) mapping [25], can be added to the classical sequences at diagnosis and at pre-operative assessment to increase conspicuity for small lesions, both Wilms’tumour and nephrogenic rests, and possibly (research) to get the prediction of histological risk group by providing an estimate of the cell density of the tumour tissue and thus information on apoptotic effects of chemotherapy [26 - 28]. Preliminary findings of serial assessment of abdominal paediatric cancers, including 4 Wilms tumour patients (2 with bilateral tumours), on a standard 1.5-T clinical MRI scanner have been published [29]. By recognising patterns of shift in the ADC distribution between the primary (diagnostic) scan and the post- chemotherapy scan, areas of necrosis and of residual viable tumour were defined, which correlated well with the histology of the resected specimen. Such results need to be validated by a large cohort of patients to clearly show the benefits of this technology for better and earlier characterization of paediatric renal tumours. In combination with biological markers such results may allow a more individualized therapy prior to surgery. New guidelines for the performance of abdominal MRI have been developed in a two-tier system: 1. Standardized anatomical imaging for initial diagnosis including 3D volumetric assessment of response to chemotherapy and surgical road-mapping, 2. Quantitative imaging by exploring DWI of abdominal MRI as a non-invasive biomarker, with the potential to refine risk-stratification and help individualise care. All renal tumour patients will benefit from central radiology review. It is expected that tier 1 is implemented as a standard of care, replacing abdominal CT in participating centres with ready access to MRI. Tier 2 will be implemented as a clinical research effort by centres where the required acquisition is possible and deemed feasible by the local radiologist. The assessment of imaging data will be performed in conjunction with pathological, molecular and proteomic data. The central radiology review will be done on a national level. Quality control and independent quality check will be performed by the exchange of images of selected cases within the panel of radiologists. The feedback from pathology in every case is essential to develop accurate criteria for tumour diagnosis and the correlation between lung lesions and histology in case of surgical excision of such lesions.

UMBRELLA Protocol SIOP 2016

5 Samples collection and storage Sample collection and storage have been pursued in SIOP 2001 but up to now, no virtual database is established to collect the information from all registered patients in SIOP-RTSG about stored biomaterial. In SIOP 2001, only half of all patients had biomaterial collected. The diagnostic and molecular research of the SIOP 2016 UMBRELLA protocol will include all paediatric renal tumour types, including non-WT, i.e. MRTK, RCC, CMN, and CCSK. It is critical to store biomaterial (including tumor, normal kidney tissue, urine) from all registered patients and germline DNA of the patient and if possible from parents as well. The quality of the biomaterial is of utmost importance. We provide guidelines for optimal retrieval, transport and storage of samples that needs to be set up in all participating countries/centres. Given the heterogeneity of kidney tumours it is recommended that biomaterial from different tumour areas is stored for each patient. Each of these specimens needs a mirror block to be analysed by pathology to allow the correct correlation between histology and corresponding molecular findings. Only biomaterial where the described quality control measures are fulfilled can be fully used for analysis. Such an optimisation is necessary to rely on results from molecular analysis. If the goal is to find new biomarkers for diagnosis and treatment stratification, adherence to such quality criteria is mandatory. A limited number of participating centres with appropriate research nursing expertise and capacity will collect blood and urine samples at serial timepoints from diagnosis through to end of treatment. These will be stored and used for analysis of circulating nucleic acids and proteomics/metabolomics. Different biomarker analyses will be led at a national level by respective research groups with established methodologies for analysis of cell-free circulating DNA, miRNAs, proteomics/metabolomics in serum/plasma or urine (see table X See section 7.3 for further details of sample processing logistics.

5.1 Further usage of biomaterial in upcoming research questions The analysis of biomaterial samples will continue beyond the period of the SIOP 2016 UMBRELLA protocol as these samples and their associated data will be used to further identification of additional biomarkers by NGS and usage of ex vivo expanded models such as xenografts and organoid models. For that purpose all data will be pseudonymized and stored in the data warehouse of SIOP-RTSG. In cases where valuable research materials (tumour tissue, normal renal tissue, urine and blood) remain after analysis within the SIOP 2016 UMBRELLA protocol, and in addition patients, parents or guardians have consented for inclusion of their samples in a SIOP-RTSG tissue bank, then these samples will be used for answering new research questions under the agreed legal framework of the relevant national childhood cancer tissue collection and storage.

6 Patient enrolment Every hospital that wants to participate in the SIOP 2016 UMBRELLA protocol needs to be a member of a National Group registered in SIOP-RTSG or - in the absence of a National Group - a single centre adhering to the ‘Structures and Standards’ given by the SIOP-RTSG. Each participating centre needs to register every patient with a renal tumour and provide information about responsible persons (paediatric oncologist, radiologist, pathologist, radiotherapist, surgeon) dealing with these patients and all of them have to sign that they do adhere to the UMBRELLA protocol and that they provide all requested material (imaging, pathology, biomaterial) and data.

UMBRELLA Protocol SIOP 2016

Minimal requirements for participating as a partner institution are 1. Providing full data sets by registration in ALEA/ObTiMA 2. Organised pathology review on a national / regional level 3. Organised radiology review (national / regional level) 4. Abdominal MRI (alternatively CT abdomen) and CT scan of the chest as a standard diagnostic approach for each child and for assessment of response in patients with metastatic tumours Countries that can only meet criteria 1 and 2, are welcome to register patients and to use the therapeutic guidelines. For each patient quality criteria are collected for further analysis. Central pathology review is mandatory for inclusion in any kind of analysis. Further analyses will not be possible due to selection bias if a central radiology review of images is not done.

Requirements for enrolment in the final analysis of research questions 1. In all countries with functional biobanking structures the submission of at least one sample of frozen tissue with corresponding control (blood or adjacent normal kidney in case of nephrectomy) is mandatory. In countries that do not yet have this infrastructure the minimal requirement is the submission of a representative paraffin block of tumour and matching normal kidney for biology studies. However, it is strongly recommended that biobanking is organized on a national level (fresh frozen tumour tissue as well as normal renal tissue, peripheral blood and urine, including also blood/serum from parents). Centers from countries with existing biobanking structures will provide support to guarantee the collection of materials at the best level possible. 2. Abdominal DWI-MRI at diagnosis and after preoperative chemotherapy

The requested form is given on the next page (page 26):

UMBRELLA Protocol SIOP 2016

Participation in the UMBRELLA protocol Centre Name Participant of the Address / Country following National Telephone / Fax Group: Email Herewith our centre confirms by signature that 1. We adhere to all regulations of the protocol 2. All patients with a renal tumour diagnosed at our centre will be enrolled in the UMBRELLA protocol if the patient or the parents or the legal representative provides informed consent 3. All requested baseline and serial information and data, imaging studies, biomaterial and pathological material will be provided from all patients National Principal Investigator Signature Name Address / Country Telephone / Fax Email Responsible Oncologist Signature Name Address / Country Telephone / Fax Email Responsible Radiologist Signature Name Address / Country Telephone / Fax Email Responsible Surgeon Signature Name Address / Country Telephone / Fax Email Responsible Pathologist Signature Name Address / Country Telephone / Fax Email Responsible Radiotherapist Signature Name Address / Country Telephone / Fax Email Responsible Biologist or external Signature biobanking contact Name Address / Country Telephone / Fax Email Responsible Data Manager Signature Name Address / Country Telephone / Fax Email

UMBRELLA Protocol SIOP 2016

6.1 Inclusion criteria All children, adolescents or young adults with a primary or relapsed renal tumour diagnosed in a participating SIOP-RTSG centre are eligible for inclusion in the SIOP 2016 UMBRELLA study. All registered and participating centres in the SIOP-RTSG study group have to enrol all their patients with renal tumours into this protocol, if the patient or the parent or the legal representative (guardian) gives informed consent. Consent needs to be given separately for the enrolment in the protocol, the sharing of data and the biological studies related to the protocol. The inclusion of patients is independent of the histology of the renal tumour, the age of the patient (except for RCC patients: <18 years old) or the country of residence. All participating centres agree to be compliant with the protocol (see form above) and to provide all requested material including imaging studies and biomaterial, data and other necessary information of all their enrolled patients. This includes also follow-up information on at least a yearly basis. Recruitment of patients is aimed to be complete in all participating centres with the provision of complete data sets and requested imaging and biomaterial.

6.2 Exclusion criteria The only exclusion criterion is missing informed consent. Patients who do not give or whose parents or guardians do not give consent for inclusion in the SIOP 2016 UMBRELLA study cannot be registered. As there are different consents for participation in the clinical and the biological part of the UMBRELLA protocol patients can be potentially registered if there is consent given for the clinical part only and not the biological part of the protocol. Each country in accordance with local regulations rules will provide consent forms in their language.

7 Centralized review (CR) 7.1 Central Radiology Review (CRR) With regards to imaging, the main consequences of the study are 1. To increase image quality and achieve technical uniformity 2. To centrally review all images at diagnosis and pre-operatively so that there is a standardised approach to definition of metastatic disease and response in relation to risk stratification and application of the clinical guidelines. The central review is mandatory for chest CT 3. Emphasis on quantitative (“functional”) MRI [opt-in for sites] and 4. Uploading of pseudonymised imaging to a rsearch data warehouse. The aim of CRR is to confirm the suspicion of a renal tumour and if it is bilateral and/or metastatic. In addition we will evaluate and improve image quality by giving feedback to the individual centers. Fully detailed radiological guidelines are made available as part of the protocol and can be seen in section 11.7. This will provide guidance on all radiological examination procedures and will be updated as required. Preferably an independent radiologist should do CRR.

7.1.1 Time points for CRR  Diagnosis: abdominal MRI (or CT), chest CT  Pre-surgery: abdominal MRI (or CT) and chest CT (only for Stage IV)

UMBRELLA Protocol SIOP 2016

 Week 9 of postoperative treatment depending on the treatment protocol followed (Non-CR in Stage IV)  At the end of treatment (Stage IV): chest CT only if no CR before  In any case of suspected recurrence

7.1.2 Central radiology review Each national centre needs to determine where central radiology review will be done in order to analyse the requested imaging studies in a standardized way to take part in the SIOP 2016 UMBRELLA protocol. Independent expert radiologists should do the second view. National/Regional Chairs for Central Radiology Review are the following: AIEP (Italy) Carlo Morosi Austria Karoly Lakatos Belgium Luc Breysem CCLG (UK) Oystein Olsen DCOG (The Netherlands) Anne Smets (chair) GBTR (Brazil) Henrique Lederman GPOH (Germany) Jens-Peter Schenk NOPHO (Scandinavian countries) Lena Gordon (Sweden) Poland Dorota Sosnowska SEHOP (Spain) Ana Coma SFCE (France) Hervé Brisse Switzerland Christian Kellenberger SIOP (all other countries) NN This list will be regularly updated and will be found in the Intranet of the SIOP-RTSG website. Data transfer: Local imaging-data will be transferred over an easy to use and secure IT-Infrastructure to the respective reference centre. Mail-born solutions are acceptable but 2nd choice (Delay, technical problems concerning the readability of DVD/CD). Finding: Will be accessible in the system. A notification mail will be sent to the respective collaborating centre. Fax-based response will be 2nd choice only for those centres where an IT solution is not possible. It is the goal to have a 2nd opinion within 72h – 96h excluding weekends and National holidays.

7.2 Central Pathology Review (CPR) For the purposes of this study, rapid or ‘real-time’ Central Pathological Review (CPR) has been defined as a review that takes place within 14 days after nephrectomy. This should be done by National/regional Pathology Panels (see below) and it will allow communicating the results of CPR back to the institutional team before decisions on postoperative treatment are implemented. According to the SIOP treatment guidelines, postoperative chemotherapy should start within 21 days of the last dose of preoperative chemotherapy [31]. The institutional pathologist will need to sample the tumour according to the protocol, record percentage of necrosis/chemotherapy-induced changes (after macroscopic and microscopic review) and an estimated percentage of residual blastema (and other tumour components) in the viable part of the tumour. He/she will also need to accurately stage the tumour according to the

UMBRELLA Protocol SIOP 2016

SIOP staging criteria. The primary handling of the specimen is described elsewhere (chapter 11.8.1). SIOP Pathology Panel will periodically do review of cases submitted to national/regional Panels. Each national/regional centre needs to provide rapid central pathology review in order to take part in the SIOP 2016 UMBRELLA protocol. The National/Regional Chairs for Central Pathology Review are the following:

AIEP (Italy) Paola Collini CCLG (UK) Gordan Vujanic DCOG (The Netherlands) Christina Hulsbergen-van de Kaa GBTR (Brazil) Isabela Werneck Cunha GPOH (Germany) Ivo Leuschner NOPHO (Scandinavian countries) Ivo Leuschner SEHOP (Spain) Enrique de Alava SFCE (France) Aurore Coulomb SIOP (all other countries)* Gordan Vujanic / Ivo Leuschner

For central pathology review all digital images of macroscopical examination, a complete set of H&E stained slides, a paraffin block of a representative area and a block guide should be submitted. Both slides and blocks will be kept in the archives of the national/regional review centre according to the national rules for tissue preservation of each participating country.

Nephrectomy

Instititutional pathologist does cut up according to the proposed Protocol, writes his/her report, and sends the case without any delay F4 Form for Rapid Central Pathology Review

Rapid Central Pathology Review done promptly by National /

Regional Pathology Panel and the result sent back to the F15 Form

institutional pathologist (within 1 week)

SIOP Pathology Panel review cases from National / Regional centres

F16 Form retrospectively

UMBRELLA Protocol SIOP 2016

7.3 Central Biology Logistics For biomarkers to be clinically useful in the future, they must be assessed within real time such that results can be fed back to the clinical team for consideration in treatment decisions in a future clinical trial. This study will analyse these logistics and aims to validate the prognostic value of various biomarkers identified in analyses of samples from the previous SIOP WT 2001 trial. We will also assess the feasibility of biomarker profiling of tumours within 3 weeks of surgical excision. In order to achieve this, a workflow for the submission of samples to the reference laboratory has been developed and is shown in Fig. 5.

7.3.1 Biological sample collection, processing and storage Clinical data and biological samples will be collected on a national basis from the Paediatric Oncology Centres where the majority of children are treated. Each country needs an established network which is very experienced in the provision of biological samples for the purposes of tissue collection and research. Such a network submitting frozen tumours samples from ~80% of children registered in their countries in the previous SIOP WT 2001 is already established in the UK, Germany, The Netherlands and France, and a similar structured biological sample collection has been in place in Italy since the period of the AIEOP-TW-2003 protocol. The current proposal aims to include samples from all patients per country as well as more countries that will be able to provide the required samples and data.

Reliable and reproducible results are entirely dependent on and can only be accomplished by standardisation of the methods of biological sample collection, handling, processing and transport by the use of standard operating procedures (SOPs). Such SOPs and/or guidelines will be provided to all participating centres.

Frozen tumour from up to 3 spatially distinct sites, matched normal kidney, and (where available) needle core biopsies from the time of initial diagnosis are required from all newly diagnosed patients at participating centres immediately after nephrectomy. All samples will be assessed for tumour cell content, and the DNA derived from them subjected to stringent quality control. We will determine the feasibility of isolating, assessing and reporting on nucleic acids from tumour at all participating laboratories within 2 weeks of nephrectomy, to confirm that the rapid sample collection and processing system put in place has the potential to provide assayable material within a clinically relevant timeframe. Corresponding clinical data will be collated for each patient, including histology of mirror blocks, overall review histopathology and tumour volume measurements, allowing calculation of total residual blastemal volume. 10% of samples will be submitted to a blinded quality assurance scheme with partner laboratories in other countries following the SIOP treatment protocols.

Sample collection

Tumour, control tissue and biological fluids must be processed, rapidly frozen and stored appropriately under controlled conditions according to the relevant laboratory manual. Snap freezing of tissue must be done as soon as possible after excision to ensure that there is minimal sample degradation and therefore no limitation on the types of studies that can be undertaken or any undue influence on the usefulness of data obtained from sample analysis. An updated protocol for sample collection is included (additional information figures. 4 and 5).

UMBRELLA Protocol SIOP 2016

In Wilms tumour, formalin fixed paraffin embedded (FFPE) tissues are a particularly valuable resource for applications including the molecular analysis of micro-dissected nephrogenic rests [30], tumour sub compartments, and anaplastic foci. An updated protocol for the collection of FFPE tissue is included (fig. 10). The serial collection of urine and blood during treatment for assessment of circulating biomarkers will be performed at participating centres with capacity to collect and process these samples. Collection of these bio-fluids is undertaken during normal visits to hospital. Ethical approval for the additional samples of blood and urine for research is included in the consent process for all patients.

It is important that all cases of surgery on metastases or later relapse are collected using the same protocol as such samples are extremely valuable to follow the evolution of tumors that led to a gain in therapy resistance and metastatic potential.

Tissue sample processing and storage for research Tumour tissue should be collected at diagnosis if a biopsy is performed or post-nephrectomy. 1) Snap freezing preserves the tissue integrity permitting the demonstration of features that would otherwise be lost. 2) Using a fresh sterile scalpel and forceps for each sample to avoid cross-contamination, dissect individual 1cm3 specimens from at least one and preferably two or three areas of the sectioned tumour (especially when these areas are macroscopically distinct). 3) Whenever possible, dissect an equivalent specimen from a macroscopically normal, tumour-free area of the kidney. 4) Bisect each sample and transfer half to a standard cassette for formalin fixing and paraffin embedding by standard protocols, and 5) Immediately snap freeze the remaining half. 6) Snap freezing should ideally take place in liquid nitrogen, with each sample contained in a fresh disposable polypropylene beaker within the working nitrogen vessel to avoid cross- contamination. 7) Centres participating in the additional studies to analyse serial timepoints of blood and urine for circulating biomarkers should follow the relevant laboratory manual.

UMBRELLA Protocol SIOP 2016

Fig. 4: Proposed pathway for biological specimen collection. FFPE: formalin fixed paraffin embedded.

7.3.2 Fresh Tissue Sampling for cell culture (limited centres only) As part of the continual development of methodologies and to ensure we make the best use of sample resources, selected centres will collect different pieces of tumour tissue immediately after nephrectomy and place this in a falcon tube in PBS at 40C for primary tumour culture and other investigations. The sample should be kept at 40C until collection. The sample SHOULD NOT be frozen or subject to temperatures below 40C. Samples should be sent on the same day that it is taken to the national samples collection and storage laboratory, according to the detailed laboratory manual to be supplied to the selected participating centres.

7.3.3 Blood Sample collection in MRTK Since patients suffering from MRTK might bear germ-line mutations of SMARCB1, SMARCA4 or other INI 1 involving genes, such molecular genetics analysis is mandatory at least in the blood of patients younger than 2 years of age and their parents. In addition counselling by a human geneticist is recommended after histological diagnosis of a MRTK. In all patients younger than 2 years of age it is recommended after histological diagnosis of a MRTK.

UMBRELLA Protocol SIOP 2016

7.3.4 Renal tumour biology study specimen collection protocol

Fig. 5: Workflow for submission of tissue samples.

UMBRELLA Protocol SIOP 2016

7.3.5 Analysis and quality control of DNA All DNA, RNA, miRNA and protein samples used in these analyses will be subjected to stringent quality control. In case of tumour samples tumour content will be assessed by pathologists in a thin section immediately adjacent to the material from which DNA was extracted. Exome capture and next generation sequencing technologies, as well as the tools used to analyse the data, are evolving rapidly, and alternative methods may be used if they provide significant benefits to the project (e.g. better coverage or more accurate variant calls).

7.3.6 Urine Sample Requirements (for full details, participating centres should refer to the relevant Laboratory Manual) The collection of urine samples particularly in very young children can be difficult and time consuming. Asking for parental assistance and providing collection tubes/bags/pads to parents may assist the process. 1. Urine will be collected at the time of the child’s visit to the hospital so that it can be processed immediately after collection. 2. Expected volume should be more than 4 ml, preferably collected in urine bag and decanted into centrifuge container. 3. If equipment is available the sample should be centrifuged at 5000 rpm for 10 minutes. The supernatant should be decanted into a new cryovial, being careful not to disturb the pellet. 4. The supernatant sample and the particulate sample should be carefully labelled and stored at - 80°C. If no suitable centrifuge is available sample can be stored directly at -80°C. Please indicate on sample sheets that the sample has NOT been centrifuged. 5. Depending on each national coordinating center, samples can be either sent directly or stored on site until tissue samples are available for a batched collection and sent to the national biobank together with tissue samples.

7.3.7 Blood Sample Guidelines (for full details, participating centres should refer to the relevant Laboratory Manual) Overall collect 5 ml blood for DNA, miRNA and tumour auto antigen analysis as follows: 1. Draw blood into vacutainer tube(s) ‘Sarstedt pink capped 5.0 ml EDTA tube’ (or equivalent manufacture). Draw the full volume to ensure the correct blood to anticoagulant ratio. 2. Invert vacutainer tubes carefully 10 times to mix blood and anticoagulant and keep at room temperature until centrifugation. 3. Samples should undergo centrifugation immediately. This should be carried out for a minimum of 10 minutes at 1000. Do not use brake to stop centrifuge. 4. This will yield three layers: (from top to bottom) plasma, leucocytes (buffy coat), and erythrocytes 5. Carefully remove the supernatant (plasma) at room temperature and decant in a centrifuge tube. Take care not to disrupt the cell layer or transfer any cells. 6. Inspect plasma for turbidity. Turbid samples should be centrifuged and aspirated again to remove remaining insoluble matter. 7. Aliquot plasma into cryovials and store at -80 °C. Ensure that the cryovials are adequately labelled with the relevant information. 8. The cellular fraction must be decanted into cryovials and stored at -80 °C, this can be used for constitutive DNA analysis.

UMBRELLA Protocol SIOP 2016

For miRNA PAXgeneTM Blood RNA tube tubes are needed. These tubes allow in addition the extraction of DNA and total RNA from one single tube. Fresh blood can be directly injected into these tubes and send by room temperature to the corresponding biobank.

Depending on each national coordinating center, frozen samples can be either directly send or stored on site until tissue samples are available for a batched collection and sent to the national biobank together with tissue samples. PAXgeneTM tubes need to be sent at room temperature immediately to the national coordinating center.

7.4 Central surgical review A national centralised review of each bilateral case is recommended as soon as the diagnosis is done in order to discuss the best surgical strategy for both kidney and consider Nephron Sparing Surgery (NSS) for at least one kidney. In case of nephron sparing surgery (NSS) or minimal invasive surgery (MIS) the indication needs to be discussed within the surgical panel on a National level. For further details see chapter 16.

7.5 Central radiotherapy review A national centralized review for radiotherapy is recommended to guarantee high quality of radiotherapy throughout all participant centres. This review process needs to be established in each participating National Group. For details of radiotherapy see chapter 17.

UMBRELLA Protocol SIOP 2016

8 Statistical Considerations with specific focus on 1q gain including sample size calculations

Biomarkers evaluated for clinical risk stratification must be considered in the context of any co- dependencies with existing risk factors (patient age, tumour stage and histology) and the current treatment given. To test associations between clinical outcome and the two principal novel biomarkers considered in this study, 1q gain and absolute blastemal volume, we assume the most relevant set of cases consists of Wilms tumours treated with neoadjuvant chemotherapy that have localised disease (stages I-III) and stromal, epithelial, mixed, regressive, or blastemal type histology. We specifically exclude bilateral, metastatic, anaplastic, completely regressive, and immediate nephrectomy cases. From previous experience with the SIOP WT 2001 trial and study, we estimate that approximately 215 cases of interest can be expected to be registered in the UMBRELLA study every year, and that the probability of an event in this subgroup (relapse or death) is 10%. Using existing 1q MLPA and outcome data from the SIOP series, we find that for a power of 80% and alpha = 0.05, fewer than 850 samples would be required to test the association between 1q gain and outcome, assuming a hazard ratio of 2.0 and a 1q gain frequency of 26.3% in the subgroup of interest. Using existing blastemal volume measurements from the SIOP series, fewer than 380 samples would be required to test an association between high (>20%) blastemal volume and outcome, assuming a hazard ratio of 2.6 and a high blastemal volume prevalence of 35.4%. The correlation between 1q gain and absolute blastemal volume in our current data is low (r < 0.1) and the variance inflation factor is close to 1, so we do not need to increase the sample size that needs to be analysed to consider both biomarkers as independent prognostic factors. It should therefore be possible to accrue a sufficient number of samples for prospective testing of both biomarkers in the first 4 years of the study, provided that analysable material can be obtained from all registered patients. If we assume that high quality data can only be obtained from 80% of patients (in line with previous experience), 5 years would be required for full accrual. For 1q gain, this estimate also assumes that the assay method can be applied to both frozen and formalin-fixed material, since freezing of tumour tissue is not standard practice in all countries involved in UMBRELLA. Both the established SIOP MLPA assay and an NGS panel that includes relevant copy number probes, such as that currently being tested by SIOP UK, should be suitable for this purpose. Full support for statistical analysis of the Europe-wide biomarker data generated in the current study will be available from Dr. Harm van Tinteren (Biometrics Department, Netherlands Cancer Institute), who was Senior Statistician for the previous SIOP clinical study and trial.

9 IT infrastructure and data management For the SIOP 2016 UMBRELLA nephroblastoma study, an IT infrastructure has been selected that allows joining heterogeneous data from oncologists, radiologists, surgeons, pathologists, radiotherapists and basic scientists, mainly molecular geneticists in a secure way respecting GCP regulations for trials including data protection and privacy. In addition pseudonymised data of WT patients from previous SIOP-RTSG clinical trials will be integrated allowing subgroup analysis with more power and time trend analysis. ALEA has been chosen as the leading eCRF system. This system provides tools for patient registration/randomisation, data collection, querying and (remote) monitoring. In addition, Electronic

UMBRELLA Protocol SIOP 2016

Case Record Forms (eCRFs) will be developed for baseline characteristics, treatment details, pathology and outcome measures. Methods for allowing reference centres to get access to imaging data (DICOM files) will be developed. The eCRFs will be largely based on the experience obtained from the last two SIOP studies (93-01 an 2001) and will provide a balance between a limited number of variables and sufficient information to answer as many research questions as possible. Variable checks and ‘cross-form’ checks are implemented. A technical specification’s overview of ALEA can be found in appendix 2. A Data Management Users’ manual is electronically available on request. Uploading and sharing of CT, MRI or PET scans from local PACS systems on the study site to the eCRF is demonstrated in an video at https://www.youtube.com/watch?v=Jp67nzP50wQ. Data from patients in German speaking countries will be registered both in ALEA and in another system called ObTiMA. ObTiMA is a modularized trial management application developed by different European funded projects (ACGT, p-medicine1, EURECA, CHIC, MyHealthAvatar). Using both systems will allow validation of ObTiMA and maximize the capabilities of both systems. Details about ObTiMA can be found in appendix 2. All data of the SIOP 2016 UMBRELLA Nephroblastoma study are stored in one single database located in at the statistical centre of SIOP-RTSG in Amsterdam lead by Harm van Tinteren. This database includes the data of the registries for local unilateral, bilateral, Non-Wilms tumours, relapsed tumours and for adults with nephroblastoma as well as the data for metastastatic nephroblastoma.

10 Patient Empowerment 10.1 Impact on the well-being of patients Participants in the SIOP 2016 UMBRELLA study are providing tissue and other biological samples as part of the diagnostic work-up and monitoring of disease response and follow up after surgery, and if applicable at relapse. No additional burden or risk is anticipated for patients. All samples are taken during regular diagnostic procedures and the additional blood sample volume and urine places minimal burden on the patient. For any individual patient, the maximum time period over which they would be asked to provide samples is during treatment (between 2 - 8 months, according to tumour stage) and 2 years of follow-up. Parents will be asked for consent for their child to provide blood and urine samples at the time of the child’s treatment. Patients’ blood and normal renal tissue after tumour resection will be stored and used as germline material for analysis if informed consent is given. The number of imaging episodes and the type of imaging are following what is considered good clinical care. There will be no extra imaging episodes, and specifically no additional burden of ionising radiation, as the study protocol encourages less use of ionising radiation by encouraging performing MRI instead of CT.

1 http://p-medicine.eu

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11 Diagnostic work-up All patients enrolled in the SIOP 2016 UMBRELLA protocol will be diagnosed in a standardized way. A minimal set of procedures is indicated in the following chapters. Imaging at the time of diagnosis needs to be provided to reference radiology. Blood and urine for molecular diagnosis need to be sent to the corresponding sample collection and storage-dedicated laboratory of each country. All data of the diagnostic work-up are stored in CRFs that are provided in appendix 3.

11.1 Clinical work-up Every patient will receive a basic clinical assessment, which at least includes the following parameters: History: - Symptoms/signs and reason for referral (screening, by chance, coincidence with other disease, tumour related symptoms) and duration of symptoms - Performance status, general signs and symptoms and duration of symptoms - History of in vitro fertilization and assisted reproductive fertility techniques and birth weight - Existence of a syndrome/malformations and their specification including history of syndrome related surgeries including urological surgeries and other treatments - History of prior malignancies/cancer treatment and coexisting diseases - Family history of syndromes and malignancies - Previous cancer treatment Clinical assessment: - Age, gender, bodyweight, length and dysmorphic signs - Assessment by Lantzky/Karnofsky Scale - Blood pressure - Body temperature and signs of infection - Tumour palpability: position and size if palpable

11.2 Laboratory work-up The following laboratory investigations are needed: - Blood/Serum o Full blood cell count (FBC) o Creatinine, Blood urea nitrogen (BUN), LDH, liver enzymes o Coagulation parameters (von Willebrand factor) o Blood group including Rhesus factor o Viral screening as a baseline o Biobanking (EDTA, PAXgene tube) and germline DNA of patients and parents - Urine o Urine metabolites of catecholamines (VMA/HVA) o Protein, glucose, cells o Biobanking and Cytospins (cytology)

UMBRELLA Protocol SIOP 2016

11.3 Radiological work-up Standardisation and quality improvement of imaging studies are still needed in multicentre trials and studies (35, 36, 37). Up to now imaging is not able to differentiate benign from malignant small lung nodules nor round nephrogenic rests from nephroblastoma. The North-American Children’s Oncology Group (COG) and Gesellschaft für Pädiatrische Onkologie und Hämatologie (GPOH) group reported a discrepancy rate between local image interpretation and central radiology review of around 15%. In every patient imaging studies of high quality need to be done. This includes abdominal ultrasound and preferably abdominal MRI (or CT) and chest CT scan, including an AP or PA chest X-ray, which can be the CT scanogram or the chest x-ray for the control of an implanted central catheter for comparison of future chest x-rays. Further details of the radiological workup are provided in chapter 11.7. Especially imaging studies of the chest (CT) need to be analysed by reference radiology to define metastatic disease. This needs to be done in due time, if patients with metastatic disease will be entered into a prospective randomized clinical trial in the future. A MIBG scintigraphy is only indicated, if there is a suspicion of neuroblastoma.

11.4 Genetic counselling In children with a renal tumour and an underlying syndrome counselling by a clinical geneticist is advised to rule out hereditary familiar syndromes with germ line mutations and to explain parents the risk of subsequent affected children. The following syndromes need to be taken into account:  Wilms tumour: o Particular features linked with Wiedemann-Beckwith syndrome (omphalocele and/or hypoglycaemia at birth, mental status, macroglossia, hemihypertrophy, hepatomegaly, nephromegaly) o WAGR (aniridia, genito-urinary malformations, mental retardation) o Denys-Drash syndrome (nephropathy and genital anomalies) o Neurofibromatosis Recklinghausen o Other rare syndromes (Simpson-Golabi-Behmel, Costello, Perlman, Sotos, Fanconi Anemia, etc.)  MRTK: o Germline SMARC B mutations  CCSK and CMN: o No related syndromes are known  RCC: o Clinical features linked with von Hippel-Lindau syndrome (retina or brain hemangioblastomas, renal cyts, pheochromocytoma) o Tuberous Sclerosis (epilepsy, developmental and behavorial disorders such as autism or hyperactivity, cardiac rhabdomyomas, retina hamartoas or renal angiomyolipoma) o Birt-Hogg-Dubé syndrome (fibrofolliculomas, neoplasmia, hamartomas, lung cysts, spontaneous pneumothorax)

UMBRELLA Protocol SIOP 2016

Since patients suffering from MRTK might bear germ-line mutations of SMARCB1, SMARCA4 or other INI 1 involving genes, such molecular genetics analysis is mandatory at least in the blood of patients younger than 2 years of age and their parents. Germline mutations of WT1 should be checked in patients with stromal type of WT and in bilateral cases without response to pre-operative chemotherapy [34].

11.5 Flow diagram of initial diagnostic work-up

Fig. 6: Flow diagram of initial diagnostic work-up.

UMBRELLA Protocol SIOP 2016

11.6 Timeframe of diagnostic work-up All renal tumour patients older than 7 months and younger than 16 years will be treated with chemotherapy before surgery. This indicates that 2 diagnostic moments are important, i.e. at presentation (day 0) and before surgery (day 28 in stage I-III, and day 42 in stage IV).

11.6.1 Standard investigations during the pre-operative phase in clinically localized disease

Investigations (Day 0) Day 15 Day 28

Abdominal ultrasound X (X) X

CT chest including a standard X chest X-Ray AP or PA view

MRI abdomen (DWI1) (alternative : CT, if MRI is X X not available)

Central radiology review X X

MRI cerebrum2 Histological proven MRTK or CCSK

Core needle biopsy3 X

Urine VMA/HVA, Stage I-III X (MIBG scintigraphy*)

Full Blood Count before each X X X course of chemotherapy

Serum (Urea, creatinine, X X creatinine clearance, electrolytes) Viral screen, Blood group Rhesus, X coagulation EDTA+Serum+PAXgene1, 2ml X (X) X Biobanking DNA

Urine1, 2ml Cytospins, DNA and proteomics X (X) X (biobanking) 1 research, 2 in case of MRTK and CCSK; brain US to consider in infants, 3 only in case of serious doubt of WT (based on clinical presentation and imaging, see radiology chapter). Response according to RECIST criteria (www.recist.com) * Only if imaging is not conclusive for Wilms tumour or VMA is elevated

UMBRELLA Protocol SIOP 2016

11.6.2 Standard diagnostics during the pre-operative phase in stage IV

(Day Investigations Day 15 Day 28 Day 42 0)

Abdominal ultrasound X X X

CT chest including a X standard chest X- Ray AP or X (only PA view CT)

MRI abdomen (DWI1) (alternative CT, if X X MRI is not available)

Central radiology review X X

MRI cerebrum2 Histological proven MRTK or CCSK

Core needle biopsy3 X

Urine VMA, HVA X Stage IV (MIBG scintigraphy*)

Full Blood Count before each X X X course of chemotherapy

Serum (Urea, creatinine, X X X electrolytes) Viral screen Blood group X Rhesus, coagulation) Cardiac ultrasound X X

EDTA+Serum+PAXgene1, 2ml X (X) (X) X Biobanking DNA

Urine1, 2 ml Cytospins, DNA and X (X) (X) X proteomics (biobanking) 1 research, 2 in case of MRTK and CCSK; brain US to consider in infants, 3 only in case of serious doubt of WT (based on clinical presentation and imaging). Response according to RECIST criteria (www.recist.com)

UMBRELLA Protocol SIOP 2016

11.6.3 Postoperative standard investigations after renal surgery in patients that received preoperative chemotherapy

Week 1 Week Week Week 24 (if End of Investigations after 6 10 applicable) therapy surgery

Central pathology review X 1 (national & SIOP) (week 2)

Abdominal ultrasound X (X) X (X) X

CT chest 4 X2,3 X4

MRI abdomen (alternative No longer needed, US is sufficient CT, if MRI is not available)

Full Blood Count before each X X X X X course of chemotherapy

Serum (Urea, creatinine, X X X X X electrolytes, ASAT, ALAT) Stage I-IV Cardiac ultrasound 5 X X

EDTA+Serum+PAXgene6, 2 ml X (X) (X) X Biobanking, DNA

Urine 6, 2 ml, Cytospins and X (X) (X) X biobanking

MRI cerebrum 7 X X

Technetium bone scan or X X whole body MRI or FDG-PET 8

Audiogram 9 X X X

Clinical geneticist10 X 1 Panel pathological review within 2 weeks after surgery, 2 in case of stage IV and only chest CT, 3 only in case of non-CR in stage IV after surgery and only chest CT, 4 Chest CT if still persistent disease after neoadjuvant chemotherapy otherwise X-Ray, 5 from 200 mg/m2 doxorubicin on, 6 for research purpose, 7 in MRTK and CCSK, 8 in CCSK only, 9 in case of carboplatin, 10 In case of bilateral disease or rhabdoid tumour.

UMBRELLA Protocol SIOP 2016

11.6.4 Postoperative investigations in patients after primary surgery (i.e. without pre-operative chemotherapy)

Week 1 Week 24 Diagnosis Week Week End of Evaluation after (if (Day 0) 6 10 therapy surgery applicable)

Central pathology review X 1 (national & SIOP) (week 2) Central Radiology Review X 2 X 3 CT chest X X 2,3 X 4 Standard chest X-Ray AP X or PA view Ultrasound X X X X X MRI abdomen (before X surgery) Full Blood Count before each course of X X X X X X chemotherapy Stage I-IV Serum (Urea, creatinine, X X X X X X electrolytes) Cardiac ultrasound 5 X X X EDTA+Serum 6, 2 ml X X (X) (X) X Biobanking DNA Urine 6, 2 ml, Cytospins X X (X) (X) X and DNA biobanking MRI cerebrum 7 X X Technetium bone scan or whole body MRI or FDG- X X PET 8 Audiogram 9 X X X Clinical geneticist10 X 1 Panel pathological review within 2 weeks after surgery, 2 in case of stage IV and only chest CT, 3 only in case of non-CR in stage IV at week 6 and only chest CT, 4 Chest CT if still persistent disease after neoadjuvant chemotherapy otherwise X-Ray, 5 from 200 mg/m2 doxorubicin on, 6 for research purpose, 7 in MRTK and CCSK, 8 in CCSK only, 9 in case of carboplatin, 10 In case of bilateral disease or rhabdoid tumour.

UMBRELLA Protocol SIOP 2016

11.6.5 Investigations in patients during follow-up after end of treatment The following table provides a schematic overview: of follow-up diagnostics

1st year 2nd year 3rd year 4th year 5th year After 5 years Physical examination + Every 3 Every 3 Every 4 Every 6 Every 6 Once a year blood pressure month month month month month Diagnostics to detect a relapse Every 3 Every 3 Every 4 Every 6 Every 6 Abdominal ultrasound Once a year month month month month month Chest x-ray AP or PA and Every 3-4# Every 3-4# Every 4 Every 6 Once a year lateral view month month month month Brain MRI In case of relapse of CCSK and MRTK Technetium bone scan or In case of relapse of CCSK or initial bone metastasis whole body MRI Toxicity diagnostics and surveillance Urine (Glucose, albumin, α 1/β2 microglobuline, Every 3 Every 3 Every 4 Every 6 Once a year Once a year calcium, phosphate, month month month month magnesium, erythrocytes) 1.1.1.1.1.1.1.1.1 24h urine collection In case of albuminuria 1.1.1.1.1.1.1.1.2 Blood (Full blood count, urea, creatinine, Ca++, Every 3 Every 6 Every 4 Every 6 phosphate, Mg++, Once a year Once a year month month month month albumine, ALAT, ASAT, bilirubine, TSH*) ECG/Echocardiography After anthracyclines, lung irradiation and in case of high blood pressure 24h blood pressure In case of high blood pressure Lung function After lung irradiation once a year Endocrinology In case of disorders contact paediatric endocrinologist Audiometry Once after carboplatin. In case of pathological result refer to ENT specialist Neuropsychological testing In case of syndromes with potential retardation (e.g. WAGR) Research EDTA+Serum+PAXgene, In case of relapse 2 ml Biobanking *: To be considered after irradiation to the lungs. #: in case of stage IV X-ray or CT of the lung every 2 months, depending on the local standards As most patients will have a nephrectomy surveillance of kidney function is mandatory. Referral to a Paediatric nephrologist in case of proteinuria, nephrocalcinosis, hypertonus and decreased kidney function is recommended. In every patient where anthracyclines are considered an echocardiogram should be performed before the first dose of anthracycline is administered and during follow-up.

UMBRELLA Protocol SIOP 2016

11.7 Details on radiology More technical details and guideline are given in appendix 4.

11.7.1 Imaging diagnostics 11.7.1.1 Abdominal Ultrasound Ultrasound is the mandatory first line imaging procedure in children with a suspected abdominal mass. The operator should be a radiologist or paediatrician with training and experience in paediatric ultrasound and paediatric oncology. The complete abdomen must be examined with probes adjusted to the size of the child: in general in this age group with a transducer of at least 5 MHz. The use of a high frequency probe (≥ 10 MHz) is mandatory for detailed examination of the contralateral kidney (also in prone and lateral position) in search for a bilateral tumour, nephrogenic rests or abnormalities that may affect renal function. The liver parenchyma must be screened for metastases. Ultrasound is the modality of choice for examining the renal vein and inferior vena cava in search for intravenous tumour thrombus, both with 2D-ultrasound and colour-Doppler and for real-time evaluation of the relationship of the tumour with adjacent organs. 11.7.1.2 Abdominal MRI Abdominal MRI is the first choice complementary imaging procedure to ultrasound. Because of the lack of ionizing radiation and the excellent soft tissue contrast, MRI is preferred to CT. The complete abdominal cavity must be examined (from liver dome to pelvis included). The examination protocol should be performed by MR-radiographers trained in paediatric abdominal MRI. Sedation or general anaesthesia is recommended in young children according to local practice. Administration of gadolinium is recommended but not mandatory. MRI should preferably be scheduled before biopsy of the tumour. More technical details and guidelines can be found in appendix 4. 11.7.1.3 Abdominal CT (only if MRI is not available) Abdominal CT is the second choice complementary imaging procedure to ultrasound. Administration of intravenous iodinated contrast is mandatory. A volumetric acquisition of 1 (portal venous) phase must be performed. The whole abdominal cavity must be assessed (complete liver and pelvis included). More technical details and guidelines can be found in appendix 4. 11.7.1.4 Chest CT An unenhanced chest CT scan is a mandatory diagnostic procedure to assess lung metastasis. Intravenous contrast is not mandatory (but may be used if it is combined with an abdominal CT scan instead of abdominal MRI). If pulmonary metastasis is detected at diagnosis, chest CT will be repeated before abdominal surgery. More technical details and guidelines can be found in appendix 4, 11.7.1.5 Chest X-Ray Chest X-ray with AP (or PA) will be performed at diagnosis as a mandatory baseline procedure. A chest X-ray performed for positioning of the central venous line may serve as baseline test. During follow-up after end of therapy, chest x-ray will be performed in both AP (or PA) and lateral views. 11.7.2 Optional and functional diagnostics imaging Guidelines and technical information can be found in appendix 4.

UMBRELLA Protocol SIOP 2016

11.7.3 Classification of lung lesions Pulmonary lesions different from lung metastases: - Linear-shaped pulmonary opacities consistent with atelectasis. - Ground glass, ill-defined or diffuse alveolar pulmonary opacities consistent with inflammatory or infectious disease. - Calcified pulmonary nodules consistent with granulomas. - Triangular or trapezoidal perifissural or subpleural densities, consistent with lymph nodes Classification of round solid nodules with sharp margins according to diameter: 1 – 2 mm 3 – 5 mm 6 – 10 mm > 10 mm 11.7.4 Cutting Needle Biopsy Cutting/Core needle biopsies can be used to verify the radiologic diagnosis of renal tumours with 1.6% relevant complications such as tumour bleeding, rupture and needle track recurrence [32]. Indications, limitations and procedural recommendations are detailed in appendix 4.

11.8 Pathology issues The institutional pathologist is responsible for initial histological classification and local staging. He/she is also responsible for the handling of the renal tumour for collection of material for research and biological studies. A minimal set of criteria for accurate pathological diagnosis and staging needs to be fulfilled [16, 32, 33]. One of the topics of the UMBRELLA protocol is the prospective quantification of blastema in the tumour and its prognostic significance. Therefore, pathologists’ findings will have a major impact on answering this question of the study. In order to gain all necessary data, the following standard operating procedure (SOP) needs to be followed.

11.8.1 Nephrectomy handling The intact surgical specimen should be brought to the laboratory without being opened by the surgeon. It should be dealt with by the pathologist as soon as it arrives in the lab to minimise degradation of RNA especially. The specimen should be examined as follows:

1. Weigh, measure and photograph the whole specimen (Figures 8-10). Look carefully for ruptures and fissures and locate any suspicious areas and/or ink them in different colours from the rest of the specimen. Decapsulation makes determination of growth beyond the capsule impossible and therefore should not be done. 2. Look for and dissect the peri-renal and perihilar lymph nodes. Block these separately, recording their site. 3. Identify renal vein, artery and ureter and take transverse section block of each at/near the resection margin. 4. Ink the surface of the whole specimen and renal sinus with Indian ink and let it dry before

UMBRELLA Protocol SIOP 2016

opening the specimen. This is a critical step and should always be done, otherwise it might be impossible to stage the tumour accurately and give adequate therapy. 5. Open by a longitudinal incision to bivalve the specimen and reveal the tumour and its relation to the kidney, capsule, and renal sinus (Figures 9 and 10). 6. Photograph the cut surface, and record the macroscopic appearance. It is critical to accurately measure the tumour in all three dimensions - this will be used for calculating volume of tumour and blastema. 7. Assess the percentage of a necrotic tumour (this percentage has to be filled in on the Form F4) 8. Describe and photograph the multicystic cut surface, if present. 9. Samples required for biology studies should be taken (please see below) 10. The specimen should be fixed in 10% buffered formalin for 24 to 48 hours, according to the usual procedure of the laboratory. Several additional cuts can be made parallel to the initial cut to divide the specimen into “slabs” for better fixation. 11. The samples for histological examination should include: a) at least one longitudinal slice of tumour and kidney surface, completely sampled (see Figures 10-12) (please consider using mega-blocks and cassettes (Figure 7) as it makes histological assessment much easier, and they are less time consuming for both pathologists and their labs)

Figure 7. Mega-cassette and normal-sized cassette

In addition, please sample the following: b) macroscopically different areas of the tumour c) dubious areas have to be marked by the surgeon and need special attention of the pathologist (they have to be marked with Indian ink or methylen blue); d) sinus lymph nodes when present; e) other lymph nodes; f) renal pelvis and pelvic fat, ureter and sinus vessels; especially the renal vein should be inspected for evidence of tumour thrombus; if present, it is critical to assess whether it is completely resected; g) each nodule away from the main mass (in multifocal tumours); h) tumour-kidney interface i) tumour-kidney capsule

UMBRELLA Protocol SIOP 2016

j) areas of the capsule that are suspected of being invaded by the tumour; k) areas of perirenal fat suspected for tumour infiltration (important for assessment whether the tumour is completely resected); l) areas of adhesions of the tumour to surrounding tissues; i) at least 2 blocks of the normal kidney and blocks from abnormal looking areas in the remaining renal tissue.

Please make sure to have a ‘block guide’ (as in Figures 10-12), i.e., all the samples should be numbered and their sites recorded as well as all other samples taken at the time of operation, i.e. adrenals, lymph nodes and various biopsies.

In Histopathology report, please clearly state all relevant findings and block/slide number (for example, “there is renal sinus invasion in block A7”) as it makes central pathology review easier.

In Summary - Weigh and accurately measure the specimen and tumour (in all 3 dimensions) - Take fresh samples for biological studies - Sample tumour according to the Guidelines (above) - Consider using mega-cassettes - Assess the percentage of chemotherapy-induced changes on gross and histological examination - Assess the percentages of viable tumour components - In your report, clearly state in which blocks/slides the relevant findings are

SEND THE CASE FOR RAPID CENTRAL PATHOLOGY REVIEW!

All cases submitted for Central Pathology Review should include a full set of H&E slides, meticulous (photo) block guide, and the preliminary/final centres pathological report. The central pathological reviewer will inform the institutional pathologist within 48 hours about their opinion (whenever possible), so that correct postoperative treatment can be applied immediately. This will allow harmonisation of accurate histological diagnosis and local staging in real time, which is a pre-requisite for appropriate treatment, and avoidance of under- and overtreatment of patients after surgery.

Fig. 8: Fresh tumour nephrectomy specimen. Fig. 9: Fresh tumour nephrectomy specimen after inking and bivalving.

UMBRELLA Protocol SIOP 2016

Fig. 10: Transparency applied to tumour nephrectomy specimen to show block map.

Fig. 11. An examples of block guide Fig. 12. An examples of block guide

11.8.2 Tissue for research (see also section 7.3 and Appendix 7) Only a pathologist should sample the specimen for research from different tumour areas so that there is no compromise of the gross specimen interpretation at a later point in time. 1. Tissue for research should be taken from regions that grossly appear viable, cut fresh tumour tissue and 2. Prepare frozen sections or touch imprints for staining and immediate evaluation of cellular content of this material, viability and proportion of tumour cells especially. Evaluation of these samples should be included in the final pathology report.

UMBRELLA Protocol SIOP 2016

3. If these are of good viability and ‘pure’ tumour content, snap-freeze material from these areas in cryo-vials clearly labelled with unique identifiers (sample code, see F15 and F16), in liquid nitrogen and then store at -80°C, properly logged as to location within the freezer. A mirror block of each sample taken for freezing and molecular studies needs to be send to reference pathology for histology. The sample code of the specimen has to be provided to reference pathology and the biobanks allowing to link histology with molecular findings. 4. Wherever possible the source of the material should be documented photographically and in reference to the pathology block guide. 5. Specimen should be taken in the same way from different tumour areas. 6. Additional touch imprints may be stored, following air-drying, for use in molecular cytogenetic evaluations. 7. Where available and there is local interest, tissue may be taken and placed in tissue culture media for drug testing, etc.. This is currently surplus to requirements, but is in itself also a valuable research exercise. 8. In the event of additional lesions being present and identified, e.g. hyperplastic rests, provided there is enough to allow for adequate histological evaluation, the excess should similarly be sampled for research and stored with a clear additional indication on the vial as to the nature of the material – “tumour”, “normal”, “rest” etc. 9. Apart from tumour tissues, also normal tissue is required to be collected for comparative analyses, preferable from the normal part of the kidney and peripheral blood for studying germline aberrations. This material should be taken and stored in the same fashion as tumour material.

12 Plan of integrated research A primary aim of SIOP 2016 UMBRELLA protocol is to find new biomarkers for better treatment stratification. For that purpose molecular biological research plays an important role in the protocol. Main research areas are provided in the following table chapters. During the lifetime of the protocol changes are possible.

12.1 List of some research proposals Investigators Aim Methods Brazil Mariana Maschietto Epigenetic alterations Microarrays, qRT-PCR, Beatriz de Camargo related do resistance or sequencing metastasis

France Aurore Coulomb, Linda 11p15 methylation Nucleic acid extraction, Bisulfite Dainese status and Wilms DNA treatment, TaqMan allele- tumours biological specific methylated multiplex behaviour real-time quantitative PCR (ASMM RTQ-PCR), methylation analysis and Multiplex ligation dependent probe amplification

UMBRELLA Protocol SIOP 2016

Germany Eckart Meese, Andreas miRNA pattern in blood Microarray-based microRNA Keller, Norbert Graf, and tumour as a (miRNA) expression analysis biomarker for kidney including the tumours and computational miRNA data nephroblastoma evaluation subtypes Testing of DNA/RNA of Mutation and individual tumors by Manfred Gessler epigenetic profiling of PCR/sequencing etc. tumors. High throughput array and Intratumoural sequencing analyses of Wilms heterogeneity and and non-Wilms kidney tumors tumour evolution. In vitro models for Primary tumor cultures Wilms tumor. Italy Filippo Spreafico Genetic predisposition Exome sequencing to familial Wilms Daniella Perotti Sequencing tumour Paolo Radice Genetics of recurrences Netherlands Marry M. van den Driving oncogenic NGS-sequencing: tumor and Heuvel-Eibrink events in non-Wilms liquid biopsies renal tumours and Organoids development from micro-dissected WT urine and tumour Hans Clevers/Jarno cells and genetic Drost changes associated with relapse and outcome. Marjolein Germline oncogenic (WES): Jongmans/R.Kuipers/ susceptibility Tumor/Blood(Parents&children) MM vd Heuvel-Eibrink Sweden David Gisselsson, Linda Can intratumour genetic High resolution SNP arrays Holmquist Mengelbier heterogeneity be used (Affymetrix Cytoscan HD) and as a biomarker for event exome sequencing (Illumina- free or overall survival? based platform) UK Kathy Pritchard- Somatic mutational Next Generation Sequencing Jones/William profile of tumours, Microarrays Mifsud/Richard including epigenetics Williams Intratumoural Droplet Digital PCR heterogeneity and Proteomics tumour evolution Circulating cell-free DNA Primary tumour culture Matt Murray Urinary proteomics FISH Circulating miRNAs (Cambridge Univ) Table 4: Integrated research proposals in different countries for WT. This list will be continuously updated also with projects from other members of SIOP-RTSG and participants of the UMBRELLA protocol.

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A detailed description of research projects is given in Appendix 7 for Wilms and Non-Wilms Tumours. This list will continuously be updated and can be found in the Intranet of the SIOP-RTSG homepage (http://siop-rtsg.eu).

13 References

1. Mitchell C, et al., Immediate nephrectomy versus preoperative chemotherapy in the management of non-metastatic Wilms' tumour: results of a randomised trial (UKW3) by the UK Children's Cancer Study Group. Eur J Cancer, 2006. 42(15): p. 2554-62. 2. Pritchard-Jones K, Bergeron Ch, de Camargo B, et al. on behalf of the SIOP Renal Tumor Study Group: Doxorubicin omission from the treatment of stage II/III, intermediate risk histology Wilms tumour: results of the SIOP WT 2001 randomised trial. Lancet, 2015. 386: p. 1156-1164 3. Rossi S, et al., p-Medicine: From data sharing and integration via VPH models to personalized medicine. Ecancermedicalscience, 2011. 5: p. 218. 4. Hing S, et al., Gain of 1q is associated with adverse outcome in favorable histology Wilms' tumors. Am J Pathol, 2001. 158(2): p. 393-8 5. Lu YJ, et al., Chromosome 1q expression profiling and relapse in Wilms' tumour. Lancet, 2002. 360: p. 385-6 6. Natrajan R, et al., Array CGH profiling of favourable histology Wilms tumours reveals novel gains and losses associated with relapse. J Pathol, 2006. 210(1): p. 49-58 7. Perotti D, et al., Genomic profiling by whole-genome single nucleotide polymorphism arrays in Wilms tumor and association with relapse. Genes Chromosomes Cancer, 2012. 51(7): p. 644-53 8. Gratias EJ, et al., Gain of 1q is associated with inferior event-free and overall survival in patients with favorable histology Wilms tumor: a report from the Children's Oncology Group. Cancer, 2013. 119: p. 3887-94 9. Segers H, et al., Gain of 1q is a marker of poor prognosis in Wilms' tumors. Genes Chromosomes Cancer, 2013. 52(11): p. 1065-74 10. Chagtai T, Zill C, Dainese L et al.: Gain of 1q as a prognostic biomarker in Wilms tumours treated with pre-operative chemotherapy in the SIOP WT 2001 trial: A SIOP Renal Tumours Biology Consortium Study. J Clin Oncol, 2015. In press 11. Grundy P, Breslow NE, Li S, et al.: Loss of heterozygosity for chromosomes 1p and 16q is an adverse prognostic factor in favorable-histology Wilms tumor: a report from the National Wilms Tumor Study Group. J Clin Oncol, 2005. 23(29): p. 7312-21 12. Williams RD, et al., Children's Cancer and Leukaemia Group; SIOP Wilms' Tumour Biology Group. Subtype-specific FBXW7 mutation and MYCN copy number gain in Wilms' tumour. Clin Cancer Res, 2010. 1(16): p. 2036-2045. 13. Maschietto M, Williams RD, Chagtai T, et al.: TP53 mutational status is a potential marker for risk stratification in Wilms tumour with diffuse anaplasia. PLoS One, 2014. 9(10): p. e109924 14. Wegert J, Ishaque N, Vardapour R, et al.: Mutations in the SIX1/2 pathway and the DROSHA/DGCR8 miRNA microprocessor complex underlie high-risk blastemal type Wilms tumors. Cancer Cell, 2015. 27(2): p. 298-311 15. Walz Al, Ooms A, Gadd S et al.: Recurrent DGCR8, DROSHA, and SIX homeodomain mutations in favorable histology Wilms tumors. Cancer Cell, 2015. 27(2): p. 286-97 16. Vujanic GM, et al., Central pathology review in multicenter trials and studies: lessons from the nephroblastoma trials. Cancer, 2009. 115(9): p. 1977-83.

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17. van den Heuvel-Eibrink MM, et al. Outcome of localised blastemal type Wilms tumor patients treated according to intensified treatment in the SIOP WT 2001 protocol, a report of the SIOP renal tumor study group (SIOP-RTSG). Eur J Cancer, 2015. 51(4): p. 498-506. 18. Dome JS, et al., Children's Oncology Group's 2013 blueprint for research: renal tumours. Pediatr Blood Cancer, 2013. 60(6): p. 994-1000. 19. Skounakis E, et al., DoctorEye: A multifunctional open platform for fast annotation and visualization of tumours in medical images. Conf Proc IEEE Eng Med Biol Soc, 2009. 2009: p. 3759-62. 20. Noone TC, et al., Abdominal imaging studies: comparison of diagnostic accuracies resulting from ultrasound, computed tomography, and magnetic resonance imaging in the same individual. Magn Reson Imaging, 2004. 22(1): p. 19-24. 21. Semelka RC, et al., Imaging strategies to reduce the risk of radiation in CT studies, including selective substitution with MRI. J Magn Reson Imaging, 2007. 25(5): p. 900-9. 22. Grundy PE, et al., Clinical significance of pulmonary nodules detected by CT and Not CXR in patients treated for favorable histology Wilms tumour on national Wilms tumour studies-4 and - 5: a report from the Children's Oncology Group. Pediatr Blood Cancer, 2012. 59(4): p. 631-5. 23. Smets AM, et al., The contribution of chest CT-scan at diagnosis in children with unilateral Wilms' tumour. Results of the SIOP 2001 study. Eur J Cancer, 2012. 48(7): p. 1060-5. 24. McCarville MB, et al., Distinguishing benign from malignant pulmonary nodules with helical chest CT in children with malignant solid tumors. Radiology, 2006. 239: p. 514-20. 25. McDonald K, et al., Patterns of shift in ADC distributions in abdominal tumours during chemotherapy – feasibility study. Pediatr Radiol, 2011. 41(1): p. 99-106. 26. Horger M, et al., Whole-body diffusion-weighted MRI with apparent diffusion coefficient mapping for early response monitoring in multiple myeloma: preliminary results. Am J Roentgenol, 2011. 196(6): p. W790-5. 27. 13, Humphries PD, et al., Tumours in pediatric patients at diffusion-weighted MR imaging: apparent diffusion coefficient and tumour cellularity. Radiology, 2007. 245(3): p. 848-54. 28. Li S, et al., Tumor response assessments with diffussion and perfusion MRI. J Magnetic Resonance Imsaging, 2012. 35: p. 745-63 29. McDonald K, et al., Patterns of shift in ADC distributions in abdominal tumours during chemotherapy-feasibility study. Pediatr Radiol, 2011. 41(1): p. 99-106. 30. SIOP 2001 Nephroblastoma Trial and Study Protocol, 2001. 2001. 31. Vuononvirta R, et al., Perilobar nephrogenic rests are nonobligate molecular genetic precursor lesions of insulin-like growth factor-II-associated Wilms tumours. Clin Cancer Res 2008. 14: p. 7635-7644. 32. Vujanic GM, et al., The role of biopsy in the diagnosis of renal tumours of childhood: Results of the UKCCSG Wilms tumour study 3. Med Pediatr Oncol, 2003. 40(1): p. 18-22. 33. Vujanic GM, Sandstedt B, Harms D et al., Revised International Society of Paediatric Oncology (SIOP) Working Classification of Renal Tumors of Childhood. Med Pediatr Oncol, 2002. 38: p. 79- 82 34. Lehnhardt A, Karnatz C, Jun Oh J, et al.: Clinical and molecular characterization of patients with heterozygous mutations in Wilms Tumour suppressor Gene 1 (WT1). Report of the WT1-Registry of the GPN. Clin J Am Society of Nephrology, 2015. 10: p. 825-831 35. Brisse HJ, Smets AM, Kaste SC, Owens CM. Imaging in unilateral Wilms tumour. Pediatr Radiol, 2008. 38(1): p. 18-29 36. Kaste SC, Dome JS, Babyn PS, Graf NM, Grundy P, Godzinski J, Levitt GA, Jenkinson H. Wilms tumour: prognostic factors, staging, therapy and late effects. Pediatr Radiol, 2008. 38(1): p. 2-17

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37. Owens CM, Brisse HJ, Olsen OE, Begent J, Smets AM. Bilateral disease and new trends in Wilms tumour. Pediatr Radiol. 2008. 38(1): p. 30-39

UMBRELLA PROTOCOL SIOP 2016 Part B

Part B: Guidelines for treatment

Part A: Guidelines for treatment

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14 General Treatment Guidelines 14.1 General remarks The main purpose of the SIOP-RTSG 2016 UMBRELLA protocol is a better molecular characterization of paediatric renal tumours to establish future biomarkers for these diseases. As this can only be achieved with a combined analysis of SIOP 2001 and UMBRELLA data, treatment remains unchanged compared to SIOP 2001 and the results of the randomized question [1]. Therefore the UMBRELLA protocol is not a treatment protocol. It gives detailed guidelines for the treatment of renal tumours in childhood and adolescence according to the state of the art. There is no treatment question asked. In this sense the UMBRELLA protocol is not falling under the Clinical Trial Directive 2001/20/EC. A description of the risk-adapted treatment for WT is outlined in the following chapters including recommendations for relapses and nephroblastoma in adults. In addition guidelines for the treatment of other renal tumours in childhood and adolescence are given as well. In case that better treatments with higher cure rates or less toxicity are developed or targeted therapies become available our guidelines will be changed accordingly. Such changes may result in prospective clinical trials. Treatment for nephroblastoma is based on chemotherapy and surgery. For a small number of patients radiotherapy needs to be added. According to SIOP protocols, preoperative chemotherapy is given in all patients with a renal tumour over the age of 6 months and under the age of 16 years. In children below 7 months of age a Congenital Mesoblastic Nephroma (CMN) is most often occuring, where surgery alone is curative. Nevertheless in these infants primary surgery should be evaluated by an interdisciplinary team in each case to weight the risk of tumour rupture after primary surgery against the risk of unnecessary chemotherapy. In patients over 16 years of age a Renal Cell Carcinoma (RCC) needs to be ruled out demanding primary surgery whenever possible. The treatment guidelines include localized, metastatic and bilateral WTs. Children with bilateral nephroblastomatosis need a prolonged treatment as described below. Treatment for relapses of a nephroblastoma is stratified according to risk groups that are mainly defined by pathological features of the initial tumour and previous treatment given. Adults with a nephroblastoma are most often primarily operated. In addition they do not tolerate Vincristine very well. This results in a specific guideline for adults with nephroblastoma. Precise guidelines are also given for CMN, Clear Cell Sarcoma (CCSK) and RCC. Rhabdoid tumours of the kidney should be treated according to the European Rhabdoid Registry EU-RHAB [2].

14.2 General chemotherapy guidelines No treatment course including ActinomycinD, Doxorubicin, Carboplatin, Cyclophosphamide or Etoposide should be initiated if the absolute neutrophil count is < 1.000/µl or the platelet count is < 100.000/µl. Dose modifications are given in chapter 10.5.3. 14.2.1 Drugs and dosage a. Actinomycin D Formulation: Dry powder vials to dissolve with sterile water, containing 0.5 mg dactinomycin Application: Intravenous infusion Not given in children weighing less than 5 kg. No single dose should exceed 2000 μg (2mg).

UMBRELLA Protocol SIOP 2016

Known important incompatibilities: doxorubicin, allopurinol, colchicine, probenecid, sulfinpyrazon Side effects and main toxicities: Nausea, vomiting, stomatitis, mucositis, diarrhea, myelosuppression, immunosuppression, fever, alopecia, transient increase of liver function (up to global liver failure, veno-occlusive disease (VOD) 2% of cases), hypocalcaemia, allergic reaction b. Vincristine Formulation: Ready-to-use vials, one vial contains vincristinesulfate 1mg (= 0.895 mg Vincristine) plus lactose Application: Intravenous infusion No single dose should exceed 2mg. Known incompatibilities: All solutions with a pH other than 3.5 to 5.0. Side effects and main toxicities: ONLY FOR INTRAVENOUS INFUSION, peripheral neuropathy, central neurotoxicity, constipation, VOD, poly-, dysuria, inadequate ADH secretion, transient myelosuppression, reversible hair loss, necrosis after paravenous injection, in combination with cyclosporin A potential for severe neurotoxicity. Cross-reactivity with doxorubicin, daunorubicin, Actinomycin D, metramicin and mitomycin. c. Doxorubicin Formulation: Dry powder and saline solution for dissolving, one vial contains 100mg doxorubicinhydrochlorid Application: Infusion over 2-6 hours Total cumulative dose given should not exceed 300 mg/m2. Important incompatibilities: allopurinol, aluminium, cephalotin, gancyclovir, diazepam, fluorouracil, furosemide, heparin, hydrocortisone, methotrexate, natrium-hydrogencarbonat, piperacilin, theophylin. Side effects and main toxicities: Transient myelosuppression, reversible hair loss, cardiotoxicity (acute arrhythmias and late cardiomyopathy), nausea and vomiting, mucositis, transient increase in liver function tests, allergic reactions, paravasation necrosis, in cases of doses excessive of a maximum cumulative dose 400mg/m2 the risk of cardiomyopathy arises without existing risk factors. In acute cardiomyopathy within 24 to 48 hours arrhythmias, extra systoles, EKG changes which are in general reversible. A minor side effect is red discoloration of the urine. d. Etoposide (VP-16) or Etoposid phosphate Formulation: Dry powder vials to dissolve with sterile water, 5 % dextrose or normal saline. Application: Infusion Total cumulative dose given should not exceed 2700 mg/m2. Except for patients with stage IV, arm B and C and for patients with relapse. Important incompatibilities: amphotericin B, cefepime, chlorpromazine, imipenem, methylpred- nisolone, mitomycin. Interaction with coumadin and derivatives. Side effects and main toxicities: myelosuppression, reversible hair loss, fever, hypotension, anaphylactic reactions, nausea and vomiting, diarrhea, mucositis, hepatic enzyme elevation, secondary malignant disease, rarely myalgia, central nervous system disturbances, peripheral

UMBRELLA Protocol SIOP 2016

neuropathy, in isolated cases acute leukemia, cardiac dysrhythmias, heart attacks, Stevens- Johnson-Syndrome. e. Carboplatin Formulation: Vials with 5ml, 15ml, 45ml containing carboplatinum 50mg, 150 mg, 450mg. Solution in dextrose 5% Application: Infusion Important incompatibilities: aluminium, amphotericin B, Sodium-Bicarbonate Side effects and main toxicities: Nausea, vomiting, painful gastrointestinal sensations, allergic reactions (pruritus, fever, redness, very rarely anaphylactic reaction with bronchospasm and cardio- depressive effects), transient myelosuppression, change of taste, rarely optic neuritis, auditory and peripheral neuropathy, ototoxicity, transient increase of liver function tests. Nephro-toxicity is of importance at high doses and in patients with prior renal dysfunction. Precautions: reduction of the dose in proportion to creatinine clearance. Total cumulative dose given should not exceed 3600 mg/m2, except for patients with relapse, where the cumulative dose may be higher depending on primary treatment with carboplatin. Dose reduction according to Calvert formula2: Dose [mg] = 4 x (GFR [ml/min] +15 x BSA [m2]) or for children according to the formula (modification after Newell3): Dose [mg] = 4 x (GFR [ml/min] +0,36 BW [kg]). This results in an AUC for Carboplatin of 4 mg x min/ml/day. f. Cyclophosphamide Formulation: Vials of 100mg, 200mg, 500mg, 1,000mg available, dry powder vials plus saline solution vials. Application: Infusion Total cumulative dose 5400 – 11400 (18000 maximum in HR schema for stage IV) mg/m2 (depending on schema). Important incompatibilities: amphotericin B, benzyl alcohol, induction of microsomal liver enzymes by phenobarbital, phenytoin, benzodiazepines, chloralhydrate or dexamethasone resulting in increased activity of cyclophosphamide, increased cardiotoxicity with simultaneous application of anthracyclines. Side effects and main toxicities: Transient myelosuppression, reversible hair loss, nausea and vomiting, hemorrhagic cystitis due to accumulation of acrolein in the urine, water retention, cardiotoxicity in high doses, VOD in high dose approaches, secondary malignancy, infertility. Mesna (Uromitexan®) needs to be given. g. Ifosfamide Formulation: Dry powder vials to dissolve with sterile water or vials with 4% Ifosfamide solution, vials as dry powder available 200, 500, 1,000, 2,000, 3,000 mg

2 Calvert AH et al.: Carboplatin dosage: prospective evaluation of a simple formula based on renal function. J Clin Oncol 1989;7:1748-56 3 Newell DR et al.: Carboplatin pharmacokinetics in children: the development of a pediatric dosing formula. J Clin Oncol 1993;11:12314-23

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Application: Infusion Total cumulative dose given should not exceed 36 g/m2. Important incompatibilities: none Side effects and main toxicities: transient myelosuppression, reversible hair loss, nausea and vomiting, hemorrhagic cystitis, encephalopathy (10% with agitation, nightmares, loss of consciousness and/or seizures), transient increased liver function tests, Fanconi-syndrome, CNS toxicity in up to 12% in phase II studies, in isolated cases cardiotoxicity. Mesna (uromitexan ®) needs to be given. h. Irinotecan Formulation: Vials 40 mg (2ml), 100 mg (5ml) or and 100mg (5ml) Application: Infusion Important incompatibilities: neuromuscular blocking agents, CYP3A-inducing anticonvulsant drugs (e.g., carbamazepine, phenobarbital, phenytoin) leads to reduced exposure to irinotecan, co- administration of ketoconazole resulted in a decrease in the AUC, drugs known to inhibit (e.g., ketoconazole) or induce (e.g., rifampicin, carbamazepine, phenobarbital, phenytoin) drug metabolism by CYP3A4. Side effects and main toxicities: The most significant adverse effects of irinotecan are severe diarrhea and extreme suppression of the immune system. Recommendation regarding diarrhoe: 5- 7 days of cefixim and loperamide. Irinotecan recipients with a homozygous (both of the two gene copies) polymorphism in UGT1A1 gene, to be specific, the *28 variant, should be considered for reduced drug doses4. i. Melphalan Formulation: Dry powder vials to dissolve with solution vials, vials as dry powder available: 50 mg Application: Infusion over 1 hour (high dose chemotherapy regimen) Important incompatibilities: Nalidixic acid: hemorrhagic enterocolitis, kidney function reduction if cyclosporine is given after stem cell transplantation. Side effects and main toxicities: myelosuppression, reversible hair loss, nausea and vomiting, transient increased liver function tests, icterus, veno-occlusive disease (VOD), myalgia, rhabdomyolysis, exanthema, hemolytic anemia, interstitial pneumonia, lung fibrosis, arrhythmias. Drugs should be stored and reconstituted according to the instructions given by the manufacturer. Adequate hydration should be given to all patients receiving chemotherapy, especially those under 1 year of age, as one factor to avoid veno-occlusive disease (VOD). G-CSF In case of severe neutropenia (high risk treatment, relapse treatment) G-CSF can be given (5 μg/kg/daily; subcutaneous) starting 5 to 6 days after the last dose of chemotherapy and given until ANC ≥ 1000 and

4 Camptosar® irinotecan hydrochloride injection August 2010: http://labeling.pfizer.com/ShowLabeling.aspx?id =533

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past the nadir of myelosuppression or a minimum of 1 week. G-CSF should be stopped for 48 hours before starting of the next chemotherapy.

14.3 Toxicity 14.3.1 Hematological toxicity Hemoglobin level, WBC and platelet counts should be performed before each course of chemotherapy.  Neutropenia: absolute neutrophil count (ANC) has to be above 1000/mm3 to start a course with actinomycin D or doxorubicin, cyclophosphamide, ifosfamide, carboplatin. Vincristine may be continued without taking the ANC into account if the patient is clinically well.  Thrombocytopenia: platelet count has to be > 100.000/mm3 to start a treatment course. The course in progress should be interrupted if the platelet count falls below 50.000/mm3 and in case of such a sudden fall, the patient should be monitored carefully for signs of VOD or sepsis/line infection, with daily full blood count and liver function tests. Transfusion of platelets is indicated always in case of hemorrhages.  Anemia alone should be treated by transfusion if necessary (Hb <7 g/l). Anemia is not a reason to modify the treatment schedule.

If a course of treatment results in a nadir WBC count below 1500/mm3 or in a nadir ANC below 1000/mm3, associated with mucositis and/or fever or in a nadir platelet count below 50.000, associated with marked enlargement of the liver or haemorrhages: The doses on the next course can be reduced to 2/3 and if the next course of chemotherapy is well tolerated full doses will be tried again in subsequent ones.

14.3.2 Neutropenic fever Definition: Temperature (rectal) > 38,5° C or 4 x > 38,0° C within 24 h with interval of more than 4 h and neutrophil count < 500/μl Diagnosis: Blood cultures each central line separately! Stool cultures, urinalysis Throat, skin and mucosa (incl. anal) cultures Virus isolation from lesions, stool and urine Chest X-ray if respiratory symptoms, sonography of abdomen Beside intensive diagnostics it is mandatory to start systemic antibiotic therapy immediately. The combination of antibiotics has to be selected according to typical pathogens of the hospital/institution. If pulmonary symptoms persist despite broad-spectrum antibiotic therapy for 72 hours bronchial lavage may be considered according to national guidelines. In case of suspected fungal disease add antifungal treatment. G-CSF can be given according to international recommendations. 14.3.3 Isolated gastrointestinal complications  Vomiting particularly occurs for a few hours after the injection of actinomycin D or doxorubicin. It can usually be treated symptomatically and rarely requires treatment modifications.

UMBRELLA Protocol SIOP 2016

 Diarrhea with or without vomiting particularly may occur after irradiation of the whole abdomen in young children. This may require the treatment to be withheld for a few days and sometimes irradiation has to be abandoned. Supportive treatment may be required. In case of diarrhea after irinotecan loperamide might be needed.  Constipation is common with vincristine. One has to see that loose stools are produced by prescribing laxatives. The drugs should be omitted in case of paralytic ileus and restarted at a 50% dose. 14.3.4 Hepatic complications Hepatic complications may occur with actinomycin D, vincristin or doxorubicin. Risk factors are nephroblastoma of the right kidney and irradiation of the whole abdomen or the right flank associated with mainly actinomycin D. Patients with signs of liver dysfunction should be monitored carefully. Patients with venous occlusive disease (VOD) do need supportive treatment including the administration of defibrotide. Defibrotide is an anticoagulant with a multiple mode of action. Actinomycin D should not be given until the main abnormalities have returned to normal and half the dose should be given for the first following course. If the symptoms reappear during actinomycin D treatment, this drug should be withdrawn permanently. Vincristine may enhance hepatopathy. If there are problems in interpreting or applying the protocol in children with hepatic disease, the National PI should be contacted for advice. 14.3.5 Exposure to infection with varicella or herpes Patients who develop varicella or herpes should receive aciclovir or valaciclovir and chemotherapy should not be started until one week after the control of the rash. 14.3.6 Cardiac toxicity There are no generally accepted guidelines for dose modifications of doxorubicin available. Monitoring with echocardiography should be done before the first administration of doxorubicin and thereafter at least every 200 mg/m2 cumulative dose. Interruption of doxorubicin must be considered if fractional shortening falls below 28% or a reduction of > 10% is seen between two consecutive administrations. If seen, do not delay chemotherapy by giving VA alone. Repeat echo after 3 weeks and if improved, proceed with doxorubicin, but perform echocardiography before each administration of doxorubicin. A reduction above 20% of baseline is a reason to withhold doxorubicin until the fractional shortening has normalized to its initial value. Beware of anemia that may influence the fractional shortening. Cardiac toxicity is more prone to occur in a patient who has received thoracic radiotherapy and has a left sided nephroblastoma stage III. We recommend measuring Fractional Shortening, Ejection Fraction and if possible the End Systolic Wall Stress to evaluate increasing afterload, which is a consequence of the wall muscle thinning, due to cardiomyocyte damage. 14.3.7 Neurological toxicity Muscular weakness and hyporeflexia are the main side effects of vincristine. Jaw pain, pain on swallowing and hoarseness may occur. In case of peripheral nerve palsies, foot drop, and severe neuritis one or two injections of vincristine should be omitted and the next dose decreased to 2/3. Vincristine neuropathy is more common in elderly patients.

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In case of ifosfamide induced CNS toxicity Methylenblue (MB) is the treatment of choice until resolution or a significant improvement of symptoms is achieved. An EEG should be performed to confirm the typical anomalies related to ifosfamide. MB administration does not affect ifosfamide pharmacokinetics. The recommended dose of intravenous MB for treatment of ifosfamide-induced encephalopathy is 50 mg every 4 hours (1% aqueous solution over 5 minutes), whereas the dose for secondary prophylaxis of ifosfamide-induced encephalopathy is 50 mg every 6 hours, either intravenously or orally. 14.3.8 Bladder and renal toxicity Cyclophosphamide and ifosfamide can cause haemorrhagic cystitis if the details for its prescription are not met. The regular use of Mesna is indicated at 120 – 150% of the cytostatic drug dosage. In case of macroscopic and repetitive hematuria chemotherapy needs to be stopped. To increase diuresis infusion hyperhydration (3 l/m2) and a diuretic drug (furosemide, mannitol) should be considered. Dose modifications due to increasing serum-creatinin-levels or in case of tubulopathy should be considered. Generally the following steps are conceivable for ifosfamide: 1. Application of ifosfamide over 24 hours instead of short infusion 2. Dose reduction of ifosfamide of about 1/3 3. Give cyclophosphamide in exchange for ifosfamide Similar strategies are possible in case of ifosfamide induced CNS-toxicity (see 14.3.7). In case of carboplatin and reduction of the creatinine-clearance dose should be adjusted according to the Calvert or Newell formula. 14.3.9 Gonadotoxicity Gonatotoxicity occurs mainly after alkylating agents and radiotherapy of the abdomen. Fertility counseling and fertility preservation should always be considered. This is especially necessary in those patients with whole abdominal irradiation (after spill) and before high-dose chemotherapy with stem cell rescue. 14.3.10 Major intolerance during pre-operative therapy If during the pre-operative chemotherapy period the following complications occur, the patients should proceed to nephrectomy once they have recovered from the toxicity, assuming surgery is deemed feasible with acceptable risk, otherwise alternative pre-operative chemotherapy drugs may be considered. Usually, post-operative chemotherapy should still be given according to tumour stage and histology, unless the tumour is low risk histology and low stage and/or the clinical situation makes it unacceptable to continue further chemotherapy. Alternative chemotherapy drugs to be considered should be discussed with the national coordinator. In some instances, (e.g. Act D) it may be acceptable to re-expose with reduced doses, for other drugs where cumulative dose is the key driver of toxicity (e.g. doxorubicin), the drug should be discontinued permanently. a. Profound thrombocytopenia (thrombocytes < 50x109/l) with or without heamorrhage associated with VOD: abdominal pain with diarrhea, ascites, edema, marked enlargement of liver, oliguria, fever and jaundice b. Or with cutaneous erythema with desquamation compatible with Stevens Johnson syndrome c. Severe neurological complications as intolerable paraesthesia with paralysis, convulsion, or amaurosis. If any of the above occurs, they should be immediately reported to the National coordinator.

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14.4 Supportive care The physician can prescribe whatever he thinks is appropriate for pain, vomiting, constipation etc. However, other antimitotic agents than the recommended chemotherapy and any kinds of immunotherapy are not allowed to be prescribed. Laxatives should be prescribed when vincristine is given to prevent constipation. A diet containing no lactose, saccharose and gluten can be given as a prophylactic measure during abdominal irradiation. Pneumonitis prevention: Patients receiving the high-risk regimen, during relapse treatment and those who are treated with lung irradiation should receive oral cotrimoxazole or pentamidine nebulization for prevention of pneumocystis jiroveci pneumonia. G-CSF: especially in the high-risk regimens it might be necessary to use G-CSF if bone marrow recovery if not sufficient before the next course. In the high-risk regimen for stage IV WT, G-CSF is highly recommended. Also in case of severe infection in aplastic patients one could decide to use G-CSF. For patients treated according to non-high-risk regimens, supportive treatment with growth factors is permitted but not considered as essential. Transfusion: Erythrocyte and platelet transfusions may be given according to national or centre recommendations.

14.5 Modifications for small children or children below 7 months of age 14.5.1 General remarks In infancy, especially under the age of 7 months, the exact epidemiology and incidence of WT and of other renal tumours, like CMN) and malignant Rhabdoid Tumour of the kidney (MRTK) is not well known. From retrospective studies it has become clear that evidence based guidelines for the management of these young patients are difficult to retrieve, and that general recommendations are difficult to set up due to incompleteness of datasets. Especially data on incidence of non-WT, data on dosages of radiotherapy and chemotherapy and information about short and long-term toxicity are difficult to retrieve. In the past, the evaluation of patients with renal tumours under the age of 7 months has been hampered by incomplete registration as most study groups considered very young patients as study patients and not as protocol patients. Also, a selection bias may have occurred in the registration of less malignant diseases like CMN, which may have been registered relatively less frequently as compared to the relatively more malignant renal tumours. In order to get insight into the disease characteristics, treatment, toxicity and outcome of very young children with renal tumours the SIOP Renal Tumour Study Group decided to also prospectively register and evaluate all these patients under the age of 7 months. The aim is to design a prospective SIOP trial for these patients with the ultimate goal to improve the outcome and to limit acute toxic side effects and long-term sequelae of treatment in this young infant group. Recently, a retrospective collaborative study [3] was conducted among patients registered in the most recent protocols from SIOP, NWTSG, GPOH and UKCCSG. In the registry of SIOP9, SIOP93-01, NWTSG 3&4 and 5, COG and UKWT3, which altogether included 10.430 patients with a renal tumour, 750 patients were identified under the age of 7 months. The incidence of the several histological subtypes of renal tumours for this age group is shown in figure 14.5.1. Despite the fact that most of the patients

UMBRELLA Protocol SIOP 2016

with CMN are below 7 months of age the majority of patients had a Wilms tumour with an age rising increasing incidence whereas the incidence of CMN is falling with age (fig 14.5.2).

Figure 14.5.1. The distribution of renal tumours in children aged less than 7 months in the retrospective cohort from NWTSG 3&4, SIOP 9, SIOP 93-01 and UKCCSG-WT3.

Figure 14.5.2. Correlation between histology and age (in months) in infants with kidney tumours below 7 months of age.

UMBRELLA Protocol SIOP 2016

In the cohort of 750 patients, 523 patients were treated with primary surgery, 103 patients received preoperative chemotherapy, and in 124 cases it was unknown what treatment was given upfront. The overall survival in this retrospective cohort was 87% (WT 94%, CMN 96%, CCSK 51%, MRTK 16%). Information of the dosages of the drugs was available only in the minority of patients treated with chemotherapy. In general, it was recommended that in SIOP, patients should receive 66% and in NWTSG/COG 50% of the advised dose. Data on toxicity were scarce as well. Only in the group of 77 WT patients, younger than 7 months treated according to SIOP93-01 toxicity data were available. 30% grade III and IV hematologic toxicity was reported. There were no severe infections, no cardiologic, renal, gastrointestinal and neurological toxicity, whereas 4% of the patients developed VOD. In comparison, older WT children, treated according to SIOP 93-01, showed 35% hematological toxicity, 6% severe infections, less than 1% cardiologic and renal toxicity, 3% neurological toxicity and 5% VOD. A very prudent conclusion may be that the toxicity did not exceed the toxicity in the older age group. Finding general recommendations for effective dosages of chemotherapeutic drugs with a limited toxicity in this very young age group is a challenge and recommendations for such young children from other study groups need to be taken into consideration.

14.5.2 Treatment recommendations for patients with renal tumours below 7 months of age It is advised to follow the UMBRELLA part B protocol guidelines, with the following adjustments. Wilms tumours: a. As primary surgery is recommended in every patient below 7 months of age, each patient needs to be discussed within the multidisciplinary team to consider preoperative chemotherapy before going to primary surgery b. If radiotherapy is considered the indication should be discussed with the national coordinator and the radiotherapist for fixing the dose and the radiation field c. Stage II high risk WT patients: discuss radiotherapy with the national coordinator d. Stage I intermediate risk patients: discuss with the national coordinator if a wait and watch policy without post-operative chemotherapy is possible, as done by COG in favorable stage I histology with a tumour below 550 gram. Histological classification of WT into low, intermediate and high risk is different for patients, who received pre-operative chemotherapy from those who underwent primary surgery. This is important to notice, as most of the children aged below 7 months will be treated with immediate surgery.

Non-Wilms Tumours: Follow guidelines of the UMBRELLA part B protocol guidelines, with adjustment of the dosage of drugs according to age and body weight.

UMBRELLA Protocol SIOP 2016

14.5.3 Chemotherapy dose adjustment for infants and/or < 12 kg body weight

< 5 kg 5 - 12 kg > 12 kg (full dose) Vincristine* 0.033 mg/kg (50%) 0.05 mg/kg (66%) 1.5 mg/m2 Actinomycin D Omit # 30 µg/kg (66%) 45 µg/kg Doxorubicin 1.1 mg/kg (50%) 1.66 mg/kg (66%) 50 mg/m2 Carboplatin 4.4 mg/kg (50%) 6.6mg/kg (66%) 200 mg/m2 (x3) Cyclophosphamide 10 mg/kg (50%) 15 mg/kg (66%) 450 mg/m2 (x3) Ifosfamide 50% 66% 100% VP-16 Omit # (because of ethanol), 5 mg/kg (66%) 150/m2 (x3) Etopophos possible Melphalan 6.6 mg/kg 200 mg/ m2 * No reduction of vincristine dose after primary surgery in stage I favorable histology; #: discuss with the National coordinator; no actinomycin D or doxorubicin below 3 months of age.

14.5.4 Radiotherapy If possible radiotherapy should be avoided in these young infants, because of the increased risk of serious long-term toxicity. In case of diffuse anaplasia and/or local stage 3 disease it is advised to discuss the indication, radiation field and dose with the national coordinator before radiotherapy is administered.

14.6 References 1. Kathy Pritchard-Jones, et al., Doxorubicin omission from the treatment of stage II/III, intermediate risk histology Wilms tumour: results of the SIOP WT 2001 randomised trial. Lancet, 2015. 386: p. 1156-1164 2. Frühwald M: European Rhabdoid Registry. A multinational registry for rhabdoid tumours of any anatomical site. V3, 2014 3. van den Heuvel-Eibrink MM, et al.: Characteristics and Survival of 750 Children with a Renal Tumour in Infancy (0-6 months). A collaborative retrospective Study of the SIOP/GPOH/SFOP, NWTSG, and UK-CCSG. Pediatr Blood & Cancer, 2008, 50: p. 1130-1134

UMBRELLA Protocol SIOP 2016

15 Treatment guidelines for Wilms Tumours 15.1 Introduction / background Wilms’ tumour treatment is regarded as one of the success stories of paediatric oncology, based on the excellent treatment results due to interdisciplinary and international collaboration. Today, in subgroups with high survival rates the focus has shifted towards reducing acute toxicity and late effects of treatment. The results of SIOP2001 suggest that doxorubicin can be safely omitted from treatment regimens for localized intermediate risk stage II and III patients [1]. Even the majority of relapsed patients can be rescued today after initial treatment for low or intermediate risk disease. Nevertheless there are still subgroups of patients who have a poor outcome, like those with stage IV diffuse anaplasia and those with relapses after intensive primary treatment. For these patients new treatment options need to be defined. For that reason the sampling and analysis of biomaterial is of utmost importance within the SIOP 2016 UMBRELLA study to gain new knowledge for better treatments. Differences still exist between the two major treating groups (SIOP and COG) in terms of using preoperative chemotherapy. The Northern America based COG group remains committed to initial nephrectomy. They are the first group that started using biomarkers (LOH 1p and 16q) for treatment stratification. The European based SIOP group continues to administer pre-operative chemotherapy in an attempt to downstage the tumour and to reduce surgical complications resulting in less postoperative treatment intensity and in decreasing the number of patients who require radiotherapy. In addition in vivo response to preoperative chemotherapy can be used as a stratification parameter in the SIOP. Patients with a blastemal type WT after preoperative chemotherapy have a significantly poorer outcome than patients with intermediate risk tumours. Whilst the different initial treatments of COG and SIOP make direct comparison of results difficult, outcome in terms of event free survival and overall survival for the whole group of patients are very similar. The intention of the SIOP Renal Tumour Group (RTSG) is to treat patients according to the results of the SIOP 2001 WT trial. This allows the analysis of biomarkers in a large cohort of uniformly treated patients. A subgroup of patients showing persistence of chemotherapy-resistant blastemal cells after preoperative chemotherapy remains at high risk of relapse. New biomarkers for these tumours need to be defined. While this work is ongoing a true estimate of the volume of remaining blastema after preoperative chemotherapy seems to be important for outcome. To quantify this volume better, it is essential that tumour volumes are measured and documented at diagnosis and at the time of nephrectomy. In addition new imaging technologies, like DWI-MRI, may contribute to a better classification in the future. In the era of improved information technology, an opportunity exists to combine the continued collection of clinical data with heterogeneous research data in individual patients. European projects with the participation of members of SIOP-RTSG are ongoing and will provide such technologies to pate the study group. This will enable consistent central review and assignment of patients to risk groups and best treatment options. These treatment guidelines are recommendations and they are not a clinical trial. They are based on the available knowledge of today and can be regarded as state of the art treatment. However, if better treatments with higher survival rates or less toxicity become available these recommendations will be replaced by the new one to guarantee the best available treatment to all patients with renal tumours in childhood and adolescence.

UMBRELLA Protocol SIOP 2016

15.2 Treatment guidelines for localized Wilms Tumours (stage I – III) 15.2.1 Treatment recommendations localized WT  In children below the age of 7 months, primary surgery is only advised after an interdisciplinary evaluation of the individual risks of tumour rupture against preoperative chemotherapy.  Fine needle aspiration or tru-cut (core) biopsy, should be considered in case of serious doubt of WT. For details see appendix 4, section 19.4.8.

15.2.1.1 Pre-operative chemotherapy Two drugs (vincristine (VCR) and actinomycin D (ACT)) x 4 weeks (Figure 1)  Vincristine: 1.5 mg/m2 (max 2 mg) weeks 1, 2, 3, 4 (5th dose can be given if week 5 falls before planned surgery)  Actinomycin D: 45 µg/kg (max 2 mg), weeks 1, 3 Both drugs are given by intravenous bolus.

ACT 45 g/kg   VCR 1.5 mg/m2         Weeks 1 2 3 4 Surgery

Dose reductions (see table in section 14.5.3): Reassessment of the tumour by imaging at week 4 Surgery should be planned for week 5-6. In case of any delay (which is not advised) an extra dose of VCR is recommended.

15.2.1.2 Post-operative treatment After surgery, the different histological subtypes of nephroblastoma and local stage of the tumour can be determined. Combined with the volume of the tumour (pre-operative scan), these prognostic factors will dictate post-operative treatment (table 15.2.1). In this SIOP protocol, tumour volume has been added as a risk stratification factor for a subgroup of nephroblastomas. Patients with a tumour volume of > 500 ml after preoperative chemotherapy and non-stromal or non-epithelial intermediate risk histology and local stage II or III will be treated more intensively with AVD. This decision is based on analyses of patients from SIOP 2001, which demonstrated that tumour volume is a significant risk factor. (see fig 15.2.1). Furthermore, patients with a high tumour volume had a significantly poorer outcome, if randomized to only AV compared with AVD (see fig. 15.2.2). For patients with stage I intermediate risk

UMBRELLA Protocol SIOP 2016

or any stage with epithelial and stromal subtype, large tumour volume (500 ml) after pre-operative chemotherapy did not significantly affect the outcome.

Fig. 15.2.1: The martingale residuals and the Kaplan-Meier curves show that there is a significant association between tumour volume after preoperative chemotherapy and the risk of an event.

Fig. 15.2.2: Analysis of patients randomized to receive AV or AVD (SIOP2001-Trial): EFS and OS according to treatment given and tumour volume.

In summary, all patients with localized intermediate risk tumours are treated according to SIOP 2001. Only patients with stage II and III mixed type, regressive type and focal anaplasia will receive AVD if their tumour volume after preoperative chemotherapy is larger than 500 ml.

UMBRELLA Protocol SIOP 2016

Tumour Stage I Stage II Stage III volume after preoperative chemotherapy

Low Risk (only CN) All No further treatment AV2 AV2

Intermediate Risk ≤ 500 ml AV1 AV2 AV2 + RT

Intermediate Risk* > 500 ml AV1 AVD AVD + RT

BT All AVD HR-1 HR-1 + RT High Risk DA All AVD HR-1 + flank RT HR-1 + RT Table 15.2.1: Overview of postoperative treatment. (*with the exception of stromal and epithelial type, they are always treated independent of tumour size: AV1 in stage I and AV2 in stage II and III); CN: completely necrotic; A= actinomycin D, V= vincristine, D =doxorubicin (cumulative dose 250 mg/m²), HR = high risk histology; BT: blastemal type; DA: diffuse anaplasia; RT: radiotherapy; Note that for Stage 1 tumours with low risk histology, no postoperative chemotherapy is given. All tumours in this category should be send for urgent pathological central review (< 2 weeks for result))

Stage I, Low Risk Histology: No further treatment In WT with low risk histology and stage 1 no further treatment is given. It is important to have the result of central pathology review before any decision about postoperative treatment is made. In case of delay one further vincristine can be given.

Stage I, Intermediate Risk Histology: Regimen AV1 Important note: This treatment is given to all patients with local stage I. In GPOH in case of focal anaplasia, mixed and regressive type with a tumour volume > 500 ml after preoperative chemotherapy treatment is given according to stage I high risk (AVD).  Vincristine: 1.5 mg/m2 (max 2mg) weekly for 4 weeks (4 doses in total). The first dose is to be given once peristalsis is established following surgery and within 21 days of the last dose of preoperative chemotherapy.  Actinomycin D: 45 µg/kg (max 2mg), at week 2 (day 7) of postoperative regimen. Delayed until the absolute neutrophil count is >1.0 x109/l or platelet count >100 x 109/l Both drugs are given by intravenous bolus.

ACT 45 g/kg  VCR 1,5 mg/m2         Weeks 1 2 3 4

UMBRELLA Protocol SIOP 2016

Dose reductions (see table in section 14.5.3): Stage I, High Risk Histology: Regimen AVD Important note: This treatment is also given for patients with stage I, II and III and focal anaplasia, mixed or regressive type with tumour volume > 500 ml after preoperative chemotherapy. The total duration of the postoperative chemotherapy is 27 weeks.  Vincristine: 1.5 mg/m2 (max 2 mg) commenced when peristalsis established following surgery and within 21 days of pre-operative chemotherapy. Give weekly for 8 weeks (8 doses) and then on day one of weeks 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27 (20 doses in total)  Actinomycin D: 45 µg/kg (max 2mg), at week 2, 5, 8, 11, 14, 17, 20, 23, 26 (9 doses in total) Both drugs are given by intravenous bolus.  Doxorubicin: 50 mg/m2 in 2-6 hours infusion every 6 weeks to start in week two concurrently with the first dose of Actimomycin D and the second dose of Vincristine. Subsequent doses are given at weeks 8, 14, 20 and 26 (5 doses in total - total cumulative dose: 250 mg/m2) Actinomycin D and Doxorubicin should be delayed if the absolute neutrophil count is <1.0 x109/l or platelet count <100 x 109/l.

ACT 45 µg/kg      VCR 1.5 mg/m²             DOX 50 mg/m²                    Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Dose reductions (see table in section 14.5.3):

Stage II/III Low and Intermediate Risk Histology: Regimen AV-2 Important note: This treatment is given for all patients with local stage II and III. In GPOH in case of focal anaplasia, mixed and regressive type with a tumour volume > 500 ml after preoperative chemotherapy doxorubicin is added to AV-2 (treatment with AVD as for high risk stage I). The total duration of the postoperative chemotherapy is 27 weeks.  Vincristine: 1.5 mg/m2 (max 2 mg) commenced when peristalsis established following surgery and within 21 days of pre-operative chemotherapy. Give weekly for 8 weeks (8 doses) and then on day one of weeks 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27 (20 doses in total)  Actinomycin D: 45 µg/kg (max 2mg), at week 2, 5, 8, 11, 14, 17, 20, 23, 26 (9 doses in total) Both drugs are given by intravenous bolus. Treatment should be delayed if the absolute neutrophil count is <1.0 x109/l or platelet count <100 x 109/l.

UMBRELLA Protocol SIOP 2016

ACT 45 µg/kg      VCR 1.5 mg/m²                             Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Dose reductions (see table in section 14.5.3): Note: Stage III tumours do need local radiotherapy (see chapter 17)

Stage II/III High Risk Histology: High Risk Regimen HR-1 Total duration of postoperative treatment is 34 weeks. There are two alternating courses of chemotherapy given at 21-day intervals. Both combinations consist of 2 drugs. The first course starts as soon as the patient has recovered from surgery and clinical condition allows. This should be the case within 21 days after end of preoperative chemotherapy. Each cycle commences when absolute neutrophil count is > 1.0x109/l and platelet count >100 x 109/l provided rising WBC values.  Course 1: Cyclophosphamide and Doxorubicin Cyclophosphamide: 450 mg/m2 on days 1, 2 and 3 of weeks 1, 7, 13, 19, 25 and 31 (6 courses in total) Doxorubicin: 50 mg/m2 on day 1 of weeks 1, 7, 13, 19, 25 and 31 (6 courses in total) Doxorubicin can be started after the first dose of cyclophosphamide.  Course 2: Etoposide and Carboplatin Etoposide (VP16): 150 mg/m2 on days 1,2,3 of weeks 4, 10, 16, 22, 28 and 34 (6 courses in total) Carboplatin: 200 mg/m2 (or AUC = 2.65 see 14.2.1e) on days 1,2,3 of weeks 4, 10, 16, 22, 28 and 34 (6 courses in total). Consider dose reduction for carboplatin to 150 mg/m2 in case of hematotoxicity after previous course. Treatment should be delayed if the absolute neutrophil count is <1.0 x109/l or platelet count <100 x 109/l. G-CSF can be given if delays of treatment or grade 4 neutropenia did occur after the first 2 – 4 cycles. Use of Cotrimoxazole is recommended for HR regimens as PCP prophylaxis.

VP16 150 mg/m2   CARBO 200 mg/m2   CYCLO 450 mg/m2    DOX 50 mg/m2      RT             

Weeks  1------2------3----- 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 

 = Echocardiography: at start of treatment, before week 19, 31 and at end of treatment  = GFR (measure at every third course, or more frequently if there is evidence of renal dysfunction.

UMBRELLA Protocol SIOP 2016

Dose reductions (see table in section 14.5.3): Note: Only stage II tumours with diffuse anaplasia do need local radiotherapy (not for stage II blastemal type. All stage III tumours do need local radiotherapy (see chapter 17)

15.2.2 Surgical recommendations For surgical recommendations, see chapter 12.

15.2.3 Radiotherapeutic recommendations For radiotherapeutic recommendations see chapter 13.

15.2.4 Chairs and members of the Stage I-III WT Panel

Name Profession Country Email

Chair Norbert Graf Oncologist Germany [email protected]

Marry van den Oncologist The m.m.vandenheuvel-eibrink@ Co-Chair Heuvel-Eibrink Netherlands prinsesmaximacentrum.nl

Tomás Acha Oncologist Spain [email protected]

Christophe Bergeron Oncologist France [email protected]

Beatriz Camargo Oncologist Brazil [email protected]

Jan Godzinski Surgeon Poland [email protected]

Rhoikos Furtwängler Oncologist Germany [email protected]

Kathy Pritchard- Oncologist UK [email protected]

Jones Members Christian Rübe Radiotherapist Germany [email protected]

Anne Smets Radiologist The [email protected] Netherlands

Filippo Spreafico Oncologist Italy [email protected]

Harm van Tinteren Statistician The [email protected] Netherlands

UMBRELLA Protocol SIOP 2016

Arnauld Verschuur Oncologist France [email protected]

Gordan Vujanic Pathologist UK [email protected]

UMBRELLA Protocol SIOP 2016

15.3 Treatment guidelines for metastatic Wilms Tumours (stage IV) 15.3.1 General remarks These guidelines do not cover all possible clinical situations. If necessary, contact your national PI or the international PIs for discussing your patient with stage IV Wilms tumor. Description of lung lesions is made as outlined in Part A Section 11.7.3 and 19.4.4 of this protocol. Biopsy of lesions suspected of metastasis at other site than in the lungs, including extra-abdominal lymph nodes, should be considered if reasonably feasible but is not mandatory (Part A Section 11.7.4). All lesions have to be re-assessed prior to nephrectomy. In case of incomplete response surgical clearance should be attempted where feasible and safe without risk of long-term morbidity (For detailed information see chapter 16). If there is a discrepancy between the histology group of the renal tumour versus that of the resected nodules/metastases, the therapeutic strategy should be adapted to the “highest” risk of histology. In principle and where possible flank and pulmonary irradiation should be administered at the same time (if indicated) to minimize toxicity of overlapping fields.

15.3.2 Pre-operative chemotherapy Preoperative treatment for patients having lung nodules is stratified according to the size of lung nodules (see below). The same reasoning applies not to other nodules (e.g. liver, etc.). Nodules of 1-2 mm are not classified as lung metastases and will be treated with 4 weeks of preoperative AV, as are localized tumours. They need to be reassessed prior to nephrectomy with chest CT. Nodule(s) of 3-5 mm are classified as lung metastases. These patients are to be treated with preoperative AVD. Re-assessment of the lung lesions prior to tumour nephrectomy is indicated in order to direct postoperative treatment. In case of persisting nodules, it is recommended – wherever possible - to perform excision of nodules in order to examine the histology and achieve a clearance of the chest disease if reasonable possible. If a complete clearance is not achievable, it is recommended to remove representative and accessible nodules. This will help directing therapy in the post-operative phase. If the histology of resected nodules rules out viable or necrotic malignant tissue, consider treating as localized tumour. If histology shows viable or necrotic malignant metastasis or biopsy is not feasible, continue according to Stage IV recommendations according to stage of local tumour (at least postoperative treatment according to stage II), histology group and complete/incomplete resection of nodules (see below). If a complete clearance of nodules is achieved at tumour nephrectomy in patients with LR or IR histology, this group of patients can receive a reduced cumulative dose of doxorubicin during postoperative treatment (AVD150). Nodules ≥ 5 mm, are classified as lung metastases. These patients are to be treated with preoperative AVD chemotherapy and reassessed prior to nephrectomy to direct postoperative treatment according to the recommendations for Stage IV. Patients having metastasis at any other site are to be treated with preoperative AVD and reassessed prior to nephrectomy. In case of incomplete response surgical clearance should be aimed for if feasible, safe and without long-term morbidity (see also chapter 16).

UMBRELLA Protocol SIOP 2016

15.3.2.1 Pre-operative Reassessment of metastasis This table per se is not decisional for the treatment strategy but is useful for clinical-radiological qualification and analysis of outcome.

Target lesion Non-target lesion Overall response (≥ 5 mm) (initially < 3 mm)* CR CR CR No lesions > 2 mm and Non PD or SD and VGPR no new lesion no new lesions > 30% response and Non PD and PR no new lesion no new lesions SD and Non PD and SD no new lesion no new lesions > 20% increase or PD or PD new lesions new lesions Table 15.3.1: Definition of metastatic response after preoperative chemotherapy: WT-Absolute-RECIST- Merge ("WARM") CT slice thickness 1mm, otherwise 2 x CT-slice thickness. Target lesions must be at least twice the size of the CT-slice thickness. Relative response is calculated based on the sum of all target-lesion’s diameters initially and at re-assessment. In case of doubt, contact national Principal Investigator (PI). ).* For target lesions of 3-5 mm, the only response categories are CR and non-CR.

Reassessment imaging of local tumour and metastases/nodules should be performed after preoperative chemotherapy and before surgery (see radiology guidelines, section 11.7) using the same technique as at diagnosis. Response criteria for metastasis to preoperative chemotherapy are defined in Table 15.3.1 and are needed to direct further treatment.

15.3.3 Surgery Nephrectomy and metastasectomy must be carried out as detailed in the surgery guidelines see Chapter 16. Intralesional excisions with potential tumour spill must be avoided at all instances.

15.3.4 Post-operative chemotherapy Surgical and radiotherapy guidelines are given in chapters 16 (surgery) and 17 (radiotherapy). Table 2 in this chapter shows a flow chart of strata according to metastatic response.

15.3.4.1 Post-operative chemotherapy for patients with nodules of <3 mm Post-operative chemotherapy is given according to the results of the chest CT after pre-operative AV:

UMBRELLA Protocol SIOP 2016

 If the chest CT shows no nodules, then postoperative treatment is given according to local stage and histology as in localized disease. Imaging controls of the chest is recommended every 8 - 12 weeks for the first two years after diagnosis (Part A Section 11.7.3).  In case of persisting nodules, at least a representative resection of nodules should be performed if feasible. o Only if histology rules out any viable or shows only necrotic tumour, continue with treatment for localized disease according to stage and histology. o If biopsy shows viable tumour treat with AVD 250 (meaning 150 mg/m2 of cumulative dose of doxorubicin as no preoperative doxorubicin was given; (see chapter 15.3.3 and 15.3.5) and reassess at W10. If persisting micronodules at that time whole lung RT is recommended (see chapter 17.7). o In case of diffuse anaplasia whole lung irradiation is indicated if metastasis are histologically verified (please contact the national PI). o If biopsy is not feasible, continue with treatment for localized disease according to histology with a minimum of AV2, regardless of stage. Reassess at W10 and contact PI if persisting nodules.  In case of increasing size of nodules, at least a representative resection of nodules has to be performed if feasible. o Only if histology rules out any viable or shows only necrotic tumour, continue with treatment for localized disease according to histology with a minimum of AV2. o If biopsy shows viable tumour treat with AVD 250 (meaning 150 mg/m2 of cumulative dose of doxorubicin as no preoperative doxorubicin was given; (see chapter 15.3.3 and 15.3.5) and reassess at W10. If persisting micronodules at that time whole lung RT is recommended (see chapter 17.7). o In case of diffuse anaplasia whole lung irradiation is indicated if metastasis are histologically verified (please contact the national PI). o If biopsy is not feasible, continue with treatment for localized disease according to histology AVD, regardless of stage. Reassess at W10 and contact PI if persisting nodules.

15.3.4.2 Post-operative chemotherapy for patients with nodules of > 3 mm There are four post-operative scenarios: A Metastasis/nodules absent (CR or VGPR as defined in table 15.3.1) or completely removed by the surgeon and IR or LR histology. A1 Lung nodules ≥ 3 and ≤ 5mm at diagnosis and LR/IR histology. A2 Lung nodules > 5 mm at diagnosis and LR/IR histology. A3 Complete surgical removal of only non-malignant tissue: If viable or necrotic malignant tissue is ruled out and other non-malignant histology has been shown (no proof of previous metastases) and complete resection of lesions is performed, proceed with postoperative treatment according to local stage (localized disease, see chapter 15.2).

UMBRELLA Protocol SIOP 2016

B Metastasis/nodules ≥ 3mm at diagnosis incompletely removed or multiple inoperable nodules and LR histology of the primary tumour. C Metastasis/nodules ≥ 3mm at diagnosis incompletely removed or multiple inoperable nodules and IR histology of the primary tumour. D Patients with high-risk histology of the primary tumour (including those that are in CR after preoperative chemotherapy and surgery) or histologically proven progressive disease of metastases during preoperative treatment (Exception: stromal predominant histology - discuss with national PI).

15.3.4.3 Post-operative treatment of group A1 Group A1: Local Stage I/II/III, low and intermediate Risk Histology. Metastatic Clearance (CR) of lung nodules ≥3-5mm obtained by chemotherapy or completely removed by surgeon. Recommended treatment: Regimen AVD150 Of Note: If complete response is achieved by resection of nodules and viable tumour is found radiotherapy is recommended (see chapter 17). Otherwise contact PI. AVD150 is still recommended provided there has been radiological response to preoperative AVD. Patients with local stage III IR receive flank/abdominal irradiation.

15.3.4.4 Post-operative treatment of group A2 Group A2: Local Stage I/II/III, low and intermediate Risk Histology; Clearance (CR/VGPR) of nodules > 5 mm at diagnosis obtained by chemotherapy or completely removed by surgeon. Recommended Treatment: Regimen AVD250 Of Note: If complete response is achieved by resection of nodules and viable tumour is found radiotherapy is recommended (see chapter 17). Otherwise contact PI. AVD250 is still recommended provided there has been radiological response to preoperative AVD. Patients with local stage III IR receive flank/abdominal irradiation.

15.3.4.5 Post-operative treatment of group A3 Group A3: Local Stage I/II/III, low or intermediate risk histology with complete surgical clearance of other non-malignant histology: Recommended Treatment: According to local stage for localized disease (see 15.2). Note: Patients with local stage III IR receive flank/abdominal irradiation. No pulmonary irradiation is indicated. For details go to chapter 17.

15.3.4.6 Post-operative treatment of group B Group B: Local Stage I/II/III, Low Risk Histology with residual nodules/metastasis after chemotherapy and surgery:

UMBRELLA Protocol SIOP 2016

It is recommended to resect (one)/multiple representative nodules. Postoperative treatment is recommended according to histology. The following options are possible:  If no proof of metastases (i.e. viable or necrotic malignant tissue has been ruled out), consider proceeding with treatment according to local stage (provided multiple, representative nodules have been resected and remaining nodules seem doubtful for metastases). Contact the national PI to decide on postoperative chemotherapy.  If no viable tumour but necrotic nodules in a representative number of metastases, proceed with regimen AVD150 postop for 27 weeks with cumulative dose of doxorubicin of 150 mg/m2 (see 15.3.5.2). Repeat CT assessment in week 10. If nodules are still visible: reconsider complete resection. No pulmonary radiotherapy.  If viable tumour in resected lung nodules, proceed with regimen AVD250 (see 15.3.5.3) and radiotherapy to the lungs (see chapter 17). In that case it is not a LR tumour and a switch to CDCV regimen may be considered (contact national PI).  If representative lung nodules cannot be resected, proceed with regimen AVD250 postop for 27 weeks with cumulative dose of doxorubicin of 250 mg/m2 (see 15.3.5.3) and carry out reassessment CT at week 10. If nodules are still visible, reconsider resection of at least 1 nodule. In addition consider lung irradiation since the lung nodules may not be LR histology. Of note: Local and lung irradiation should be given simultaneously to avoid overlapping fields. Irradiation may be postponed to week 10 for this purpose. However the indication of RT in LR tumours should be discussed with national PI since mostly not indicated.

15.3.4.7 Post-operative treatment of group C Group C: Local Stage I/II/III, Intermediate Risk Histology with residual metastatic disease. It is recommended to resect (one)/multiple nodules. The indication of obtaining a complete surgical remission is depending on the number, size and location of the nodules (see 16.2.2). At least, if the number of nodules at diagnosis and at surgery is limited (<10 at diagnosis, < 6 at surgery) a complete resection of lung nodules should be discussed with reference surgeons. Postoperative treatment is recommended according to histology and achieved clearance of metastasis. The following options are possible:  If no proof of metastases (i.e. viable or necrotic malignant tissue has been ruled out), consider proceeding with treatment according to localized disease (provided multiple nodules have been resected and remaining nodules seem doubtful for metastases). Contact the national PI to decide on postoperative chemotherapy. If multiple nodules disappeared after preoperative AVD and remaining nodules are non-malignant, postoperative AVD250 can still be indicated.  If no viable tumour but necrotic nodules in a representative number of nodules is found, proceed with regimen AVD250 postop for 27 weeks with cumulative dose of doxorubicin of 250 mg/m2 (see 15.3.5.3). Carry out chest CT in week 10. If nodules are still visible: reconsider complete resection or pulmonary radiotherapy. In case of persistent viable lung metastasis at week 10 pulmonary radiotherapy is indicated.  If viable tumour in resected nodules is found, proceed with 4 drugs regimen postoperatively for 34 weeks (see 15.3.5.4) and reassess at post-OP week 10. Pulmonary radiotherapy is indicated even if CR can be achieved at week 10 (see chapter 17).

UMBRELLA Protocol SIOP 2016

 If representative nodules cannot be resected, proceed with 4 drugs regimen postop for 34 weeks (see 15.3.5.4) and reassess at post-OP week 10. Pulmonary radiotherapy is indicated if persisting nodules. In case of local stage III it is highly recommended to combine radiotherapy to the flank and to the lung to avoid overlapping fields (see chapter 17). This can be delayed to post op week 10 if necessary. Of note: Other sites of metastasis than the liver and lungs receive radiotherapy independent of response, if they cannot be resected completely. Patients with local stage III IR receive flank/abdominal irradiation.

15.3.4.8 Post-operative treatment of group D Group D: Local Stage I/II/III with high risk histology regardless of metastatic status and patients with progressive disease and intermediate risk histology (stromal predominant excluded) and histologically proven metastasis. This group has a particularly poor outcome despite intensive 4-drug treatment and therefore there has been reconsideration of the approach to treatment. Only few patients per year will fall into this group D and therefore local centres should ask the PI of stage IV to get advice for the best current treatment approach. Experts’ opinion of the SIOP-RTSG board considers the approach as depicted in Appendix 6 as the best available treatment option, but alternative treatment options can be discussed.

15.3.5 Treatment schedules for Stage IV 15.3.5.1 Preoperative AVD Group: All Stage IV with lung nodules >2 mm (See 15.3.1) Three drugs (vincristine (VCR), actinomycin D (ACT) and doxorubicin) x 6 weeks  Vincristine: 1.5 mg/m2 (max 2 mg) weeks 1, 2, 3, 4, 5, 6  Actinomycin D: 45 µg/kg (max 2 mg) weeks 1, 3, 5  Doxorubicin: 50 mg/m2 weeks 1, 5 Vincristine and actinomycin D are given by intravenous bolus, doxorubicin as a 4-6 hours infusion. ACT 45 g/kg    VCR 1.5 mg/m2       DOX 50 mg/m2         Weeks 1 2 3 4 5 6 Surgery

Dose reductions (see table in section 14.5.3): Treatment in weeks 1, 3 and 5 is blood count dependent and delay is indicated if neutrophils are <1000/µl and/or blood values are still decreasing. In case of severe and unexpected thrombocytopenia consider VOD-diagnostics and treatment. In exceptional cases, a week 7 dose of vincristine can be administered if there is some delay of surgery. However, nephrectomy should preferably be performed in week 7 and not later than week 8.

UMBRELLA Protocol SIOP 2016

Reassessment imaging of local tumour and metastases should be performed at week 6/7 (see radiology guidelines) using the same technique as at diagnosis. Response criteria to preoperative chemotherapy are defined in Table 15.3.1 (see above).

15.3.5.2 Regimen AVD150 Group A1 Patients The total duration of the postoperative chemotherapy is 27 weeks.  Vincristine: 1.5 mg/m2 (max 2 mg) commenced when peristalsis established following surgery and within 21 days of pre-operative chemotherapy. Give weekly for 8 weeks (8 doses) and then on day one of weeks 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27 (20 doses in total)  Actinomycin D: 45 µg/kg (max 2mg), at week 2, 5, 8, 11, 14, 17, 20, 23, 26 (9 doses in total) Both drugs are given by intravenous bolus.  Doxorubicin: 50 mg/m2 in 4-6 hours infusion once in week 2 concurrently with the first dose of Actimomycin D and the second dose of Vincristine. Subsequent doses are not given! (1 dose in total - total cumulative dose including pre-operative treatment: 150 mg/m2)

ACT 45 µg/kg      VCR 1.5 mg/m²             DOX* 50 mg/m²     RT              Weeks 1 2< ---- 3------4----- >5 6 7 8* 9 10 11 12 13 14* 15 16 17 18 19 20* 21 22 23 24 25 26* 27 28 * No doxorubicin in weeks 8, 14, 20 and 26, cumulative dose 150 mg/m2 Echocardiogram in week 1, if abnormal before each doxorubicin, and at end of treatment Actinomycin D and doxorubicin should be delayed if the absolute neutrophil count is <1.0 x109/l or platelet count <100 x 109/l. Chemotherapy cycles can be adapted to avoid use of Doxorubicin within 14 days of radiotherapy. Reduce dose of ACT by 1/3 when RT is given within 14 days of this administration.

Dose reductions (see table in section 14.5.3): Note: Patients with local stage III receive abdominal/flank irradiation. No pulmonary irradiation is indicated, unless viable malignant tissue is found in the completely resected lung nodules. For details go to chapter 17.

15.3.5.3 Regimen AVD250: Groups: A2, B, and C only in the case of resection of representative and exclusively necrotic nodules The total duration of the postoperative chemotherapy is 27 weeks.

UMBRELLA Protocol SIOP 2016

 Vincristine: 1.5 mg/m2 (max 2 mg) commenced when peristalsis established following surgery and within 21 days of pre-operative chemotherapy. Give weekly for 8 weeks (8 doses) and then on day one of weeks 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27 (20 doses in total)  Actinomycin D: 45 µg/kg (max 2mg), at week 2, 5, 8, 11, 14, 17, 20, 23, 26 (9 doses in total) Both drugs are given by intravenous bolus.  Doxorubicin: 50 mg/m2 in 4-6 hours infusion every 6 weeks to start in week 2 concurrently with the first dose of Actimomycin D and the second dose of Vincristine. Subsequent doses are given at weeks 8 and 14 (3 doses in total - total cumulative dose including pre-operative treatment: 250 mg/m2)

ACT 45 µg/kg      VCR 1.5 mg/m²             DOX* 50 mg/m²       RT              Weeks 1 2< ---- 3------4----- >5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20* 21 22 23 24 25 26* 27 28 * No doxorubicin in weeks 20 and 26, cumulative dose 250 mg/m2 Echocardiogram in week 1, if abnormal before each doxorubicin, and at end of treatment Actinomycin D and doxorubicin should be delayed if the absolute neutrophil count is <1.0 x109/l or platelet count <100 x 109/l. Chemotherapy cycles can be adapted to avoid use of Doxorubicin within 14 days of radiotherapy. Reduce dose of ACT by 1/3 when RT is given within 14 days of this administration.

Dose reductions (see table in section 14.5.3): Note: Patients with local stage III receive flank/abdominal irradiation. Irradiation to metastasis of other sites than lungs is indicated, if not resected or viable metastasis. Pulmonary irradiation is indicated in case of viable lung metastasis at any point. For details go to chapter 17.

15.3.5.4 4-Drug-Regimen (HR-2) Groups: C where nodules are viable and incompletely resected or representative resection is not feasible Total duration of the postoperative treatment is 34 weeks. There are two alternating courses of chemotherapy given at 21-day intervals. The first course starts as soon as the patient has recovered from surgery and clinical condition allows. This should be the case within 21 days after the end of the last preoperative course of chemotherapy. Each cycle commences when absolute neutrophil count is > 1.0x109/l and platelet count >100 x 109/l provided rising WBC values.  Course 1: Cyclophosphamide and Doxorubicin in weeks 1, 7, 19 and 31 (4 courses in total) Cyclophosphamide: 450 mg/m2 on days 1, 2 and 3; infusion time 1 hour Doxorubicin: 50 mg/m2 on day 1; infusion time 2 - 6 hours Cumulative dose of doxorubicin including pre-operative chemotherapy 300 mg/m2

UMBRELLA Protocol SIOP 2016

Doxorubicin to be started after the first dose of Cyclophosphamide  Course 2: Etoposide and Carboplatin in weeks 4, 10, 13, 16, 22, 25, 28 and 34 (8 courses in total) Etoposide (VP16): 150 mg/m2 on days 1, 2, 3; infusion time 1 hour Carboplatin: 200 mg/m2 on days 1, 2, 3; infusion time 1 hour, Consider dose reduction for carboplatin to 150 mg/m2 in case of hematotoxicity after previous course.

VP16 150 mg/m2    CARBO 200 mg/m2    CYCLO 450 mg/m2   DOX 50 mg/m2           < ---- --RT------ >    Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Echocardiogram before first doxorubicin in week 1, if abnormal before each doxorubicin and at the end of treatment. Radiotherapy for local stage III is postponed to week 10 (= assessment of persisting nodules) in order to combine with pulmonary RT if required. If viable metastases were confirmed at time of nephrectomy, RT to this organ is indicated. If at week 10, complete metastatic clearance has been observed, omit the last dose of doxorubicin in order to limit the cumulative cardiotoxicity of RT and doxorubicin. Treatment should be delayed if the absolute neutrophil count is <1.0 x109/l or platelet count <100 x 109/l. G-CSF can be given if delays of treatment or grade 4 neutropenia occurs. Use of Cotrimoxazole is recommended for this regimen as PCP prophylaxis. Dose reductions (see table in section 14.5.3): Note: All stage III tumours do need local radiotherapy (see chapter 17).

15.3.6 Recommended Treatment adjustments – Steering during treatment Actinomycin D, Doxorubicin, Cyclophosphamide, Etoposide and Carboplatin should be delayed if:  The absolute neutrophil count is <1,0 x109/l and not-rising tendency or  Platelet count <100 x 109/l Actinomycin D should be delayed if:  Bilirubine > 1.5 x ULN  ALAT > 5 x ULN  Clinical signs of VOD Doxorubicin should be delayed if:  Bilirubine > 2 x ULN  ALAT > 5 x ULN  Grade I mucositis

UMBRELLA Protocol SIOP 2016

If a next course is delayed for > 1 week: consider dose reduction of 33% for the next course. Consider giving G-CSF in case of HR treatment. If kidney function is severely damaged contact the National PI for further advice. 15.3.7 Radiotherapy For a detailed radiotherapy guideline for both primary and metastatic site see chapter 17. Please note recommendation to delay flank irradiation to be given with pulmonary irradiation where possible.

15.3.8 Follow-up Detailed information on follow-up is given in section 11.6.5.

15.3.9 Treatment Recommendation Overview The following table (Table 15.3.2) gives an abridged overview of most clinical situations with Stage IV nephroblastoma. This table does not substitute the more detailed information in the respective chapters!

Overall Metastasis Nephro- Treatment Res- Surgery blastoma ponse histology

CR/ VGPR LR/IR & lung AVD150, no pulmonary RT unless complete resection of still nodules 3-5 mm viable metastasis  pulmonary RT LR/IR & lung AVD250, no pulmonary RT unless complete resection of viable nodules >5 mm metastasis  pulmonary RT or other site

LR/IR No evidence of Treatment as localized (Chapter 15.2) Surgical Surgical complete resectionneeded if viable tumour PR/SD LR Viable AVD250, lung/metastasis RT, CT at week 10 metastasis  if remaining nodules  surgery

confirmed recommended to achieve CR if feasible Completely AVD150, CT at week 10  if remaining necrotic nodules  surgery recommended to achieve metastasis CR if feasible LR/IR No evidence of Contact principal investigator potentially viable tumour treatment as localized (Chapter 15.2) or AVD250, CT week 10  if remaining nodules  surgery recommended to achieve CR if feasible, no RT to metastatic site(s) IR Viable 4 drug regimen, RT to metastasis. CT week 10 metastasis  if remaining nodules  surgery confirmed recommended to achieve CR if feasible

Representativenodule resection feasible Completely AVD250 regimen, CT week 10  if remaining necrotic nodules  surgery recommended to achieve metastasis CR if feasible

UMBRELLA Protocol SIOP 2016

Resection LR AVD250, CT week 10  reconsider not feasible resection and discuss RT to metastasis IR 4 drug regimen,  if remaining nodules RT to metastasis indicated PD IR Metastasis 4 drug regimen, metastasis RT. CT week 10  confirmed if remaining nodules  surgery

recommended to achieve CR if feasible

No evidence of AVD250, CT week 10  if remaining nodules easible

f viable or  surgery  if viable metastasis  CDCV +

Representative necrotic tumour RT to metastases indicated: contact PI (these noduleresection situations will be very rare). All All HR Ask PI for advice, metastasis RT  if remaining nodules consider resection if feasible Mixed Indicated Confirm metastatic disease by histology  if metastasis  treat according to worst histology and worst response

Table 15.3.2: Treatment overview

15.3.10 Chairs and members of the Stage IV WT Panel

Name Profession Country Email

Chair Arnauld Verschuur Oncologist France [email protected]

Vice- Rhoikos Oncologist Germany Rhoikos.Furtwaengler@unikliniku Chair Furtwängler m-saarland.de

Marry van den Oncologist The m.m.vandenheuvel-eibrink@ Heuvel-Eibrink Netherlands prinsesmaximacentrum.nl

Lisa Howell Oncologist UK [email protected]

Claudia Pasqualini Oncologist France [email protected]

Norbert Graf Oncologist Germany [email protected]

Members Annalisa Serra Oncologist Italy [email protected]

Christian.Ruebe@uniklinikum- Christian Rübe Radiotherapist Germany saarland.de

[email protected] Steven Warmann Surgeon Germany tuebingen.de

UMBRELLA Protocol SIOP 2016

The Anne Smets Radiologist [email protected] Netherlands

Hervé Brisse Radiologist France [email protected]

Harm van Tinteren Statistician The [email protected] Netherlands

Gordan Vujanic Pathologist UK [email protected]

UMBRELLA Protocol SIOP 2016

15.4 Management of bilateral disease (stage V) and bilaterally-predisposed unilateral Wilms Tumour The aim of these recommendations is to consider all bilateral diseases since the goal is to spare as much renal function as possible by preserving as much normal renal tissue as possible. This guideline deals with bilateral WT, bilateral nephroblastomatosis (NB), unilateral WT with NB on the other side, and WT in a single kidney or in a horseshoe kidney. In addition patients with a unilateral WT and a syndrome that is associated with a predisposition of bilateral tumour development will be managed according to this guideline.

15.4.1 BACKGROUND 15.4.1.1 Results of prior studies Synchronous bilateral WT (BWT- Stage 5) is reported to account for 5% of all nephroblastoma patients. A major challenge in BWT is to achieve high cure rates while preserving as much functional renal tissue as possible for preserving a renal function sufficient for normal growth and development. The outcome of children with BWT has improved with therapeutic advances in SIOP and COG protocols. In 1989, Coppes et al. reported a 10-year overall survival of 69% for patients with synchronous BWT treated with neoadjuvant chemotherapy and surgery [1]. More recently, different studies reported a long-term OS about 80 % [2-4]. The most significant risk factor for overall survival (OS) and event free survival (EFS) is local stage with stage III tumours doing worse than stage I and II [1, 3]. The excellent outcome is the result of a strong cooperation between different specialists in international studies and individual treatment approaches according to the tumour response. The most significant morbidity is the risk of end-stage renal disease (ESRD) in this sub-group of patients with an incidence of 9% to 12% [3-6]. The risk of ESRD increases in patients with greater than 50% loss of renal tissue. Aronson et al. [7] have observed that functional renal outcome is significantly improved after bilateral nephron sparing surgery (NSS) compared to other types of surgery. Independently of the type of treatment, children with WAGR, Denys-Drash or other syndromes associated with WT1 gene mutations, are at higher risk of ESRD [6]. Therefore avoiding total nephrectomy at initial surgery is advised. In order to facilitate this goal SIOP and COG have recommended the same strategy over the past two decades: • Front-line chemotherapy to decrease tumour volume as much as possible • Nephron-sparing surgery as often as possible • Adjuvant chemotherapy adjusted to the highest histological type and local stage of the tumours

15.4.1.2 Rationale for the current guideline in BWT Factors that could contribute to improved outcomes of patients with BWT are: 1. Increasing efficiency of diagnostic imaging tools Bilateral involvement is diagnosed by imaging in > 95% of the cases [3, 8-9]. It is very important to perform a complete radiological examination to establish number, size, aspects and extension of the tumours at diagnosis and during follow up. The same exam with the same sequences needs to be done during all follow-up imaging studies. CT scan and MRI are very sensitive modalities to detect small

UMBRELLA Protocol SIOP 2016

lesions [10]. MRI (with sequences before and after contrast agent administration and with T1, T2 and STIR sequences) seems more sensitive to differentiate nephroblastomatosis (NB) and Wilms tumour [11] and to detect nephrogenic rests [11]. In patients with multicentric bilateral tumours, it has become apparent that many lesions thought to be WT are indeed hyperplastic NR. Quantitative MRI techniques (diffusion Weighted imaging with calculation of ADC) might help in terms of early prediction of treatment response [10]. Moreover, it is important to consider the ionizing hazards of CT in these patients who will need many imaging studies. The same is true for patients with a predisposition syndrome under surveillance of developing a WT. These are more than 20% of patients in this group. In conclusion for this population, MRI seems to be the best choice for staging and assessment of treatment response. If renal MR Angiograpy is not feasible CT scan with 3D reconstruction may be recommended for the preoperative planning in order to visualize the vascular pedicles and assess feasibility of NSS. In rare cases PET-CT may help to distinguish between active lesions and scars. In such cases the National coordinator should be contacted. 2. Neoadjuvant chemotherapy a. For BWT, in order to facilitate NSS In SIOP 2001, the recommended chemotherapy following imaging diagnostic is the standard preoperative chemotherapy (VCR + Actinomycin D). The response to this therapy was evaluated for the first time after 4 weeks of chemotherapy, then every 4 weeks. If tumour volume decreased further after 8 weeks [12], the GPOH group has reported an increased risk of progression when chemotherapy is prolonged over 3 months [13]. Moreover, the French experience in SIOP 93 demonstrated that 3 months of preoperative chemotherapy allow performing NSS in 67% of the kidneys [3]. In non-responsive tumours, many studies demonstrated that it is ineffective to prolong or change the neoadjuvant chemotherapy regimen. Many of these patients have a stromal subtype that is related to WT1 mutations or they have an unfavorable histology that needs to be ruled out. In that situation definitive surgery is recommended or if only bilateral tumour-nephrectomies are possible a biopsy should be done to determine histology and to continue with adequate further treatment [2-3, 14]. In the UMBRELLA protocol the optimal duration of preoperative chemotherapy and the strategy to adopt in non-responsive tumour will be addressed. Time intervals for evaluation are fixed to 6 weeks to be comparable with COG who do start with a three-drug regimen (AVD). b. For NB, in order to prevent Wilms tumour development and preserve renal units Nephroblastomatosis (NB) or diffuse hyperplastic perilobar nephrogenic rests (DHPLNR) is recognized as a pre-neoplastic proliferative process associated with a high risk of developing WT. There is a lack of data regarding the outcome of children with this diagnosis. Nevertheless, there are many data, which strongly support the fact that chemotherapy reduce the number of cells capable of malignant transformation and reduce the risk of developing WT in both kidneys [15, 16]. In a study of 52 patients, Perlmann et al. found that patients, who received adjuvant chemotherapy and more or less surgery, had a lower risk to develop WT than those who did not receive chemotherapy [24%]. The SIOP WT 2001 trial recommended a treatment plan with prolonged chemotherapy with vincristine and actinomycin D, 2 agents recognized as effective against NB in children [17]. Prasil et al. emphasize the importance of duration of chemotherapy and the risk of leaving lesions in the kidney [18]. If, despite chemotherapy, children with Diffuse Hyperplastic Perilobar Nephrogenic Rests (DHPLNR) show no response in images or even a progression of the lesions, as well as persisting heterogeneity of the lesions, or development of spheric lesions, they are suspected to develop WT and immediate surgery is needed. Nephron sparing surgery (NSS) may safely allow disease-free survival with sparing a

UMBRELLA Protocol SIOP 2016

maximum of healthy parenchyma in both kidneys [19]. This conservative management is preferred to nephrectomy because of a real renal function advantage. Sometimes, chemotherapy alone can cure some patients. 3. Optimizing surgical recommendations BWT should be treated with a tailored individual approach according to tumour response and the possibility of NSS. The main surgical aim is to perform, if possible, bilateral NSS to preserve as much functional renal tissue as possible. The less involved kidney should be operated on first. Ritchey et al. reported that complete nephrectomy on one side with NSS on the opposite side can be a good solution, providing that enough functional renal tissue can be preserved [20]. Different techniques of NSS have been described [9; 21-22]. Surgical resections can be multiple and repeated for one kidney. NSS can also be repeated in case of local recurrence with acceptable oncologic outcome and preservation of renal function [23]. Further details are given in chapter 16.2.3. The documentation of NSS should be done according to chapter 16.1.2, where the classification of NSS is described for unilateral cases. This classification will be used also for bilateral cases. The rarity of stage V tumours, unilateral tumour associated with nephroblastomatosis and unilateral disease with predisposition syndrome, raises the question, to refer these patients to few highly experienced surgical centers to offer these patients the highest possibility of bilateral NSSs. 4. Optimizing radiotherapy indications and doses In previous SIOP protocols, radiotherapy is indicated for local stage III or stage II disease with diffuse anaplastic histology. Doses, as low as 15 Gy, when given after actinomycin D, have resulted in the development of clinical radiation nephritis [24]. Moreover, a study of 100 COG patients who had undergone unilateral nephrectomy followed by irradiation to the remaining kidney or surgical bed and chemotherapy showed that renal complications developed only in those patients receiving dose > 12 Gy [25]. Thus, it is recommended that the dose to the remaining kidney should not exceed 12Gy. A remaining question is the efficiency of radiotherapy for positive resection margins. Sometimes partial nephrectomy with safe margins is not possible and bilateral nephrectomy might seem the only way out. In the experience of the American Pediatric Surgery Association, local recurrence rates after NSS were not affected by surgical margin status although all patients with positive margins received Radiotherapy and strategy with incomplete surgery followed by radiotherapy (10Gy) and chemotherapy may result in long-term remission [26]. This option should always (including blastemal predominant subtype after neoadjuvant chemotherapy) be taken into account with the exception of diffuse anaplasia, where in case of positive margins a complete nephrectomy should be strongly considered followed by radiotherapy. Further details are given in section 17.6. 5. Update biology knowledge Germline WT1 mutations (locus 11p13) have been described in WT and Huff et al. provide direct molecular data supporting the two-hit mutational model for WT [27]. Loss of heterozygosity (LOH) for DNA markers located at chromosome 11p13, 11p15, 16q, and 1p has been reported to occur in a minority of WTs. Grundy et al [28] found significant associations between the presence of intralobar nephrogenic rests and LOH for both 11p13 and 11p15. LOH for chromosome 16q and 1p were identified as associated with a significantly worse outcome [29]. These genetic factors need to be analyzed prospectively also in BWT to allow a better risk stratification and find better treatment options in BWTs patients. When relevant biomarkers will be validated, the stratification of bilateral Wilms tumour and unilateral tumours should go in parallel.

UMBRELLA Protocol SIOP 2016

To gain further insight into different genetic events and their corresponding biological role, molecular cytogenetic techniques such as array comparative genomic hybridization (array CGH) need to be performed. Such a technique could lead to identify new chromosomal regions involved in Wilms’ tumourigenesis and more particularly in bilateral disease. More details are given in sections 4.1, 5, 7.3 and 19.7. 6. Optimizing the renal follow-up Patients with BWT are at increased risk of ESRD. There are some experimental studies that suggest that the remaining nephrons after subtotal nephrectomy are subject to chronic hyperfiltration, leading to azotemia, proteinuria and glomerulosclerosis [30]. Patients with less than 50% of preserved renal tissue can have adequate renal function but develop hypertension or microalbuminuria requiring treatment [31]. Some patients may require transplantation. Thus, patients with BWT or unilateral tumour and predisposing syndrome should be long-term monitored for microalbuminuria, proteinuria, hypertension and level of renal function (serum creatinine and glomerular filtration rate). This follow-up should be done by a specialist in nephrology. More details are given in chapter 11.6.5. The incidence of cardiac impairment and hypertension in bilateral cases will be analysed to drive recommendations for further usage of anthracyclines in bilateral cases.

15.4.2 GOALS AND OBJECTIVES (scientific aims of the study) 1. To evaluate the impact of limiting duration of neoadjuvant chemotherapy to 12 weeks. The following evaluation criteria will be considered:  Five-year OS and EFS for all patients and comparison according to local stage and histology  Local stage repartition in tumours operated at week 12  Percentage of NSSs done and the level of preservation of renal parenchyma according to the classification of NSS. We want to keep the same rate of NSSs as in the previous protocol. 70% of NSSs is expected.  Long-term renal function depending on NSS 2. To evaluate a new strategy for patients with non-responsive tumours after 6 weeks of VA/V that is defined as follow: in case of stable or progressive disease at first assessment (week 6), an interdisciplinary discussion needs to take place. An early modification of the chemotherapy regimen can be decided and two courses of etoposide + carboplatin performed. The following evaluation criteria will be considered:  Histology repartition found in these patients with non-responsive disease  Percentage of partial or very good partial response to etoposide-carboplatin courses  Five-year EFS and OS in this subgroup of patients  Long-term renal function 3. To evaluate the efficacy of two-drugs chemotherapy in preventing WT development in children with DHPLNR and preserving renal parenchyma. To establish the optimal duration of this chemotherapy. The following evaluation criteria will be considered:

UMBRELLA Protocol SIOP 2016

 Percentage of patients presenting WT development during chemotherapy or during post- treatment follow-up.  Histology repartition of WT in these patients  Percentage of no surgery, NSS and nephrectomy performed in these patients  Duration of chemotherapy necessary to let all lesions disappear on imaging  Long-term renal function  Five-year OS and EFS 4. To facilitate NSS in lieu of nephrectomy in children with unilateral tumours and Beckwith- Wiedemann, WAGR, Deny-Drash syndrome, aniridia, genitourinary anomaly or hemi-hypertrophy by prolonging neoadjuvant 2 drugs chemotherapy as in BWT. The following evaluation criteria will be considered:  Duration of neoadjuvant chemotherapy  Percentage of NSS  Percentage of metachronous tumours  Five-year OS and EFS  Long-term renal function 5. To evaluate accuracy of MRI in distinguishing NR, NB and WT and its utility in predicting conversion of NR in WT. For this aim, a prospective central review of images will be performed. 6. To facilitate NSS by prospective central review with surgical expertise. Surgery should only be performed at centers specialized in the treatment of this disease. Each patient needs to be discussed in the context of a national surgical meeting to take decisions with the advice of national experts. Patients with BWT should benefit from an experienced surgeon in NSS. 7. To perform biology studies on biomaterial from blood, urine and tumour pieces of all patients (at least 75%) with bilateral disease as described in the UMBRELLA protocol. The objective is to identify prognosis factors, possible factors associated with renal toxicity and to better understand tumour genesis. 8. To homogenize renal function follow-up, particularly with EDTA clearance and systematic annual consultations by nephrologists in order to determine the prevalence and type of renal dysfunction.

UMBRELLA Protocol SIOP 2016

15.4.3 TREATMENT PLAN 15.4.3.1 General Recommendations

1. Radiology guidelines These are detailed in the main protocol UMBRELLA (Chapter 11.7)

2. Chemotherapy guidelines Doses and administration of all drugs are as recommended in the protocol for unilateral tumours. In case of non-response (SD, PD or NSS is not possible) in the preoperative phase a change of treatment to carboplatin / etoposide is recommended according to the following schedule: VP16 150 mg/m2   CARBO 200 mg/m2        Weeks 1 2 3 4 5

3. Surgical guidelines Patients with BWT should benefit from an experienced surgeon in renal parenchyma-sparing procedures. Thus:  Each patient should be discussed in the context of a national surgical meeting to take decision with the advice of national experts.  Surgery will be performed only at centers specialized in the treatment of this disease. Further details are given in chapter 16.2.3.

4. Radiotherapy guidelines Recommendations for doses and site are similar to the recommendations for unilateral disease. When indicated, it shall begin concurrent with the initiation of adjuvant chemotherapy. Further details are given in section 17.6.

5. Pathology and Biology guidelines Recommendations for pathologists for the study of surgical specimens are similar to the recommendations for unilateral disease. A central pathology review is needed too. Details are given in section 11.8.

UMBRELLA Protocol SIOP 2016

15.4.4 Treatment plans In all of the following treatment plans drugs and doses are according to unilateral WT. Even if NSS is possible after the first assessment, it needs to be discussed during a multidisciplinary meeting if further shrinkage with chemotherapy would be beneficial to spare more normal kidney.

15.4.4.1 Patients with bilateral Wilms Tumour

UMBRELLA Protocol SIOP 2016

15.4.4.2 Patients with unilateral Wilms Tumour and contralateral nephroblastomatosis

UMBRELLA Protocol SIOP 2016

15.4.4.3 Patients with bilateral or unilateral nephroblastomatosis

UMBRELLA Protocol SIOP 2016

15.4.4.4 Patients with unilateral tumour and predisposition syndrome as cited previously These patients are at high risk of metachronous bilateral tumour and need to benefit from a strategy with NSS if possible. Therefore they are included in treatment options for bilateral disease.

UMBRELLA Protocol SIOP 2016

15.4.5 Chairs and members of the Bilateral WT Panel

Name Profession Country Email

Christophe Oncologist France [email protected] Chair Bergeron

Hélène SUDOUR- Oncologist France [email protected] BONNANGE

Georges Audry Surgeon France [email protected]

Monica Terenziani Oncologist Italy [email protected]

Davide Biasoni Surgeon Italy [email protected]

The Lieve Tytgat Oncologist [email protected] Netherlands

Rhoikos Rhoikos.Furtwaengler@uniklinikum- Oncologist Germany Furtwängler saarland.de

Dietrich von [email protected] Surgeon Germany Schweinitz muenchen.de

Jörg Fuchs Surgeon Germany [email protected]

[email protected] Jens-Peter Schenk Radiologist Germany

Members heidelberg.de

Ivo Leuschner Pathologist Germany [email protected]

Gema Ramirez- Oncologist Spain [email protected] Villar

Rosa Cabello Surgeon Spain [email protected]

Kathy Pritchard- Oncologist UK [email protected] Jones

Harm van Tinteren Statistician The [email protected] Netherlands

The Geert Jansen Radiotherapist [email protected] Netherlands

NN Geneticist

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15.4.6 References

1. Coppes MJ, deKraker J, vanKijken PJ, et al.: Bilateral Wilms’ tumor: Long-term survival and some epidemiological features. J Clin Oncol, 1989. 7: p. 310-315 2. Thomas E. Hamilton, MD, Michael L. Ritchey, MD, et al.: The Management of Synchronous Bilateral Wilms Tumor: A Report from the National Wilms Tumor Study Group. Ann Surg, 2011. 253: p. 1004–1010 3. Sudour H, Audry G, Schleimacher G, et al. Bilateral Wilms Tumors (WT) Treated With the SIOP 93 Protocol in France: Epidemiological Survey and Patient Outcome. Pediatr Blood Cancer, 2012. 59: p. 57–61 4. Indolfi P, Jenkner A, Terenziani M et al. Synchronous Bilateral Wilms Tumor. A Report From the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP). Cancer, 2013. 119: p. 1586-1592 5. Ritchey ML, Green DM, Thomas P, et al.: Renal failure in Wilms tumor. Med Pediatr Oncol, 1996. 26: p.75-80 6. Breslow N, Collins AJ, Ritchey ML, Grigoriev YA, et al. End-stage renal disease in patients with Wilms tumor: results from the national Wilms Tumor Study Group And the United States renal data system. J Urol, 2005. 174: p. 1972-1975 7. Aronson DC, Slaar A Heinen RC, et al. Long-term outcome of bilateral wilms tumors. Pediatr Blood Cancer, 2011. 56: p. 1110-1113 8. Kumar R, Fitzgerald R, Breatnach F. Conservative surgical management of bilateral Wilms’ tumor: Results of the United Kingdom Children’s cancer Study Group. J Urol, 1998. 160: p. 1450– 1453 9. Horwitz JR, Ritchey MR, Moksness J, et al. Renal salvage procedure in patients with synchronous bilateral Wilms’ tumor: A report from the National Wilms’ Tumor Study Group. J Pediatr Surg, 1996. 31: p. 1020–1025 10. Owens CM, Brisse HJ, Olsen ØE et al. Bilateral disease and new trends in Wilms tumor. Pediatr Radiol, 2008. 38: p. 30–39 11. Gylys-Morin V, Hoffer FA, Kozakewich H et al. Wilms tumor and nephroblastomatosis: imaging characteristics at gadolinium-enhanced MR imaging, Radiology, 1993. 188: p. 517-521 12. Lemerle J, Voute PA, Tournade MF, Rodary C, et al. Effectiveness of preoperative chemotherapy in Wilms tumor : results of an international society of paediatric Oncology (SIOP) clinical trial. J Clin Oncol, 1983. 1: 604-609 13. Graf N, Reinhard H, Bürger D, et al. for the SIOP 93-01/GPOH study committee. Bilateral Wilms tumor treated according to SIOP 93-01/GPOH. Oral communication at the 35th Congress of the International Society of Paediatric Oncology (SIOP), Cairo, Egypt, 8–11 October 2003. Med Ped Oncol, 2003. 41: SIOP XXXV Meeting Abstracts: O040 , p. 267 14. Shamberger RC, Ritchey Ml, Hamilton TE, et al. Bilateral Wilm’s Tumor with progressive disease or non responsive disease. J Pediatr Surg, 2006. 41: p. 652-657 15. Cozzi F, Zani A, Cozzi DA, Schiavetti A. Letter to the editor. Management of hyperplastic nephroblastomatosis. Pediatr Blood Cancer, 2006. 46: p. 263 16. Perlman EJ, Faria P, Soares A, Hoffer F, et al. Hyperplastic perilobar nephroblastomatosis: long- term survival of 52 patients. Pediatr Blood Cancer, 2006. 46: p. 203-221 17. Kumar AP, Pratt CB, Coburn CP, Johnson WW, et al. Treatment strategy for nodular renal blastema and nephroblastomatosis associated with Wilms’tumor. J Pediatr Surg, 1978. 13: p. 281-285 18. Prasil P, Laberge JM, Bond M, Bernstein M, et al. Management decisions in children with nephroblastomatosis. Med Pediatr Oncol, 2000. 35: p. 429

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19. Cozzi F, Schiavetti A, Cozzi DA, Morini F, et al. Conservative management of hyperplastic and multicentric nephroblastomatosis. J Urol, 2004. 172: p. 1066-1070 20. Ritchey Ml, Coppes MJ. The management of synchronous bilateral Wilms tumor. Hematol Oncol Clin North Am, 1995. 9: p. 1303-1315 21. Gruner M, Audry G, Heloury Y. Partial nephrectomy in nephroblastoma. Chir Pediatr, 1985. 26: p. 300-304 22. Fuchs J, Wunsch P, Flemming P, et al. Nephron-sparing surgery in synchronous bilateral Wilms tumors. J Pediatr Surg, 1999. 34: p. 1505-1509 23. Kieran K, Williams MA, McGregor LM, Dome JS, Krasin MJ, Davidoff AM. Repeat nephron-sparing surgery for children with bilateral Wilms tumor. J Pediatr Surg, 2014. 49: p. 149-53 24. Bond J. Radiation nephropathy. Lancet, 1976. 2: p. 207 25. Mitus A, Tefft M, Fellers F. Long-term follow-up of renal function of 108 children who underwent nephrectomy for malignant disease. Pediatrics, 1969. 44: p. 912-921 26. Kieran K, Williams MA, Dome JS, et al. Margin status and tumor recurrence after nephron- sparing surgery for bilateral Wilms tumor. J Pediatr Surg, 2013. 48: p. 1481–1485 27. Huff V, Miwa H, Haber DA et al. Evidence for WTI as a Wilms Tumor (WT) Gene: Intragenic Germinal Deletion in Bilateral WT. Am J Hum Genet, 1991. 48: p. 997-1003 28. Grundy P, Telzerow P, Moksness J, Breslow NE. Clinicopathologic correlates of loss of heterozygosity in Wilm's tumor: a preliminary analysis. Med Pediatr Oncol, 1996. 27: p. 429-33 29. Spreafico F, Gamba B, Mariani L et al. Loss of Heterozygosity Analysis at Different Chromosome regions in Wilms Tumor confirms 1p Allelic Loss as a Marker of worse prognosis: A Study from the Italian Association of Pediatric Hematology and Oncology. J Urol, 2013. 189: p. 260- 267 30. Anderson S, Meyer TW, Brenner BM. The role of hemodynamic factors in the initiation and progression of renal disease. J Urol, 1985. 133: p. 363-368 31. Kubiak R, Gundeti P, Ransley PG, Wilcox DT. Renal function and outcome following salvage surgery for bilateral Wilms tumor. J Pediatr Surg, 2004. 39: p. 1667-1672

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15.5 Treatment guidelines after primary surgery The following treatment recommendations are based on the United Kingdom experience in UKW1 & 2 studies [1, 2]. Modifications to the length of therapy, total dose of anthracycline and treatment of focal anaplasia have been made in the light of published data from NWTS 4 and 5 study [3-7]. Note that the recommended management of infants less than 7 months of age or teenagers above 16 years with a primary intrarenal tumour is immediate nephrectomy. Risk group assignment in cases treated with immediate nephrectomy is based solely on tumour stage and presence of unfavourable histology (anaplasia). Blastemal predominant type belongs to favourable histology. 15.5.1 Staging Initial and follow-up diagnostics during treatment is given in section 11.6.4. Definition of tumour stage will be as for tumours receiving pre-operative chemotherapy except that the concept of “regressive changes/necrotic tumour” will not be applicable. It is of particular importance to assign correct local stage. Lymph nodes must be adequately sampled at time of nephrectomy (see surgical guidelines, chapter 16). 15.5.2 Histological classification The SIOP pathological risk group B applies to tumours that have not received pre-operative chemotherapy. Note that the presence of large amounts of viable blastema is of no prognostic significance in immediate nephrectomy specimens. Histological classification after immediate nephrectomy is done according to Vujanic et al. [8] and shown here: Pathological risk group for primary operated cases [8]: I LOW RISK TUMOURS - Mesoblastic nephroma - Cystic partially differentiated nephroblastoma II INTERMEDIATE RISK TUMOURS - Non-anaplastic nephroblastoma and its variants - Nephroblastoma - focal anaplasia III HIGH RISK TUMOURS - Nephroblastoma – diffuse anaplasia - Clear cell sarcoma of the kidney - Rhabdoid tumour of the kidney

15.5.3 Post-operative chemotherapy regimens for tumours having primary excision Patients with low risk tumours do not receive postoperative chemotherapy even in stage II and III. Please contact the National Coordinator in case of low risk and stage III for further advice. For dose modifications see section 14.5.3

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15.5.3.1 Regimen 1 (intensive VCR): Stage I, intermediate risk (excluding focal anaplasia).  Vincristine: 1.5 mg/m2 (maximum dose 2 mg) weekly for 10 weeks (10 doses in total). The first dose is to be given once peristalsis is established following surgery.

VCR 1.5 mg/m²           | | | | | | | | | | Week 1 2 3 4 5 6 7 8 9 10 Total duration of therapy: 10 weeks

Note: Infants and children < 12 Kg should be given full dose vincristine when the drug is used alone, unless there are signs of toxicity in which case the subsequent dose should be reduced by 50% followed by cautious increases if tolerated.

15.5.3.2 Regimen 2 (AV): Stage I, focal anaplasia and Stage II, intermediate risk  Vincristine: 1.5 mg/m2 (maximum dose 2 mg) weekly for 11 weeks and then three weekly, at weeks 14, 17, 20, 23 and 26 (16 doses in total)  Actinomycin D: 45 µg/kg (maximum dose 2 mg) at weeks 2, 5, 8, 11, 14, 17, 20, 23 and 26 (9 doses in total)

VCR 1.5 mg/m²            ACT-D 45 µg/kg _     | | | | | | | | | | | Week 1 2 3 4 5 6 7 8 9 10 11 14 17 20 23 26 Total duration of therapy: 26 weeks

Note: Infants and children < 12 Kg receive lower doses of both drugs when given in combination (see section 14.5.3).

15.5.3.3 Regimen 3 (sequential AVD): Stage III intermediate risk (includes focal anaplasia)  Vincristine: 1.5 mg/m2 (maximum dose 2 mg) weekly for 10 weeks and then three weekly, at weeks 13, 16, 19, 22, 25 and 28 (16 doses in total)  Actinomycin D: 45 µg/kg (maximum dose 2 mg), 50% dose at week 2* then full dose at weeks 10, 16, 22, 28 (5 doses in total).  Doxorubicin: 50 mg/m2 at weeks 7, 13, 19, 25 (4 doses (200 mg/m2) in total). Each dose to be infused over 4-6 hours

VCR 1.5 mg/m²          ACT-D 22.5 g/kg * DOX 50 mg/m²  | |<-15Gy | RT---> | | | | | | Weeks 1 2 3 4 5 6 7 8 9

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VCR 1.5 mg/m²     ACT-D 45 g/kg   DOX 50 mg/m²   | | | | | | | | | | Weeks 10 11 12 13 14 15 16 17 18 19 22 25 28 *Note 2/3 of dose because of closeness to radiotherapy

Total duration of therapy: 28 weeks. Note: Infants and children < 12 Kg receive lower doses of both drugs when given in combination (see section 14.5.3). *Abdominal radiotherapy (14.4 Gy) to be given weeks 2 – 4.

15.5.3.4 Stage IV patients Stage IV patients having immediate nephrectomy should be few in number and confined to patients presenting as surgical emergencies with unrecognised lung or liver metastases. They should be treated with the three drug “preoperative” chemotherapy for stage IV tumours. Metastatic response should be evaluated at week 6 by chest CT. Subsequent chemotherapy is dictated according to whether or not metastatic complete remission has been achieved by chemotherapy +/- surgery, as per the main protocol recommendations including indications for radiotherapy (see chapter 17). Note: Blastemal predominant tumours after immediate surgery are belonging to the intermediate risk group. 15.5.3.5 Post-operative chemotherapy for high risk histology tumours having primary excision Diffuse anaplasia and CCSK Stages I - IV - SIOP ‘high risk’ post operative chemotherapy (see chapter 15.2 and 18.1). Abdominal radiotherapy is given to local abdominal stages II and III. Lung radiotherapy is given to all stage IV cases with lung metastases, regardless of metastatic response to chemotherapy/surgery. MRTK Include these patients into the EU-RHAB Protocol (see chapter 18.3).

15.5.3.6 Guidelines for radiotherapy after immediate nephrectomy The guidelines given in chapter 17 for radiotherapy apply also for patients with immediate surgery. On case local radiotherapy is needed it should start within 10 days after surgery. Despite the fact that a delay of radiotherapy for more than 10 days did not significantly influence flank or abdominal tumour recurrence in patients with favorable histology WT treated on NWTS-3 and NWTS-4 they were unable to test for a meaningful difference, because of the concentration of RT delay was close to 10 days [9].

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15.5.4 Chairs and members of the primary surgery panel

Name Profession Country Email

Chair Jan Godzinski Surgeon Poland [email protected]

Tomás Acha Oncologist Spain [email protected]

Christophe Oncologist France [email protected] Bergeron

Beatriz Camargo Oncologist Brazil [email protected]

Jan Godzinski Surgeon Poland [email protected]

Marry van den Oncologist The m.m.vandenheuvel-eibrink@ Heuvel-Eibrink Netherlands prinsesmaximacentrum.nl

Rhoikos Oncologist Germany [email protected] Furtwängler

Norbert Graf Oncologist Germany [email protected]

Ivo Leuschner Pathologist Germany [email protected]

Kathy Pritchard- Oncologist UK [email protected]

Jones Members

Christian Rübe Radiotherapist Germany [email protected]

Anne Smets Radiologist The [email protected] Netherlands

Serena Catania Oncologist Italy [email protected]

Harm van Statistician The [email protected] Tinteren Netherlands

Cees van de Ven Surgeon The [email protected] Netherlands

Arnauld Oncologist France [email protected] Verschuur

Gordan Vujanic Pathologist UK [email protected]

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15.5.5 References 1. Pritchard J, Imeson J, Barnes J, Cotterill S, Gough D, Marsden HB, Morris-Jones P, Pearson D. Results of the United Kingdom Children’s Cancer Study Group first Wilms tumour study. J Clin Oncol, 1995. 13: p. 124-133. 2. Mitchell C, Morris Jones P, Kelsey A, Vujanic G, Marsden B, Shannon R, Gornall P, Owens C, Taylor R, Imeson J, Middleton H & Pritchard J for the UKCCSG. The treatment of Wilms tumour: results of the UKCCSG second Wilms tumour study. Brit J Cancer, 2000. 83: p. 602-608. 3. Green D et al. Effect of duration of treatment on treatment outcome and cost of treatment for Wilms tumour: a report from the NWTSG. J Clin Oncol, 1998. 16: p. 3744-3751. 4. Green D et al. Comparison between single dose and divided dose administration of dactinomycin and doxorubicin for patients with Wilms tumour: a report from the NWTSG. J Clin Oncol, 1998. 16: p. 237-245. 5. Dome JS, Cotton CA, Perlman EJ et al. Treatment of anaplastic histology Wilms’ Tumor: results from the fith National Wilms’ Tumor Study. J Clin Oncol, 2006. 24: p. 2352-8 6. Malogolowkin M, Cotton CA, Green DM et al. Treatment of Wilms tumor relapsing after initial treatment with vincristine, actinomycin D, and doxorubicin. A report from the National Wilms Tumor Study Group. Pediatr Blood Cancer, 2008. 50: p.236-41 7. Grundy PE, Green DM, Dirks AC et al. Clinical significance of pulmonary nodules detected by CT and Not CXR in patients treated for favorable histology Wilms tumor on national Wilms tumor studies-4 and -5: a report from the Children's Oncology Group. Pediatr Blood Cancer, 2012. 59: p. 631-5 8. Vujanic GM, Sandstedt B, Harms D et al., Revised International Society of Paediatric Oncology (SIOP) Working Classification of Renal Tumors of Childhood. Med Pediatr Oncol, 2002. 38: p. 79-82 9. Kalapurakal JA, Li SM, Breslow NE et al. Influence of radiation therapy delay on abdominal tumor recurrence in patients with favorable histology Wilms' tumor treated on NWTS-3 and NWTS-4: a report from the National Wilms' Tumor Study Group. Int J Radiat Oncol Biol Phys, 2003. 57: p. 495-9

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15.6 Treatment guidelines for relapsed Wilms tumours 15.6.1 Introduction and background The outcome for children with WT has improved significantly with the refinement of multimodal therapy, and overall survival (OS) rates are approaching 90% [10, 11, 15, 16, 63]. Approximately 15% of patients with intermediate risk WT and 50% of patients with anaplastic or post-chemotherapy blastemal-type WT experience recurrence [33]. Most recurrences occur within two years of diagnosis, although in rare cases relapses have developed later [19]. The general profile of relapse site shows that the lungs and pleura alone account for 50-60%; abdominal recurrences make up to 30% of relapses (isolated abdomen or combined to other sites), while other sites (brain or bone) are involved alone in 10-15% of cases [13, 34, 35]. Whereas the salvage rate for patients with recurrent favorable-histology WT was historically 25 to 40% [8, 21], overall modern treatment combinations have improved outcome after recurrence up to 60% [5]. However, the evidence for the optimal retrieval therapy for recurrent WT comes from limited experiences, more often concerning unselected groups of patients. Because recurrent WT is infrequent, early phase clinical trials on novel agents are scanty and no randomized questions have been answered comparing different promising regimens. A general principle for the treatment of relapsed WT is to use agents not used for primary therapy. Phase 2 trials demonstrated efficacy of ifosfamide (52% objective responses) [31], etoposide (42% responses) [24], and carboplatin (52% responses) [4] either as single agents or as combinations [26; 64]. In a review of 54 cases involved in consecutive trials at St. Jude Children’s Hospital, Dome et al. underline that outcome has improved noticeably since around the mid-eighties, when these drugs became available [5]. More recently, investigators at St. Jude Children’s Research Hospital (Memphis) documented the activity of topotecan (48% responses in favorable histology WT) [20]. The effective use of high-dose therapy with autologous stem cell rescue (ASCR) for the treatment of recurrent WT has been reported by several groups, with survival rates up to 70% [23]. In 1994 the European Bone Marrow Transplant (EBMT) group published a first series of 25 children [65]. In the following period other European and American experiences have been reported [2, 14, 23, 29]. Since the first EBMT report, the number of WT patients registered in the EBMT registry treated with ASCR has grown to more than 300 cases (personal communication 2015, data from the EBMT registry). Nevertheless, the best combination, dose-intensity and -density, and duration of chemotherapy regimens remain poorly explored.

15.6.2 Prognostic factors and risk stratification at relapse As in the case of newly diagnosed WT, what emerges from the results with recurrent WT is that they are clinically and biologically heterogeneous. A number of potential prognostic features influencing outcome post-recurrence have been analyzed, but it is somehow difficult to separate whether these factors are independent of each other (Table 15.5.1) [reviewed in 66]. Moreover the prognostic factors appear to be changing over time as therapy for primary and recurrent WT evolves. In addition, risk assessment to manage upfront WTs is evolving differently among different protocols (SIOP or COG), and this will likely render more difficult to adopt a common method to stratify all patients at relapse. Grundy and co-workers provided a comprehensive analysis of prognostic indicators after relapse on National Wilms Tumour Studies (NWTS)-2 and 3 [13]. The time to recurrence was predictive of survival, with those patients who relapsed early (0 to 5 months from nephrectomy) having worse outcome than those who relapsed later than 6 months. Other adverse factors were (focal and diffuse) anaplastic

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histology, advanced tumour stage, and non-localized relapse outside the lung. In addition, the group of patients treated initially with 2-drugs fared better than patients treated initially with 3 drugs, indicating that initial treatment remains a powerful prognostic factor. The SIOP initially identified adverse prognostic factors for relapsed WT including initial stage 4 disease, anaplastic histology (at that time the blastemal type WT was not yet identified as an adverse prognostic histology, and focal and diffuse anaplasia were treated the same), time to recurrence 6 months of less after diagnosis and recurrence in multiple organs or a previously irradiated field [25, abstract]. The German group analyzed their cohort of 170 relapsed patients and in accordance with the previous studies initial stage III or IV, high-risk histology (according to the SIOP classification [36]), early relapse (usually 6 months from nephrectomy) and combined site of relapse emerged as relevant prognostic indicators [28]. The explanation for the difference in outcome between pulmonary and abdominal relapses may be that many of the abdominal recurrences occur in irradiated fields, whereas most lung recurrences developed in unirradiated sites. In addition, it is sometimes difficult to extrapolate the precise recurrence site in the published reports, with “lung relapses” category including not only pulmonary parenchymal relapses but also those involving the mediastinum. Abdominal recurrence generally involves the original tumour bed (kidney area), but can correspond to retroperitoneal lymph nodes, liver, peritoneum or contralateral kidney as well. Most “recurrences” in the contralateral kidney probably represent second primary tumours rather than true relapses. For these reasons, the site of relapse should deserve more attention and analysis before being definitively considered for patient risk grouping.

Factor Significant Not Significant

Treatment received NWTS-2,-3 [13]

Relapse within the RT field SIOP93-01 [23]

Early relapse (0-5 months) GPOH [7]; NWTS-2,-3 [13]; St. Jude [5]; AIEOP [29]; NWTS-5 [12, 18] SIOP93-01 [23]

Initial stage IV SIOP93-01 [23], GPOH [7, 28]

Site of relapse (combined vs local) NWTS-2, -3 [13]; SIOP93-01 [23], GPOH St Jude [5]; NWTS-5 [12, 18]; AIEOP [7, 28] [29]

High risk histology GPOH [7]; NWTS-2,-3 [13]; St Jude [5]; SIOP [23]

Age at relapse GPOH [7, 28]

Gender (male) NWTS-2,-3 [13] GPOH [7] Table 15.6.1: Potential prognostic features influencing outcome post-recurrence analyzed.

Overall, features that are clearly associated with a worse outcome after relapse are diffuse anaplasia (little is known about focal anaplasia) or SIOP high-risk histology (diffuse anaplasia and blastemal type)

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and initial chemotherapy including doxorubicin [13, 25, 27, 59]. It is less clear whether time to recurrence remains prognostically significant with contemporary therapy. Interestingly, the more recent experience from NWTS-5 showed that time to recurrence and site of recurrence were no longer prognostically significant [12, 18]. However, it is likely that factors originally identified as predictors of survival, like combined site of relapse or short recurrence-free interval, can lose their significance when more aggressive and effective regimens, like the ones adopted in recent years, are applied at relapse. Furthermore, the changes in initial disease therapy, now evolving towards less intense treatment, may eventually influence risk categories at relapse and therefore outcome at recurrence. The more conservative upfront use of radiation in the recent era may have allowed for increased use at relapse, contributing to the improved patient outcome. Worthy of note is the fact that even some stage I or II tumours also received radiation therapy in older protocols. Based on current most consistent data, three risk categories for recurrent WT have been proposed and retrospectively analyzed, relying on the initial treatment received more than on histological type of the primary tumour (table 15.5.2) [59, 66]: 1) Standard risk (SR): defined as patients suffering from relapse after initial therapy with only vincristine and/or actinomycin-D (no doxorubicin a/o radiotherapy given). These patients usually correspond to stage I or II non anaplastic WT or tumour classified as stage I or II, low/intermediate risk histology in the more recent SIOP 2001 protocol. This group is expected to have event-free survival (EFS) estimates in the 70-80% range, as the results reported for the NWTS-5 or United Kingdom Children’s Cancer and Leukaemia Group (UKCCLG) recurrent protocols (UKW-R) [12, 37]. These situations account for 30% of recurrences. 2) High risk (HR): defined as patients with relapse after therapy with three or more agents. These patients, accounting for 45-50% of the children with WT who relapse, are expected to have survival rates in the 40-50% range [18]. 3) Very high risk (VHR): defined as patients with recurrent diffuse anaplastic or post-chemotherapy blastemal-type WT, originally receiving ≥ 4 drugs. These patients have an unfavorable prognosis, and are expected to have survival rates in the 10% range [28, 37]. This group accounts for 10- 15% of all WT relapses.

Risk group Reason % of all N. Expected relapses cases/year EFS?

Standard Initially received vincristine ± 30% 15 70% actinomycin D

High Doxorubicin already given 50-60% 30-35 50%

Very high Anaplastic histology 10-15% 6-8 10% Blastemal-type after CT (SIOP) (HR-CT already given) Table 15.5.2: Published risk stratification at relapse according to the most consistent prognostic features

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15.6.3 Rationale for treatment of relapsed WT with standard-risk features from previous studies Published experiences focusing on relapsing patients matching the above discussed criteria for the SR group definition are limited. The NWTS group treated a cohort of 58 relapsing children initially given only vincristine and actinomycin D, regardless of site or timing of relapse. Relapse treatment included surgical excision, when feasible, radiation therapy and alternating courses of vincristine/doxorubicin/cyclophosphamide and etoposide/cyclophosphamide (stratum B of the NWTS-5 relapse protocol) [12]. Post-relapse 4-year EFS was 71.1% and 4-year OS was 81.8% for all patients. Sixty-four percent of these children relapsed within the first 11 months after nephrectomy, but this did not negatively affect outcome. The UKW-R protocol was the other prospective analysis on relapsed patients classifiable as SR. Preliminary data displayed OS of 88% in 20 patients, of whom 10 children, who initially received only vincristine, were rescued with intensive vincristine, actinomycin D and doxorubicin, and an additional 10 children, initially receiving two-drug chemotherapy, were rescued with 8 alternating courses of doxorubicin/cyclophosphamide and cyclophosphamide/etoposide [37]. Despite there are anecdotal experiences with rescue therapies including only vincristine, actinomycin D and doxorubicin in very selected children relapsing after initial two-drug therapy, to our knowledge this is the only reported results.

15.6.4 Rationale for treatment of relapsed WT with high-risk features from previous studies More recent experiences on HR recurrent WT, in series ranging between 11 and 60 cases, seem to support the rationale for dose-response strategies, though there is no consensus on whether or not high-dose chemotherapy with ASCR can account for the improvement in outcome. Important to note, in the published experiences the definition of risk categories at relapse, that guided the adoption of more intensive therapies, were somehow heterogeneous, and not always overlapping with the above shared classification scheme. As a consequence, it is sometime difficult to extrapolate information on solely HR or VHR relapsed patients, because in some papers the distinction between these two categories was less clear. Recent trials with high-dose chemotherapy followed by ASCR obtained a better outcome than in historical controls, with 3 or 4-year OS rates ranging from 60 to 73% [2, 14, 23, 29]. However, other investigators reached comparable results adopting intensive conventional chemotherapy, with a combination of etoposide and carboplatin with either ifosfamide or cyclophosphamide [1, 18, 30] (summarized in table 15.5.3). Tannous et al. reported on the treatment of 66 patients with two cycles of the drug pair cyclophosphamide/etoposide (CyE) followed by two cycles of carboplatin and etoposide (Carbo/E) [30]. Patients who achieved complete response of the tumour received maintenance therapy with further 5 cycles of alternating CyE and Carbo/E, while those with partial response or stable disease received ablative chemotherapy followed by ASCR. The 3-year EFS were 59% ± 9% and 40% ± 14% for the maintenance and ASCR subgroups, while the 3-year OS were 64%± 8% and 42% ± 14%, respectively. Abu-Gosh et al. reported on 11 children treated with ICE (ifosfamide, carboplatin, etoposide) chemotherapy, obtaining a 63.6% EFS and OS at 3 years [1, 3]. Doses for ifosfamide were 1800 mg/m2/d x 5 days, carboplatin 400 mg/m2/d x 2 days, and etoposide 100 mg/m2/d x 5 days. The ICE regimen was demonstrated to be extremely efficacious in determining second responses (82% objective response rate). It is significant that persistent nephrotoxicity was moderate, as remarked also by other groups [3,

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29]. Malogolowkin et al. reported for the NWTS on 60 homogeneously treated children who relapsed after initial three-drug treatment: 4-year EFS and OS were 42.3 and 48% respectively for all patients [18]. These results were obtained using alternate cycles of CyE and Carbo/E. The regimen was 90 weeks long, and many children had discontinuation of therapy due to prolonged hematological toxicity. In a previous experience, Malogolowkin et al. treated 27 patients with alternating Carbo/E and ifosfamide/doxorubicin. The EFS and OS at three years were 58% [17].

Author Group Year No. Induction CT ASCT Follow- EFS DFS OS up (%) (%) (%)

Pein1 1998 SFOP 1998 28 various MEC 48 3-yr 60 [23] months “conventional” 50

Abu-Gosh CCG 2002 11 ICE no 4.2 3-yr 63.6 2002 [1] years 63.6 (1-12) (2)

Kremens2 GPOH 2002 20 various MEC 58 48.2 60.9 2002 [14] months ± CARBO, ETO (15/20)

Campbel 2004 Chicago 2004 13 7/13 ≠ 30 4-yr 73 [2] months CPM/ETO/C (1 or 2) 60

Malogolowkin NWTS-5 2007 60 CPM/ETO no 4-yr 48 2008 [18] 42.3 CARBO/ETO

Spreafico AIEOP 2008 20 ICE MEC 36 4-yr 4-yr 62 2008 [29] months (15/20) (8/15) 54 57

Hale 2008 UK 2008 45 CPM/ETO Melphalan 2-yr 66 [37] CCLG CARBO/ETO

Table 15.5.3. Published experiences on patients with recurrent WT classified as high risk. ( 1exclusion of 10 progressing patients before ABMT (only CR or PR); 2inclusion of 4 patients in 1st CR; exclusion of progressing patients before ABMT; M: Melphalan, E: etoposide, C: carboplatin, I: Ifosfamide, CPM: cyclophosphamide, ASCT: autologous stem cell transplantation)

Other authors have investigated the role of high-dose chemotherapy and ASCR. Pein et al. originally reported on 28 HR chemotherapy-responsive patients receiving high-dose MEC chemotherapy (melphalan, etoposide, carboplatin) regimen, and the 3-year OS and DFS were 60 and 50%, respectively [23]. Kremens et al. described 23 cases treated with high-dose chemotherapy and ASCR (18 children had the MEC course), after various reinduction regimens [14]: the OS was 60.9%, and the EFS 48.2%. Campbell et al. showed 4-year EFS and OS rates of 60 and 73%, respectively, in 13 patients who underwent single or double ASCR after various conditioning regimens [2]. Spreafico et al. reported on 20 patients with HR features at recurrence: all patients received an intensive-dose chemotherapy

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induction, most of them adopting ICE-based therapy, and 15/20 receiving high-dose chemotherapy and ASCR as consolidation [29]. This group electively reduced the drug dosage of the ICE and MEC associations vis-à-vis the doses used by others, in an attempt to reduce the expected toxicity without jeopardizing outcome. Three-year DFS and OS rates were 56% ± 12% and 55% ± 13%, respectively. Neither recurrence within 12 months of nephrectomy nor extra-pulmonary recurrence negatively affected outcome. A survival advantage was demonstrated in patients without disease evidence prior to transplant. The UKCCLG has recently closed its non-randomised, risk-stratified treatment trial for relapsed renal tumours [37]. The UKW-R protocol for HR relapses was based on re-induction dose-intense regimen and a consolidation with high-dose chemotherapy and ASCR. The re-induction chemotherapy alternated Carbo (750 mg/m2/d) and etoposide (200 mg/m2/d x 3 days) with Cy (500 mg/m2/d twice a day x 2 days) and etoposide (200 mg/m2/d x 3 days). After 6 chemotherapy courses, responding patients received high-dose single-agent Melphalan (200 mg/m2/d) with ASCR. The majority of high-dose chemotherapy regimens used in the previous reports were high dose alkylating drugs based therapy, which can be complicated by regimen related toxicity for the heavily pretreated patients. Severe mucositis and acute renal toxicity were observed in a significant proportion of cases treated with a CEM based regimen. Since many patients with recurrent WT will have only one kidney and will have been heavily exposed to carboplatin prior to the high-dose chemotherapy regimen, many patients may have low GFR prior to the CEM regimen requiring reductions in the carboplatin dose.

Figure 15.5.1: Survival Data by type of preparative regimen from EBMT Solid Tumours PDWP and from Paediatric Haematology/Oncology Association BMT Registries

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Melphalan has been extensively evaluated as a component of autologous transplant for high risk and relapsed WT. Single agent melphalan has been used in an attempt to reduce renal toxicities associated with the use of CEM conditioning regimen. In a review of the data from EBMT Solid Tumours PDWP they showed that the survival was superimposable (or even highest) for patients who received single agent melphalan when compared with other regimens (figure 15.5.1; Dallorso, personal communication). Furthermore, preliminary results of the recently closed UKW-R protocol showed that the single-agent melphalan regimen was well tolerated and effective [37].

15.6.5 Comparison between high-dose therapy and standard-dose therapy There are no randomized trials comparing conventional dose to high-dose chemotherapy. The above mentioned reports dealt with diverse inclusion criteria for patient selection (responding or not to miscellaneous re-induction chemotherapy, different disease status at the time of transplant, different histologic types of renal tumours), and various high-dose chemotherapy regimens (single or tandem courses, different agents). Direct comparisons are limited by these differences. It is not clear which regimen is superior, but it does seem that there is a survival advantage in patients whose recurrent disease was chemo-sensitive and those without disease evidence prior to transplant. In an effort to improve evidence on the role of ASCR in the setting of relapsed WT, an international analysis has attempted a synthesis of relevant published information, however remaining conscious of constraints that the underlined heterogeneity imposes on conclusions drawn [59]. The authors have summarized EFS and OS experience of patients with relapsed or refractory WT with the objective of comparing patients who received high dose therapy (HDT) with those that did not (NoHDT), following a Bayesian framework for comparison. A total of 19 publications concerning 1,226 patients were identified (5 HDT, 6 NoHDT, 8 both). Further 12 of these provided some individual patient data, and 7 summary figures. OS and EFS rates were combined in a weighted manner to derive hazard ratios (HR). Pooling all studies suggested an advantage to HDT with a hazard ratio (HR) for EFS of 0.87 and 0.94 for OS. A stratified analysis confined to studies that provided individual patient data on both HDT and NoHDT gave HRs of 0.83 and 0.92. Further, analyses of those of HR suggested a HR of 0.90 (95% CI 0.62 to 1.31), and for the VHR 0.50 (CI 0.31 to 0.82). The authors concluded that the evidence was suggestive of the value of a high dose option, particularly in the highest risk relapse group. However in summary, evidence from the literature suggests a great deal of uncertainty concerning the role of HDT in patients having relapsed after treatment for their WT. We are aware that a randomised trial to compare standard and more intensive options should lead to an improved level of certainty, but the main criticism remains the low number of such rare cases and thus a not achievable time frame to conduct such a trial if a classical statistical trial framework will be adopt.

15.6.6 Topoisomerase inhibitors Topotecan, a camptothecin analogue that interacts with DNA topoisomerase I, demonstrated antitumour activity in different childhood cancers including WT [22, 47, 48]. The schedule of administration was probably important in determining its activity, with the protracted schedule being more effective than an intermittent high-dose regimen. Investigators at St. Jude Children’s Research Hospital studied its activity on WT, both in pre-clinical models and in clinical phase I and II trials [6, 32]. In the WILTOP study, a St. Jude-based phase II study, a response rate of 48% was obtained in 25 evaluable heavily pretreated favorable-histology WT patients (12 patients reached partial remission, 6

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patients had stable disease, and 7 patients had progression) [20]. The encouraging results obtained in the WILTOP study however differed from previous trials [22, 32], and were ascribed to the protracted topotecan schedule (the action of poisons during S phase are optimized by longer exposure). Importantly, topotecan seemed to be less effective in anaplastic tumours [20]. Based on the St. Jude’s experience, topotecan has been variably included into salvage strategies for high-risk recurrent WT patients in the past few years, but outside controlled clinical trials. Recently the SIOP group aimed at retrospectively reviewing the available data on the use of topotecan in relapsing WT patients within Europe. Data on 30 patients who heterogeneously received topotecan as part of their salvage regimens were analysed. Concluding observations were that among unfavorable histology (either diffuse anaplasia or blastemal type after chemotherapy) 15 out of 16 patients eventually died of unresponsive tumour, while objective responses to topotecan were observed in 4 out of 14 patients with favorable histology WT, and overall 6 patients survived [67]. Prolonged and short schedules, as well as single infusions of irinotecan alone or in combination with other agents, have been investigated in the traditional phase II setting in various pediatric tumours such as neuroblastoma, Ewing sarcoma, WT, and certain malignant brain tumours [42, 50-58]. A recent Children’s Oncology Group (COG) trial on rhabdomyosarcoma revealed no differences in response rates between two different schedules of irinotecan (prolonged versus short: daily for 5 days v daily for 5 days, 2 days off, and then an additional 5 days, respectively), disproving the preclinical prediction of superior activity with a prolonged schedule. Despite there were no response to single-agent irinotecan in previous early phase clinical trials, in a recent report 4 patients with multiply relapsing WT were treated with a combination of vincristine, irinotecan, temozolomide and bevacizumab. Two had a complete response, and two had a partial response to treatment [68]. Importantly, vincristine shows synergism with the camptothecins [42, 48]. The respective dose-limiting toxicities were myelosuppression and diarrhea. The (COG) AREN0321 renal tumour study investigated the response to irinotecan in combination with vincristine (VI regimen) in patients with newly diagnosed stage IV diffuse anaplastic measurable WT in a upfront window [69]. Preliminary data seem to support the use of the VI combination in diffuse anaplastic tumours, since 11/14 (79%) patients treated with VI window had partial remission (and then incorporated VI into an intensive regimen containing carboplatin, etoposide, cyclophosphamide and doxorubicin, named UH-1). Despite the difficulties in extrapolating results in the context of a complex treatment, 4-year EFS for the 14 patients treated with VI window was 57% compared to 33% for the 10 patients treated with UH-1 upfront.

15.6.7 Relapsed WT with very high risk features Patients with relapsed advanced-stage diffuse anaplastic tumours have dismal long-term survival rate regardless of the site of relapse [5, 10, 70]. Patients with blastemal-type WT after primary chemotherapy (as defined in SIOP-2001, and treated accordingly with the high-risk chemotherapy regimens) who relapse displayed a dismal prognosis after recurrence, comparable to diffuse anaplastic tumours [28]. Overall, very poor responses to any drug or combination have been reported in these patients. This is the reason why, despite some evidence suggestive of the value of a high dose chemotherapy and ASCR option in this group, the problem for these patients is to obtain a second remission before high-dose chemotherapy.

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In the analysis by Pinkerton, only one out of 7 patients with anaplastic histology responded to second- line chemotherapy [64]. In the WILTOP study (see above) two partial responses to topotecan were observed out of 11 diffuse anaplastic recurrent tumours [20]. No survivors were registered among 9 anaplastic WT patients, who relapsed in the retrospective analysis of the St. Jude Hospital’s survey [5]. Whether for SR or some HR relapsed WT the major cause of initial treatment failure was relative under- treatment as opposed to the development of drug resistance, we can speculate that for the VHR the intrinsic resistance to drugs is the main cause of failure at relapse as well. Taking a look at the more recent early phase published trials emerges a relative lack of promising novel agents in WT. Drugs recently tested included biological agents such as anti-angiogenic compounds, tyrosine kinase, aurora-A-kinase, EGFR, heat shock protein 90, mTOR inhibitors, agents targeting IGF1R, differentiating agents, as well as cytotoxic drugs (e.g. mitotic inhibitors -like paclitaxel or docetaxel; folate antimetabolite, and topoisomerase inhibitors). Because VHR relapsed patients will have received most conventional active agents in their initial therapy, inclusion into trials of novel agents is justified for these patients. One of the goals of a wider international cooperation on recurrent WT might be to systematically channel WT cases into a limited number of active experimental studies. This is particularly true for VHR relapsing tumours and subsequent (following first one) failures of HR relapses. In general, these children should be referred to centres that are conducting early-phase research trials on novel agents in the treatment of children with solid tumours (http://www.itcc-consortium.org/).

15.6.8 “Local” therapies: role for surgery and radiation therapy Surgical removal of operable recurrent tumours (regardless of the risk group) is probably helpful, but its influence has not been examined prospectively. However, current „local” approach to recurrent tumour is often intuitive or „personal experience based” and not structured. The NWTS group suggested that surgical removal of all pulmonary metastases is unlikely to improve post-relapse survival compared with treatment with whole-lung radiation therapy (RT) and chemotherapy [9]. However, in the retrospective analysis by Dome et al., patients who underwent a complete surgical resection of recurrent tumour had a higher probability of survival than did patients who had a partial resection or no surgery [5]. Because the lungs are the most frequent sites of first recurrence, more information on the therapeutic role of surgical resection of pulmonary metastases, and on which techniques to adopt, might be important. The therapeutic role of solitary lung metastasis resection in place of RT, seeking to avoid the toxicity of whole lung radiation therapy, is poorly explored. The German data on surgical aspects for liver metastases or recurrences suggested that complete surgical resection of liver recurrences improves survival. Fuchs reported that in children with a recurrence in the liver, those with a complete resection survived, whereas the patients with an incomplete resection all died [40]. Overall, it is tempting to speculate that surgery plays an important role in treating recurrent disease, but we cannot exclude the bias that patients who underwent a complete surgical resection had less aggressive disease. Further details are given in section 16.2.4. Similar to surgery, the discussion on indications, dose and modality of RT in children with recurrent WT has been rarely addressed. Most of the information we gather on RT is extrapolated from unselected groups of patients or case discussions. In the St. Jude study, the administration of RT to a previously unirradiated field was associated with a higher probability of survival [5]. In the NWTS-5 relapse

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protocol, the committee electively decided to administer RT at higher doses compared to the ones used at initial diagnoses, even if primary therapy did not include RT [12]. Table 15.5.4 summarizes the current available indications on how to administer RT at relapse. Detailed information is given in section 17.9.

Abdomen Total Lung Irradiation Notes Reference

NWTS-5 CR: 21.6 Gy 12 Gy Abdomen: 12.6-18 Gy if Green 2007 age ≤ 12 months; PR: boost up to 30 Gy (± boost 7.5 Gy if [12] persistent small disease) TLI: 9 Gy if < 18 months

AIEOP 15-21 Gy 12 Gy Spreafico 2008 [29]

UKW-R 20 Gy ± 10 Gy boost if 12 Gy ± 7.5 Gy boost Group C: 30 Gy UKW-R Protocol persistent disease These doses are for [37] patients who have not received prior RT Table 15.5.4: summary of available indications on radiation therapy at time of tumour recurrence. (CR: complete remission, PR: partial remission, TLI: total lung irradiation)

Based on all these observations, the present protocol aims to give a stronger guidance also for local disease re-therapy to achieve complete remission.

15.6.9 Biological studies at relapse To date, gene-expression analyses have given inconsistent results in the identification of prognostic signatures in WTs, perhaps due to the cellular heterogeneity found in the tumours. Contrariwise, whole- genome analyses seemed to be more promising in the discrimination of relapsing tumours, and there are increasing data on molecular signatures present in WTs at diagnosis, which are possibly associated with prognosis [44, 45, 71]. There is an urgent clinical need to define which pathways are involved in progression or relapse of WT, for which targeted therapies could already exist. However, due to the difficulty in recruiting pairs of primary and recurrent tumours of the same patients, there is little information regarding the genetic events acquired between primary tumours and subsequent relapses. Such information could allow the gain of insight into the molecular basis of tumour progression or relapse, and into the mechanisms through which tumour cells home into the different sites of relapse. The analysis of 10 paired primary and recurrent WTs found that acquired alterations occurring in more than one relapsed samples included the gain of 5p, 8p12, 15q (where the IGF1R gene is mapped), 16p and 20q, and loss of 17p (where the TP53 gene is mapped) [44]. IGF1R copy number gain and overexpression may be considered as steps leading to relapse, opening to a possible role for anti-IGF1R in the therapy for recurrent WT.

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Furthermore, there is scanty data on the specific cellular pathways involved in tumour progression or metastasis, mainly relating to presence of TP53 mutations [41]. TP53 mutations are variably associated with anaplasia [42, 61]. Whether anaplasia and/or 17p/TP53 acquired anomalies also confer an additional higher attitude to disseminate is not clear. Therapy directed at replacing TP53 function might be expected to reverse the chemo-resistant phenotype of the TP53 mutation bearing tumours [43].

15.6.10 Combined rationale for the proposed therapy recommendations: summary In recent years, effort has been made trying to approach patients with recurrent WT with homogeneous therapies following recognised risk factors, however never shared between international groups. The current protocol will represent the first attempt to give efforts and resources to treat and study recurrent WT basing on a wide cooperation, as it is in SIOP-RTSG. Treatment regimens will be designed to include drugs that are possibly not used during initial chemotherapy, using a risk-stratified approach. Therapy of recurrent disease will mainly depend on the nature of initial treatment (in turns reflecting recognized prognostic factors inherent in the primary tumour). Highly effective chemotherapy regimens include ICE, CyCE, Carbo/E, CyD, which are considered first treatment choice for recurrent disease and will represent the backbone of the present guidelines. Summarizing the above available and discussed experiences, the backbone on which research/guidelines on recurrent WT patients are developed is here synthetized:  In those patients who have received only one or two agents (vincristine, actinomycin D) and no RT before relapsing (regardless of timing and site of recurrence), it is reasonable to use an alkylating agent, doxorubicin, etoposide, and RT. The combination of these drugs was already tested in two comprehensive experiences, the UKW-R protocol and the NWTS-5 relapse protocol, respectively. In general, the concern of potential nephrotoxicity of ifosfamide led to the preferential use of Cy, in doses with equivalent alkylating activity. The results from early phase trials with carboplatin supports the assumption that its inclusion might increase objective response rate, and with known toxic profile.  In those patients suffering from WT relapse with more unfavorable characteristics than the previous group, studies conducted by different researchers demonstrated that ICE is an effective retrieval regimen. Although acceptable response rates have been seen with the CyE and Carbo/E pairs as well, ifosfamide shows the higher response rate in early phase trials (table 5). However, in an effort of reducing the ifosfamide-related nephrotoxicity, cyclophosphamide will be used to alternate it with ifosfamide. o The heterogeneous settings, and inconclusive results, in which the role of high-dose chemotherapy and ASCR has been explored, render it useful to propose this approach at the discretion of the treating physician, and describe the results in a prospective observational fashion. Emerging new evidence in this field might help supporting the generation of a phase III randomized trial with a Bayesian framework. o Overall, one purpose for the current retrieval scheme is to reinforce our knowledge of an intensive standard backbone of effective drugs (like CyCE/ICE), that was rarely prospectively tested and that will allow to incorporate biological agents or novel drugs, if available in the future.  Recurrent tumours among the advanced-stage diffuse anaplastic and blastemal-type after preoperative chemotherapy are likely to develop chemo-resistant disease to conventional

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drugs/regimens, and novel therapeutic strategies are necessary to cure these patients. There is an urgent need to channel patients from this group into new-drug early-phase trials.  Evidence on how to properly administer surgery and radiotherapy at relapse is more fragmentary. After proven reduction of recurrent disease post-chemotherapy, resection could be considered - independently of histological subtype or risk group- when radical surgery seems possible or when it is useful to evaluate histological tumour response, i.e. in the case of stable disease. a. Whether applying radiotherapy to initially non-irradiated infra-diaphragmatic sites is uniformly accepted, arguments like the approach to already-irradiated cases or to pulmonary irradiation is much less standardised. Other issues, like the timing of pulmonary radiotherapy in patients, who are candidates to receive high-dose chemotherapy and ASCR, needs to be addressed, due to the cumulative respiratory toxicity in conjunction with high- dose chemotherapy.

Objective No. Drug Notes Reference responses patients

Many different Tournade 1988 [31] 52% IFO 40 schedules (Pratt 1989 [72], Pinkerton (8 CR, 11 PR) (2-9 g/m2/course) 1985, [73])

27% Finklestein 1969 [74], Pinkel Cy 37 (10 PR) D [77], Sutow WW [78]

ETO 42% 10 patients 31 Pein 1993 [24] 200 x 5 (2 CR, 11 PR) Stage I-II

40% Carbo (560) 5 Ettinger 1994 [75] (2 PR)

53% Carbo (550) 15 De Camargo 1994 [4] (4 CR, 4 PR)

ETO + Carbo 73% 10 patients 26 Pein 1994 [26] (100 x 5 – 160 x 5) (8 CR, 11 PR) Stage I-II

86% ETO + Cy + VCR 7 1 toxic death Carpenter 1997 [76] (1 CR, 5 PR)

48% No response in Topotecan 25 Metzger 2007 [20] (12 PR) anaplasia Table 5. Phase II trials concerning the most active agents in Wilms tumour.

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15.6.11 Specific objectives The SIOP RTSG seeks to promote for the first time an international shared approach for recurrent WT, to further investigate factors implicated in prognosis post-relapse and in the potential biological differences between primary and recurrent tumours, as well as continuing to obtain information on treatment and outcome data. One purpose is to reinforce our knowledge of standard backbones of effective drugs in a risk-adapted fashion, with surgical and radiotherapy guidelines, with which possibly incorporating in the future biological agents or novel drugs, if available. Important note: the SIOP-RELAP-2016 protocol has been designed to be used in conjunction to and under the UMBRELLA of SIOP RTSG 2016. General objectives of this study are (specific objectives for each risk group will be presented separately): 1. To provide all participants of the SIOP-RTSG with the best available treatment option to recurrent WT. 2. To introduce more standard approaches for surgery and radiation therapy. 3. To ensure adequate data collection of factors potentially relating to post-recurrence outcome, in order to prospectively validate the proposed risk stratification at relapse, and exchange potentially useful information with the colleagues of the Children’s Oncology Group. 4. To better characterise recurrent tumour from a molecular “omics” profile, encouraging the systematic biomaterial sample banking from relapses. 5. To promote European wide access to new agent phase I/II trials in patients with dismal prognosis at relapse by working closely together with ITCC.

15.6.12 Diagnostic and staging investigations at relapse The diagnostic and staging investigations for a child with suspected recurrent WT are detailed below. Biopsy, to obtain histological diagnosis, is strongly recommended in clinically doubtful situations. These may include: 1. Late recurrence, occurring > 24 months from end of treatment; 2. Isolated single lung or liver nodule; 3. Important note: In all the other situations in which the histological confirmation of relapse might be helpful in guiding the clinical approach and judged with a limited risk of morbidity, tumour biopsy is advised, also to implement our biological knowledge on relapsing tumours. Noteworthy to remind, in future new agent clinical trials, it might be important to have a molecular profile of a relapsed tumour before registering patients. Increased emphasis is placed on the quality and standardisation of imaging. 3D volume measurements of the tumour, both at diagnosis of the relapse and to document response to chemotherapy, are important. MRI or CT scanning are the recommended standard for abdominal imaging. MRI studies with new protocols (ADC mapping) allow assessment of tumour composition, which may be used as a biomarker for response to chemotherapy. CT thorax scan is recommended to lung and mediastinum re-staging.

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Centralised radiology review is introduced to improve standardisation, as well as better defining metastatic and bilateral disease, both at diagnosis and at relapse (see chapter 6, and especially 6.7). All children with a suspected tumour recurrence should have:  Clinical history and examination  Measurement of blood pressure and urinary protein  Abdominal ultrasound  3D cross sectional imaging of abdomen (either MRI or CT)  CT scan chest, with documentation of number and largest size of any visible lung nodule  Recommended: submit imaging for real time central review as detailed in UMBRELLA protocol  Functional imaging of the kidneys (DMSA scan) prior to re-treatment  Echocardiogram for patients receiving doxorubicin, or receiving pulmonary radiotherapy or doxorubicin already given  Blood and urine samples for biomarker studies at diagnosis and during follow-up (see section 7.3 and Appendix 7, chapter 19.7)  Consent for national tumour banking  Consent for UMBRELLA Protocol

15.6.13 Eligibility and risk stratification 15.6.13.1 Eligibility criteria Considering the nature of this protocol, aimed at providing each patient with the best available treatment for relapse, all patients should be registered, and no formal exclusion criteria are considered. We suggest to discuss with the national coordinator the management of patients with relapse if they present with significant impairment of renal or cardiac function. Main inclusion patients will be: i. All patients with first relapse of WT ii. Patients with refractory (tumour progressing on first line therapy) WT iii. Patients with second and subsequent relapses

Note 1: Age is not a criterion for exclusion (patients older than 18 years suffering from relapsing WT are also eligible). Note 2: Since patients suffering from metachronous contralateral tumours might mask subsequent primary tumours in the context of WT predisposing syndromes, we suggest to discuss these cases (and in general cases with initial bilateral disease suffering from relapse) with the national coordinator, rather than considering metachronous tumours as truly relapsing disease.

15.6.13.2 Risk grouping Three risk categories for recurrent WT will be identified: 1) Group AA: Patients with initial stage I or II low/intermediate risk SIOP-2001 histology WT, with relapse after therapy including only vincristine (V) and/or actinomycin-D (A), and no RT. Also

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patients fulfilling the same criterion of not having received previous drugs apart from VA, and receiving up-front nephrectomy will be included (see Italian patients, or SIOP patients receiving primary surgery). This group is expected to have EFS estimates in the 70-80% range, and account for 30% of all recurrences. 2) Group BB: Patients without initial diffuse anaplasia, or blastemal-type after pre-operative chemotherapy, treated initially with ≥3 chemotherapy drugs with or without radiation. Patients with second and subsequent relapses may be entered if prior therapy was according to group AA, in the absence of alternative treatment solutions by the responsible treating physician. This group, accounting for 45-50% of the children with WT who relapse, is expected to have survival rates in the 40-50% range [7, 18]. 3) Group CC: patients with initial stage II to IV diffuse anaplastic or blastemal-type histology after pre-operative chemotherapy [36]. The great majority of these patients will have already received doxorubicin, etoposide, carboplatin, ifosfamide or cyclophosphamide (according to their national protocols). These patients are expected to have survival rates in the 10% range [28, 37]. This group accounts for 10-15% of all WT relapses. Important note: histology is intended of the initial tumour (not at relapse). If anaplasia, not previously recognised in the primary tumour, turns out at relapse, we advise to discuss the case with the national coordinator.

15.6.14 Flow diagram with treatment recommendations

AA BB CC

ITCC early phase CyD ICE trials, (if available) consider CE CyCE irinotecan/vincristine

Response evaluation Response evaluation PR/SD: S (feasable) PR/SD: S (feasable) CR CR PD, regimen BB PD, alternative tretment

Complete either Complete with with HD-LPAM XRT, CyD/CE x or further 8 ICE/CyCE (+ XRT)

Figure 2: Flow diagram with treatment recommendations according to groups, AA, BB and CC. (CyD: cylophosphamide + doxorubicin; CE: carboplatin +etoposide; I: ifosfamide, S: surgery, CR: complete remission, PR: partial remission, SD: stable disease, PD: progressive disease. ITCC: Innovative Therapies for Children with Cancer: http://www.itcc-consortium.org/)

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15.6.15 Therapeutic recommendations 15.6.15.1 Group AA Specific assumptions of the recommendations for AA patients will rely on the proven experience with doxorubicin, etoposide, and cyclophosphamide, in terms of toxicity and efficacy, and that the incorporation of carboplatin, whose efficacy is well proven, will likely help to improve the OS rate (since 60-80% of failures are systemic failure).

Primary specific objectives: 1. To determine the EFS and OS rate of children from group AA recurrent risk profile treated with a chemotherapy regimen alternating cyclophosphamide/doxorubicin and carboplatin/etoposide.

Secondary specific objectives: 1. Prospective validation of this risk profile at relapse 2. Comparison of genomic profile of primary tumour vs recurrent tumour samples 3. Comparison of miRNA profiles in blood at primary diagnosis and relapse

There are two alternating courses of chemotherapy given at 21 day intervals (total 8). Both combinations consist of two drugs. Each cycle commences when absolute neutrophil count is ≥ 1.0x109/l and platelet > 75 x 109/l and rising. Use of cotrimoxazole as Pneumocystis Carinii prophylaxis is recommended, as well as use of G-CSF.

Course 1. Cyclophosphamide and doxorubicin (CyD) Cyclophosphamide: 500 mg/m2, every 12 hours as an infusion over 15 min on days 1 and 2 (total 2 g/m2/course). Mesna: i.v. bolus of 200 mg/m2 immediately prior to first dose of Cyclophosphamide followed by continuous Mesna at 1.0 g/m2/day until 12 hours after last dose of cyclophosphamide. Doxorubicin: 50 mg/m2 in 3-hour infusion on day 1. Weeks: 1, 7, 13 and 19 (total of 4 courses).

Course 2. Carboplatin and etoposide (Carbo/E) Etoposide: 150 mg/m2 in 500 ml/m2 dextrose saline in 2-hour infusion on days 1, 2 and 3. Carboplatin: 200 mg/m2 (or AUC = 2.65) in 2-hour infusion on days 1, 2 and 3. Weeks: 4, 10, 16, and 22 (a total of 4 courses).

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Treatment plan

Week 1 2 3 4 5 6 7 8 9 10 11 12

D Carbo D Carbo

Cy E Cy E

adiotherapy

urgery urgery

S R

13 14 15 16 17 18 19 20 21 22 23 24

D Carbo D Carbo

Cy E Cy E

Single doses: D: 50 mg/m2; Cy: 500 mg/m2 bd x 2 days; E: 150 mg/m2 x 3; Carbo: 200 mg/m2 x 3

Cumulative doses: D: 200 mg/m2; Cy: 8 g/m2; E: 1.8 g/m2, Carbo: 2,4 g/m2

Reassessment imaging at week 6 Surgery should be planned for week 6 Note Dose reductions for all drugs 1.Give 66% (on kg basis) of above doses for children weighting <12 kg (see 14.5.3) 2. If weight < 5kg or age < 6 months: dose reduction to 50% (on kg basis) of each drug (see 14.5.3)

Figure 3. Treatment scheme for Group AA (D: doxorubicin, Cy: cyclophosphamide, E: etoposide, Carbo: carboplatin)

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15.6.15.2 Group BB The specific assumption will be that the use of high-dose melphalan and ASCR as consolidation for those children who will have achieved a partial or complete response to induction chemotherapy will improve EFS for children of group BB.

Primary specific objectives • To determine the EFS and OS rate of children from group BB recurrent WT treated with a chemotherapy regimen including ifosfamide, cyclophosphamide, carboplatin, etoposide, and high-dose melphalan.

Secondary specific objectives • Prospective validation of this risk profile at relapse • Comparison of genomic profile of primary tumour vs recurrent tumour (to aid the identification of drugable targets) • Comparison of miRNA profiles in blood at primary diagnosis and relapse • To describe the outcomes of group BB patients treated with CyCE/ICE, who will not be able to receive high-dose chemotherapy and ASCR.

1. Induction chemotherapy There are four sequential courses of chemotherapy (alternating ICE and CyCE), administered at 21 day intervals, before HD-melphalan. Since prolonged thrombocytopenia due to carboplatin is expected, we recommend trying not to delay treatment courses, and followed the recommended cut off of 75 x 109/l platelets to start the subsequent course. Each course commences when absolute neutrophil count is ≥ 1.0x109/l and platelet > 75 x 109/l and rise. Use of Cotrimoxazole as Pneumocystis Carinii prophylaxis is recommended, as well as use of G-CSF.

Induction Week 1 4 6 7 10 13 15

I Cy I Cy HD-

melphalan

Carbo Carbo Carbo Carbo

E E E E

valuation valuation

First evaluation First PBSCharvest E surgery and

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Route of Drug Dose Day(s) administration i.v. 66.7 mg/kg/day for infants < 12 months Ifosfamide (I) 1-3 over 2 hours 2,000 mg/m² for children ≥ 12 months Cyclophosphamide i.v. 440mg/m2 (total 2.2 g/m2/course) 1-5 (Cy) over 15 min GFR Dose 560 mg/m2 > 150 ml/min/1.73m2 (18.7 mg/kg for infants) 500 mg/m2 100-150 ml/min/1.73m2 (16.6 mg/kg for infants) 370 mg/m2 Carboplatin i.v. 75-99 ml/min/1.73m2 (12.3 mg/kg for infants) 1 (Carbo) over 1 hour 290 mg/m2 50-74 ml/min/1.73m2 (9.7 mg/kg for infants) 200 mg/m2 30-49 ml/min/1.73m2 (6.7 mg/kg for infants) < 30 ml/min/1.73m2 Hold carboplatin Etoposide (E) i.v. 3.3 mg/kg/day for infants < 12 months 1-3 during ICE over 1 hour 100 mg/m2 for children ≥ 12 months Etoposide (E) i.v. 3.3 mg/kg/day for infants < 12 months 1-5 during CCE over 1 hour 100 mg/m2 for children ≥ 12 months GCSF 5 µg/kg/day subcutaneously from day 4 through ANC > 3,000/µl i.v. infusion over 3 hours during the administration of ifosfamide, at an equivalent ifosfamide dose (2,000 mg/m2/d for children > 12 months). with ifosfamide An additional Mesna 1,000 mg/m2/d dose is required in a parallel liquid infusion over 24 hours, during all the 3 days of ICE. Mesna Reduced dose for children < 12 months i.v. bolus of 200 mg/m2 immediately prior to first dose of with cyclophos- Cyclophosphamide followed by continuous Mesna at 1.0 gr/m2/day phamide until 12 hours after last dose of cyclophosphamide. Reduced dose for children < 12 months

2. High dose chemotherapy and autologous hematopoietic stem cell rescue All patients with proven responding tumour will proceed to high-dose chemotherapy with autologous PBSC rescue. Patients must have sufficient stem cells available (defined as PBSC > 3 x 106 CD34 cells/kg). Hematopoietic progenitor cells-apheresis will be done approximately at week 6 after CyCE chemotherapy for patients demonstrating responding tumour at first evaluation. Patient will receive GCSF at 10 µg/kg/day starting 48-72 hours following the completion of chemotherapy. Apheresis will be performed according to each institutional standard guideline. If the targeted CD34+ cells cannot be collected after a maximum of two procedures, the flexible nature of the proposed guidelines allows the treating physicians to decide to use alternative factors for stimulation, or to give a further cycle of ICE chemotherapy before a next attempt to collect sufficient CD34+ cells, or to decide to continue induction chemotherapy without high-dose melphalan consolidation.

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To start high dose chemotherapy the following needs to be fulfilled:  Proven partial or complete response to induction chemotherapy  Sufficient stem cells available (defined as > 3 x 106 CD34 cells/kg)  GFR > 70 ml/min/1.73 m2 using the Schwartz formula [79] or 24-hour creatinine clearance or radioisotope GFR > 40 ml/min/m2 or > 70 ml/min/1.73 m2  Cardiac function should be assessed as adequate  Consider fertility preservation

Stem cell infusion A minimum of 3 x 106 CD34 cells/kg (optimum 5 x 106 CD 34 cells/kg) PBSC should be used. Stem cells will be infused intravenously on day 0, 48 hours after chemotherapy is completed, immediately following thawing.

High-Dose Chemotherapy followed by Autologous Stem Cell Transplant:

Day

-2 Melphalan

-1

0 Autologous Stem Cell reinfusion

Drug doses during consolidation:

Melphalan 200 mg/ m² total dose over 1 hour

DAY 0: 48 hours following chemotherapy completion - infusion procedure will be by institutional standard.

GCSF infusion (5 µg/kg/day begins from day +4 after stem cell infusion and continues daily until ANC is greater for 2,000/µl for 3 consecutive days). Bactrim starts only after neutrophil recovery.

15.6.15.3 Group CC One of the goals of a wider international cooperation on recurrent WT will be to systematically channel WT patients into a limited number of active and emerging experimental studies. This is particularly true for group CC tumours and subsequent (following first one) failures of group BB relapses. As a priority these children should be referred to centres that are conducting research trials on novel agents in the treatment of children with solid tumours (see: http://www.itcc-consortium.org/ for up-to- date news).

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In the absence of available early phase clinical trials for entering WT patients, we suggest to adopt the vincristine/irinotecan (VI) regimen as investigated in the recently closed COG AREN0321 protocol [69] (see also paragraph 15.6.6 Topoisomerase inhibitors). For doses and schedule for the VI combination please refer to Appendix 6. In case of complete or very good partial remission after the VI regimen, consolidation with HD- melphalan and PBSC rescue might be considered.

15.6.16 Surgical guidelines for relapse Surgical guidelines for patients with a relapse are given in chapter 16.2.4. Please ensure familiarity of surgical guidelines in the context of the WT management guidelines. General considerations regarding surgery as described above do apply for relapses as well. The following statements for surgery in relapsed patients need to be taken into consideration: 1. The first treatment is second line chemotherapy. Exceptions are late solitary lung metastasis. The CNS metastasis may be a surgical emergency. 2. The goal of surgery should aim for clear resection margins. The tumour bed and any suspicious residual disease should be clearly described to provide detailed information for radiotherapy targeting this site. 3. It is not recommended to operate on metastases that have progressed under chemotherapy unless it is a single metastasis. 4. If the relapse occurs in the field of radiotherapy, surgery likely represents the most efficacious local treatment, and all possible efforts should be undertaken to perform a complete resection. 5. For contralateral kidney recurrence (metachronous Wilms), surgical intervention requires an experienced team. Surgery is planned after tumour reduction with chemotherapy also in these patients.

15.6.17 Radiotherapy guidelines for relapse Radiotherapy guidelines for relapse are given in detail in chapter 17.9. In case of radiotherapy during first line treatment the national coordinator should be contacted.

15.6.18 Biological studies Biological studies are important in the relapsed situation and every chance to analyse biomaterial (blood, urine, tumour) should be undertaken. We should encourage tissue sampling at time of relapse so that we can link molecular genotyping to response or signposting the very highest relapses to the appropriate targeted agent trials open at the time. The comparison of the genomic profile of the primary tumour against the recurrent tumour will help to answer the following questions: - Can we identify anomalies commonly acquired and/or present in relapsing disease? - Are they the same as those associated with relapse? - Can we identify anomalies associated to the different sites of recurrence? - Can these anomalies lead to the identification of potential molecular targets/pathways?

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For more details on biological studies go to section 7.3 and Appendix 7, chapter 19.7.

15.6.19 Chairs and members of the Relapsed WT Panel

Name Profession Country Email

Chair Filippo Spreafico Oncologist Italy [email protected]

The A.M.C.Mavinkurve- Annelies Mavinkurve Oncologist Netherlands [email protected]

Arnauld Verschuur Oncologist France [email protected]

Claudia Pasqualini Oncologist France [email protected]

The Anne Smets Radiologist [email protected] Netherlands

Lorenza Gandola Radiotherapist Italy [email protected]

Marry van den Oncologist The m.m.vandenheuvel-eibrink@ Heuvel-Eibrink Netherlands prinsesmaximacentrum.nl

Jörg Fuchs Surgeon Germany [email protected]

Maximilian.Stehr@diakonieneuendettelsa Maximilian Stehr Surgeon Germany

u.de Members Guido Seitz Surgeon Germany [email protected]

Bruce Okoye Surgeon UK [email protected]

Gordan Vujanic Pathologist UK [email protected]

Norbert Graf Oncologist Germany [email protected]

Harm van Tinteren Statistician The [email protected] Netherlands

Leo Kager Oncologist Austria [email protected]

Rhoikos Furtwängler Oncologist Germany [email protected]

Jan Godzinski Surgeon Poland [email protected]

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15.6.20 References

1. Abu-Ghosh AM, Krailo MD, Goldman SC, et al.: Ifosfamide, carboplatin and etoposide in children with poor-risk relapsed Wilms’ tumor: a Children’s Cancer group report. Ann Oncol, 2002. 13:460-469 2. Campbell AD, Cohn SL, Reynolds M, et al. Treatment of Relapsed Wilms’ Tumor With High-Dose Therapy and Autologous Hematopoietic Stem-Cell Rescue: The Experience at Children’s Memorial Hospital. J Clin Oncol, 2004. 22:2885-2890 3. Daw NC, Gregornik D, Rodman J, Marina N, Wu J, Kun LE, Jenkins JJ, McPherson V, Wilimas J, Jones DP: Renal function after ifosfamide, carboplatin and etoposide (ICE) chemotherapy, nephrectomy and radiotherapy in children with Wilms tumor. Eur J Cancer, 2009. 45:99-106 4. De Camargo B, Melarango R, Saba e Silva N, et al.: Phase II study of carboplatin as a single drug for relapsed Wilms’ tumor: experience of the Brazilian Wilms’ Tumor Study Group. Med Ped Oncol, 1994. 22:258-260 5. Dome JS, Liu T, Krasin M, et al. Improved survival for patients with recurrent Wilms tumor: the experience at St. Jude Children’s Research Hospital. J Pediatric Hematology/Oncology, 2002. 24:192- 198 6. Dome JS, Neale G, Hill DA, et al.: Anti-tumor activity of topotecan against Wilms tumor: Translation of a xenograft model to phase II study. Pediatr Blood Cancer, 2005. 45:432-433 7. Furtwängler R, Nourkami N, Alkassar M, von Schweinitz D, Schenk JP, Rübe C, Siemer S, Leuschner I, Graf N: Update on relapses in unilateral nephroblastoma registered in 3 consecutive SIOP/GPOH studies - a report from the GPOH-nephroblastoma study group. Klin Pädiatr 2011, 223:113-9 8. Garaventa A, Hartmann O, Bernard JL, et al. Autologous bone marrow transplantation for pediatric Wilms’ tumour: the experience of the European Bone Marrow Transplantation Solid Tumour Registry. Med Pediatr Oncol, 1994. 22:11-14 9. Green DM, Breslow NE, Ii Y, et al. The role of surgical excision in the management of relapsed Wilms’ tumor patients with pulmonary metastases: a report from the National Wilms’ Tumor Study. J Pediatr Surg, 1991. 26:728-733 10. Green DM, Beckwith BJ, Breslow NE et al. Treatment of children with stages II to IV anaplastic Wilms’ tumor: a report from the National Wilms’ Tumor Study Group. J Clin Oncol, 1994. 12:2126- 2131 11. Green DM, Breslow NE, Beckwith JB, et al. Comparison between single-dose and divided-dose administration of dactinomycin and doxorubicin for patients with Wilms’ tumor: a report from the National Wilms’ Tumor Study Group. J Clin Oncol, 1998. 16:237-45 12. Green DM, Cotton CA, Malogolowkin M, et al. Treatment of Wilms tumor relapsing after initial treatment with vincristine and actinomycin D: A report from the National Wilms Tumor Study Group. Pediatr Blood Cancer, 2007. 48:493-499 13. Grundy P, Breslow N, Green DM et al. Prognostic factors for children with recurrent Wilms’ tumor: results from the Second and Third National Wilms’ Tumor Study. J Clin Oncol, 1989. 7:638-647 14. Kremens B, Gruhn B, Klingebiel T, et al. High-dose chemotherapy with autologous stem cell rescue in children with nephroblastoma. Bone Marrow transplantation, 2002. 30:893-898 15. Lemerle J, Voute PA, Tournade MF, et al. Effectiveness of preoperative chemotherapy in Wilms’ tumor: results of an International Society of Pediatric Oncology (SIOP) clinical trial. J Clin Oncol, 1983. 1:604-609 16. De Kraker J, Graf N, van Tinteren H, Pein F, Sandstedt B, Godzinski J, Tournade MF. Reduction of postoperative chemotherapy in children with stage I intermediate-risk and anaplastic Wilms' tumour (SIOP 93-01 trial): a randomised controlled trial. Lancet, 2004. 364:1229-35

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17. Malogolowkin MH, Casak SJ, Feusner J, et al. Carboplatin and etoposide alternating with ifosfamide and doxorubicin for the treatment of children with recurrent Wilms’ tumors. Proceedings of ASCO, 1994. 13:424 (abstract n. 1453) 18. Malogolowkin M, Cotton CA, Green DM, et al. Treatment of Wilms tumor relapsing after initial treatment with vincristine, actinomycin D, and doxorubicin. A report from the National Wilms Tumor Study Group. Ped Blood Cancer, 2008. 50:236-241 19. Malogolowkin M, Spreafico F, Dome JS et al.: Incidence and outcomes of patients with late recurrence of Wilms' tumor. Ped Blood Cancer, 2013. 60:1612-5 20. Metzger ML, Stewart CF, Freeman III BB, et al. Topotecan is active against Wilms’ tumor: results of a multi-institutional phase II study. J Clin Oncol, 2007. 21:3130-3136 21. Miser JS, Tournade MF. The management of relapsed Wilms tumor. Hematol Oncol Clin North Am, 1995. 9:1287-1302 (1995) 22. Nitschke R, Parkhurst J, Sullivan J, et al. Topotecan in pediatric patients with recurrent and progressive solid tumors: a Pediatric Oncology Group phase II study. J Pediatric Hematol Oncol, 1998. 20:315-318. 23. Pein F, Michon J, Valteau-Couanet D, et al. High-dose melphalan, etoposide, and carboplatin followed by autologous stem-cell rescue in pediatric high-risk recurrent Wilms’ tumor: a French Society of Pediatric Oncology study. J Clin Oncol, 1998. 16:3295-3301 24. Pein F, Pinkerton R, Tournade MF, et al. Etoposide in relapsed Wilms’ tumor: a phase II study by the French Society of Pediatric Oncology. J Clin Oncol, 1993. 11:1478-1481 25. Pein F, Rey A, de Kraker J, et al. Multivariate analysis of adverse prognostic factors (APF) in children with recurrent (Rec) Wilms’ tumor (WT) after initial treatment according to SIOP-6 or SIOP-9 strategies. Med Ped Oncol, 1999. 33:170 (abstract N. 111) 26. Pein F, Tournade MF, Zucker JM, et al. Etoposide and carboplatin: a highly effective combination in relapsed or refractory Wilms’ tumor – A phase II study by the French Society of Pediatric Oncology. J Clin Oncol, 1994. 12:931-936 27. Perlman EJ. Pediatric renal tumors: practical updates for the pathologists. Pediatric and Developmental Pathology, 2005. 8:320-338 28. Reinhard H, Schmidt A, Furtwängler, et al. Outcome of relapses of nephroblastoma in patients registered in the SIOP/GPOH trials and studies. Oncology Reports, 2008. 20:463-467 29. Spreafico F, Bisogno G, Collini P, et al. Treatment of high-risk relapsed Wilms tumor with dose- intensive chemotherapy, marrow-ablative chemotherapy, and autologous hematopoietic stem-cell support: experience by the Italian Association of Pedaitric Hematology and Oncology. Ped Blood Cancer, 2008. 51:23-28 30. Tannous R, Giller R, Holmes E, et al. Intensive therapy for high risk (HR) relapsed Wilm’s tumor (WT). A CCG-4921/POG-9445 Study Report. Proceedings of ASCO, 2000. 19:588a (abstract no 2315) 31. Tournade MF, Lemerle J, Brunat-Mentigny M, et al. Ifosfamide is an active drug in Wilms’ tumor: a phase II study conducted by the French Society of Pediatric Oncology. J Clin Oncol, 1988. 6:793-796 32. Tubergen DG, Stewart CF, Pratt CB, et al. Phase I trial and pharmacokinetic (PK) and pharmacodynamics (PD) study of topotecan using a five-day course in children with refractory solid tumors: a Pediatric Oncology Group study. J Pediatr Hematol Oncol, 1996. 18:352-361 33. Kalapurakal JA, Dome JS; Pearlman EJ, et al. Management of Wilms’ tumour: current practice and future goals. Lancet Oncol, 2004. 5:37-46 34. van den Heuvel-Eibrink MM, Graf N, Pein F, et al. Intracranial relapse in Wilms Tumor Patients. Pediatr Blood Cancer, 2004. 43:737-741 35. Lowis SP, Foot A, Gerrard MP. Et al. Central nervous system metastasis in Wilms tumor: A review of three consecutive United Kingdom trials. Cancer, 1998. 83:2023-2029

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36. Vujanic GM, Sandstedt B, Harms D, et al. Revised International Society of Paediatric Oncology (SIOP) working classification of renal tumors of childhood. Med Pediatr Oncol, 2002. 38:79-82 37. Hale J, Hobson R, Moroz V, Sartori P. Results of UK Children’s Cancer and Leukemia Group (CCLG) protocol for relapsed Wilms tumor (UKWR): unified relapse strategy improves outcome. 40th meeting of International Society of Paediatric Oncology. 2008. O154;62 38. Italiano A, Sirvent N, Michiels JF, et al. Tumour response to paclitaxel in an adult with relapsed nephroblastoma. Lancet Oncol, 2005. 6:252-53 39. Ramanathan RK, Rubin JT, Ohori NP, Belani CP. Dramatic response of adult Wilms’ tumour to paclitaxel and cisplatin. Med Pediatr Oncol, 2000. 34:296-98 40. Fuchs J, Szavay P, Luithle T, Furtwängler, Graf N. Surgical implications for liver metastases in nephroblastoma – data from the SIOP/GPOH study. Surgical Oncology, 2008. 17:33-40 41. Bardeesy N, Beckwith JB, Pelletier J. Clonal expansion and attenuated apoptosis in Wilms’ tumors are associated with p53 gene mutations. Cancer Res, 1995. 55:215-219 42. Bardeesy N, Falkoff D, Petruzzi MJ, et al. Anaplastic Wilms’ tumour, a subtype displaying poor prognosis, harbours p53 gene mutations. Nat Genet, 1994. 7:91-7 43. Stock C, Ambros IM, Lion T, et al. Genetic changes of two Wilms’ tumors with anaplasia and a review of the literature suggesting a marker profile for therapy resistance. Cancer Genet Cytogenet, 2002. 135:128-138 44. Natrajan R, Little SE, Sodha N, et al. Analysis by array CGH of genomic changes associated with the progression or relapse of Wilms’ tumour. J Pathol, 2007. 211:52-9 45. Huang CC, Gadd S, Breslow N, et al. Predicting relapse in favorable histology Wilms tumor using gene expression analysis: a report from the Renal Tumor Committee of the Children’s Oncology Group. Clin Cancer Res, 2009. 15:1770-8 46. Grundy PE, Breslow NE, Li S, et al. Loss of heterozygosity for chromosome 1p and 16q is an adverse prognostic factor in favourable histology Wilms tumor: a report from the National Wilms Tumor Study Group. J Clin Oncol, 2005. 23:7312-7321 47. Creemers GJ, Lund B, Verweij J: Topoisomerase I inhibitors: Topotecan and irenotecan. Cancer Treat Rev, 1994. 20:73–96 48. Thompson J, George EO, Poquette CA, et al. Synergy of topotecan in combination with vincristine for treatment of pediatric solid tumor xenografts. Clin Cancer Res, 1999. 5:3617–3631 49. Pappo AS, Lyden E, Breitfeld P, et al. Two consecutive phase II window trials of irinotecan alone or in combination with vincristine for the treatment of metastatic rhabdomyosarcoma: The Children's Oncology Group. J Clin Oncol, 2007. 25:362–369 50. Soft Tissue Sarcoma Committee of the Children's Oncology Group, Lager JJ, Lyden ER, et al. Pooled analysis of phase II window studies in children with contemporary high-risk metastatic rhabdomyosarcoma: A report from the Soft Tissue Sarcoma Committee of the Children's Oncology Group. J Clin Oncol, 2006. 24:3415–3422 51. Bomgaars LR, Bernstein M, Krailo M, et al. Phase II trial of irinotecan in children with refractory solid tumors: A Children's Oncology Group Study. J Clin Oncol, 2007. 25:4622–4627 52. Vassal G, Giammarile F, Brooks M, et al. A phase II study of irinotecan in children with relapsed or refractory neuroblastoma: A European cooperation of the Société Française d'Oncologie Pédiatrique (SFOP) and the United Kingdom Children Cancer Study Group (UKCCSG). Eur J Cancer, 2008. 44:2453– 2460 53. Kushner BH, Kramer K, Modak S, et al. Irinotecan plus temozolomide for relapsed or refractory neuroblastoma. J Clin Oncol, 2006. 24:5271–5276 54. Osone S, Hosoi H, Tsuchiya K, et al. Low-dose protracted irinotecan as a palliative chemotherapy for advanced neuroblastoma. J Pediatr Hematol Oncol, 2008. 30:853–856

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55. Turner CD, Gururangan S, Eastwood J, et al. (Phase II study of irinotecan (CPT-11) in children with high-risk malignant brain tumors: The Duke experience. Neuro Oncol, 2002. 4:102–108 56. Wagner LM, McAllister N, Goldsby RE, et al. Temozolomide and intravenous irinotecan for treatment of advanced Ewing sarcoma. Pediatr Blood Cancer, 2007. 48:132–139 57. Casey DA, Wexler LH, Merchant MS, et al. Irinotecan and temozolomide for Ewing sarcoma: The Memorial Sloan-Kettering experience. Pediatr Blood Cancer, 2009. 53:1029–1034 58. Shitara T, Shimada A, Hanada R, et al. Irinotecan for children with relapsed solid tumors. Pediatr Hematol Oncol, 2006. 23:103–110 59. Ha TC, Spreafico F, Graf N, Dallorso S, Dome JS, Malogolowkin M, Furtwängler R, Hale JP, Moroz V, Machin D, Pritchard-Jones K. An international strategy to determine the role of high dose therapy in recurrent Wilms' tumour. Eur J Cancer, 2013. 49:194-210 60. Hayes-Jordan A, Green H, Ludwig J, Anderson P. Toxicity of hyperthermic intraperitoneal chemotherapy (HIPEC) in pediatric patients with sarcomatosis/carcinomatosis: early experience and phase 1 results. Pediatr Blood Cancer, 2012. 59:395-7 61. Popov SD, Vujanic GM, Sebire NJ et al. Bilateral wilms tumor with TP53-related anaplasia. Pediatr Dev Pathol, 2013. 16:217-23 62. S.L.M. Gooskens, E. Braakman, A.L. van den Boom, C. So-Osman, F. de Winter, M.M. van den Heuvel- Eibrink. Peripheral stem cell harvest using regular chemotherapy schedules in childhood cancer. Pediatric Transplantation, 2012. 16:758-65 63. Kathy Pritchard-Jones, Christophe Bergeron, Beatriz de Camargo, Marry van den Heuvel-Eibrink, Tomas Achas, Jan Godzinski, Foppe Oldenburger, Liliane Boccon-Gibod, Ivo Leuschner, Gordan Vujanic, Bengt Sandstedt, Jan de Kraker, Harm van Tinteren, Graf N on behalf of the SIOP Renal Tumor Study Group: Doxorubicin omission from the treatment of stage II/III, intermediate risk histology Wilms tumour: results of the SIOP WT 2001 randomised trial. Lancet, 2015. Published online; dx.doi.org/10.1016/S0140-6736(14)62395-3 64. Pinkerton CR, Groot-Loonen JJ, Morris-Jones PH, Pritchard J: Response rates in relapsed Wilms’ tumor. A need for new effective agents. Cancer, 1991. 67:567-71 65. Garaventa A, Hartmann O, Bernardt JL et al. Autologous bone marrow transplantation for pediatric Wilms’ tumor: the experience of the European Bone Marrow Transplantation Solid Tumor Registry. Med Pediatr Oncol, 1994. 22:11-4 66. Spreafico F, Pritchard-Jones K, Malogolowkin MH et al. Treatment of relapsed Wilms tumors: lessons learned. Expert Rev Anticancer Ther, 2009. 9:1807-15 67. Mavinkurve-Groothuis AMC, van den Heuvel-Eibrink MM, Tytgat GA, van Tinteren H, Vujanic G, Pritchard-Jones KLP, Howell L, Graf N, Bergeron C, Acha T, Catania S, Spreafico F: Treatment of relapsed Wilms’ tumour (WT) patients: experience with topotecan. A Report From the SIOP Renal Tumour Study Group (RTSG). Pediatr Blood Cancer, 2015. 62:598-602 68. Venkatramani R, Malogolowkin MH, Mascarenhas L. Treatment of multiply relapsed wilms tumor with vincristine, irinotecan, temozolomide and bevacizumab. Pediatr Blood Cancer, 2014. 61:756-9 69. Daw N, Anderson J, Kalapurakal J et al. Treatment of stage II-IV diffuse anaplastic Wilms Tumor: Results from the Children’s Oncology Group AREN321 Study. Pediatr Blood Cancer, 2014. 61(S2):S113, O-026 70. Dome JS, Cotton CA, Perlman EJ et al. Treatment of anaplastic histology Wilms’ Tumor: results from the fifth National Wilms’ Tumor Study. J Clin Oncol, 2006. 24:2352-8 71. Perotti D, Spreafico F, Torri F et al. Genomic profiling by whole-genome single nucleotide polymorphism arrays in Wilms tumor and association with relapse. Genes Chromosomes Cancer, 2012. 51:644-53 72. Pratt CB, Douglass EC, Etcubanas EL et al. Ifosfamide in pediatric malignant solid tumors. Cancer Chemother Pharmacol, 1989. 24(Suppl 1):S24-7

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73. Pinkerton CR, Rogers H, James C et al. A phase II study of ifosfamide in children with recurrent solid tumours. Cancer Chemother Pharmacol, 1985. 15:258-62 74. Finklestein JZ, Hittle RE, Hammond GD. Evaluation of high dose cyclophosphamide regimen in childhood tumors. Cancer, 1969. 23:1239-42 75. Ettinger JL, Gaynon PS, Krailo MD et al. A phase II study of carboplatin in children with recurrent or progressive solid tumors. A report from the Children’s Cancer Group. Cancer, 1994. 73:1297-301 76. Carpenter PA, White L, McCowage GB et al. A dose-intensive, cyclophosphamide-based regimen for the treatment of recurrent/progressive or advanced solid tumors of childhood: a report from the Australia and New Zealand Children's Cancer Study Group. Cancer, 1997. 80:489-96 77. Pinkel D. Cyclophosphamide in children with cancer. Cancer, 1962. 15:42-9 78. Sutow WW. Proceedings: Chemotherapy in Wilms’ tumor. An appraisal. Cancer, 1973. 32:1150-3 79. Schwartz GJ, Gauthier B. A simple estimate of glomerular filtration rate in adolescent boys. J Pediatr, 1985. 106:522-6

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15.7 Treatment guidelines for adults with Wilms tumours 15.7.1 Introduction Wilms tumour in adults is a very rare but well recognised phenomenon, with an incidence rate of less than 0.2 per million per year. In an average European country, between three to five cases will be diagnosed annually [1]. Most cases will be diagnosed unexpectedly, following nephrectomy for presumed renal cell cancer. Published data on the treatment of adult WT consisted, until recently, of case reports and some institutional series, which suggested that adults have worse survival than children with this tumour type [2, 3]. Better outcomes for two larger cohorts of adults with WT treated according to the paediatric protocols used in North America and in Germany were published in 2004 [4, 5]. It should be noted that the adult patients treated according to these paediatric protocols were younger than the median age at diagnosis for adult WT as a whole. Huszno et al. did a PubMed research in 2013 on clinical and histopathological features of nephroblastoma in adults [6]. They found that modern treatment regimens did improve OS up to 90% in this group of patients [6]. As a consequence it is important to prospectively treat these patients in a standardized way. This will give new knowledge about possible biological risk factors in this patient group and will help to reduce the time between diagnosis and start of adjuvant treatment, one of the risk factors for a worse outcome [7]. These guidelines are the result of discussions between representatives of all of the major paediatric WT treatment groups. They are primarily summarized in a draft in December 2005 and published in 2011 [7]. To systematically and prospectively collect data of adult patients with nephroblastoma these treatment guidelines are included in the SIOP 2016 UMBRELLA Part B protocol. Diagnostic workup, histological classification and staging is to be done according to patients with primary surgery (see sections 11.6.1, 11.6.2, 11.8, 19.5).

15.7.2 Treatment Regimens 15.7.2.1 For tumours treated with initial nephrectomy: Stage I non-anaplastic (favourable) histology (Regimen 1) These patients will be treated with vincristine and actinomycin D alone without radiotherapy, providing that all of the following criteria are met:  Staging and histology have been reviewed by a paediatric pathologist familiar with Wilms tumour and send for central pathology review  Histological examination and review has included at least one lymph node.  CT scan of the chest has excluded the presence of lung metastases  Chemotherapy will be started within 30 days of date of nephrectomy. The chemotherapy schedule is as follows: Vincristine: (1.5 mg/m2 i.v., max. single dose 2 mg) on day 1 of weeks: 1, 2, 4, 5, 7, 8, 10, 13, 16, 19, 22 Actinomycin D: (1.5 mg/m2 i.v., maximum single dose 2mg) on day 1 of weeks: 1, 4, 7, 10, 13, 16, 19, 22

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ACT 1.5 mg/m2      VCR 1.5 mg/m2                       Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Note: The administration of vincristine is not count dependent. Observe patient for signs of vincristine neurotoxicity that may require dose reduction or omission. The administration of actinomycin D is count dependent: Each dose should only be given if absolute neutrophil count > 1.0 x 109/l and platelets > 100 x 109/l. If there is marked thrombocytopenia, check liver function tests and observe carefully for signs of hepatic veno-occlusive disease (VOD).

Stage II/III non-anaplastic (favourable) histology (Regimen 2) These patients will be treated with “intensive AVD” (vincristine with simultaneous actinomycin D and doxorubicin) plus flank radiotherapy (also in stage II, as in most adults no lymphnodes are biopsied). It is important to give the first course of ‘triple’ chemotherapy as soon as possible after nephrectomy and to organise radiotherapy to commence within the next two weeks but not within 48 hours of the initial doses of actinomycin D and doxorubicin. It is not possible to be prescriptive about the exact timing of the start of radiotherapy. Therefore, the following chemotherapy scheme is written on the basis that ‘triple’ chemotherapy is given every 21 days or on count recovery (absolute neutrophil count > 1.0 x 109/l, platelets > 100 x 109/l). Timing and doses of actinomycin D and doxorubicin should be altered to fit around the administration of radiotherapy, observing the following recommendations:  Radiotherapy should not start until at least 48 hours after a dose of actinomycin D/doxorubicin;  Actinomycin D should not be resumed until 14 days after the last fraction of radiotherapy  Doxorubicin should not be resumed until 7 days after the last fraction of radiotherapy;  Further omissions or dose reductions should be considered if there is a significant volume of liver within the radiotherapy field or if the patient experiences gut or liver toxicity during or immediately following the end of radiotherapy. The chemotherapy schedule is as follows: Vincristine: (1.5 mg/m2 i.v., max. single dose 2 mg) on day 1 of weeks: 1, 2, 4, 5, 7, 8, 10, 13, 16, 19, 22 Actinomycin D: (1.5 mg/m2 i.v., maximum single dose 2mg) on day 1 of weeks: 1, 4*, 7, 10, 13, 16, 19, 22 Doxorubicin: (30 mg/m2 i.v., no maximum dose) on day 1 of weeks: 1, 4*, 7, 10, 13, 16, 19, 22 (total cumulative dose: 240mg/mg2)

ACT 1.5 mg/m2  *    VCR 1.5 mg/m2         Dox 30 mg/m2  *     < ------RT------>            Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22   = Echocardiography: at start of treatment, before week 19, and at end of treatment

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*Omit week 4 actinomycin D if the dose is due within 14 days of end of radiotherapy. Consider dose reduction of subsequent dose at week 7 and use of ursodeoxycholic acid as prophylaxis against veno- occlusive disease if a significant proportion of the liver is in the radiation field or if there is evidence of liver dysfunction or disproportionate thrombocytopenia. *Do not give doxorubicin during radiotherapy or within 7 to 14 days of end of radiotherapy. Consider omission or 50% dose reduction if there is evidence of liver or bowel dysfunction. Note: The administration of vincristine is not count dependent. Observe patient for signs of vincristine neurotoxicity that may require dose reduction or omission. The administration of actinomycin D is count dependent: Each dose should only be given if absolute neutrophil count > 1.0 x 109/l and platelets > 100 x 109/l. If there is marked thrombocytopenia, check liver function tests and observe carefully for signs of hepatic veno-occlusive disease (VOD). The administration of doxorubicin is count-dependent: each dose should only be given if absolute neutrophil count > 1.0 x 109/l, platelets > 100 x 109/l. If there is marked thrombocytopenia, check liver function tests and observe carefully for signs of hepatic veno-occlusive disease (VOD).

Stage IV, non-anaplastic histology Initial treatment starts with the same three drug chemotherapy as for stage II/III disease for 6 weeks. Prior to week 7 responses of metastases needs to be assessed by CT scan. According to the response the subsequent chemotherapy will be stratified. Patients whose metastases resolve completely by week 7 of chemotherapy remain on the same ‘intensive AVA’ chemotherapy schedule but should receive whole lung radiotherapy (12 Gy) concurrently with their flank radiotherapy, regardless of their speed of metastatic response. Patients with incomplete resolution of their metastases at week 7 will be changed to the ‘high risk’ chemotherapy schedule that includes carboplatin, cyclophosphamide, etoposide and continued doxorubicin. Lung metastases still detectable after 6 weeks of chemotherapy may be subjected to surgical excision. Even if this is successful, this should not lead to a treatment reduction. All such patients would still receive lung radiotherapy plus switch to the high risk chemotherapy regimen. If a nephrectomy has not been performed at initial diagnosis, then treatment is according to the guidelines as described in chapter 15.3.

Radiotherapy Guidelines for radiotherapy are given in chapter 17. Radiotherapy should ideally be completed within the interval between chemotherapy at week 1 and 4, ensuring that neither actinomycin D nor doxorubicin is given within 48 hours of commencing radiotherapy, that actinomycin D is not given concurrently or within 2 weeks of end of radiotherapy and that doxorubicin is not given concurrently or within 7 days of end of radiotherapy. The subsequent course of these two chemotherapy agents may need to be dose reduced or delayed if there are signs of excessive gut or hepatic toxicity. Only in those cases actually enrolled and quality ensured in the UMBRELLA protocol with an in time and exact confirmed stage II by reference pathology, including histological proven negative lymphnodes, one can discuss to avoid local radiotherapy in stage II intermediate risk tumours. In all other cases radiotherapy is advised. Doxorubicin is still necessary.

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Anaplastic histology, any stage and slowly responding stage IV non-anaplastic histology tumours (Regimen 3 = HR-1) Any tumour with anaplastic features or stage IV patients whose metastases have not resolved by week 7 of chemotherapy will be treated according to the ‘high risk’ chemotherapy protocol recommended in chapter 11.3.2.4. There are two alternating courses of chemotherapy given at 21-day intervals. Both combinations consist of 2 drugs. The first course starts as soon as the patient has recovered from surgery and clinical condition allows. This should be the case within 21 days after end of preoperative chemotherapy. Each cycle commences when absolute neutrophil count is > 1.0x109/l and platelet count >100 x 109/l provided rising WBC values.  Course 1: Cyclophosphamide and doxorubicin Cyclophosphamide: 450 mg/m2 on days 1, 2 and 3 of weeks 1, 7, 13, 19, 25 and 31 (6 courses in total) Doxorubicin: 50 mg/m2 on day 1 of weeks 1, 7, 13, 19, 25 and 31 (6 courses in total) Doxorubicin can be started after the first dose of cyclophosphamide.  Course 2: Etoposide and carboplatin Etoposide (VP16): 150 mg/m2 on days 1,2,3 of weeks 4, 10, 16, 22, 28 and 34 (6 courses in total) Carboplatin: 200 mg/m2 (or AUC = 2.65) on days 1,2,3 of weeks 4, 10, 16, 22, 28 and 34 (6 courses in total) Treatment should be delayed if the absolute neutrophil count is <1.0 x109/l or platelet count <100 x 109/l. G-CSF can be given if delays of treatment or grade 4 neutropenia did occur after the first 2 – 4 cycles. Use of Cotrimoxazole is recommended for HR regimens as PCP prophylaxis.

Weeks  1-----2------3-----4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 

 = Echocardiography: at start of treatment, before week 19, 31 and at end of treatment  = GFR (measure at every third course, or more frequently if there is evidence of renal dysfunction.

Lung metastases still detectable after 6 weeks of chemotherapy may be subjected to surgical excision. Even if this is successful, this should not lead to a treatment reduction. All such patients still receive whole lung radiotherapy.

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15.7.2.2 For tumours treated with initial biopsy, neo-adjuvant chemotherapy and delayed nephrectomy It is anticipated that this group of patients will include those presenting with lung metastases and those with localised but extensive abdominal tumours that are deemed a high surgical risk. These patients will be treated according to the Stage IV protocol for children (see chapter 15.3). Toxicity, especially vincristine neuropathy might be higher than n children. In case of vincristine neuropathy the number should be adjusted postoperatively to regimen 2 by omitting vincristine in week 3 and 6. In contrast to children all patients with initial metastases should receive whole lung radiotherapy regardless of speed of metastatic response and histology. Flank radiotherapy should be given to all patients treated with local stage II and III after neo-adjuvant chemotherapy.

15.7.3 Summary of radiotherapy recommendations Radiotherapy is given according to the guidelines provided in chapter 13. There are two exceptions: 1. All patients with stage II and III receive local radiotherapy, as retrospective analyses show that there are rarely lymph nodes biopsied in case of immediate tumour nephrectomy 2. All patients with lung metastasis will receive whole lung irradiation regardless of histology and response to treatment

15.7.4 Diagnostic procedures during and after treatment Monitoring of patients during and after treatment is done according to the guidelines provided in chapter 11.6. Consider sperm banking in males prior to receiving cyclophosphamide and carboplatin.

15.7.5 Chairs and members of the Adult WT Panel

Name Profession Country Email

Chair Raimund Stein Urologist Germany [email protected]

Filippo Spreafico Oncologist Italy [email protected]

Davide Biasoni Urologist Italy [email protected]

Marry van den The m.m.vandenheuvel-eibrink@ Oncologist Members Heuvel-Eibrink Netherlands prinsesmaximacentrum.nl

Heidi Segers Oncologist Belgium [email protected]

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Christian Rübe Radiotherapist Germany [email protected]

NN Radiologist

Ivo Leuschner Pathologist Germany [email protected]

Norbert Graf Oncologist Germany [email protected]

RhoiksoFurtwängler Oncologist Germany [email protected]

Leo Kager Oncologist Austria [email protected]

The Harm van Tinteren Statistician [email protected] Netherlands

NN Radiotherapist

15.7.6 References 1. Mitry E, Ciccolallo L, Coleman MP, Gatta G, Pritchard-Jones K on behalf of the EUROCARE Working Group. Incidence of and survival from Wilms' tumour in adults in Europe: data from the EUROCARE study. Eur J Cancer, 2008. 42: p. 2363-8 2. Terenziani M, Spreafico F, Collini P, et al: Adult Wilms' tumor: A monoinstitutional experience and a review of the literature. Cancer, 2004. 101: p-289-93 3. Kattan J, Tournade MF, Culine S, Terrier Lacombe MJ, Droz JP. Adult Wilms' tumour: review of 22 cases. Eur J Cancer, 1994. 30A(12): p. 1778-82. 4. Reinhard H, Aliani S, Ruebe C, et al: Wilms' tumor in adults: results of the Society of Pediatric Oncology (SIOP) 93-01/Society for Pediatric Oncology and Hematology (GPOH) Study. J Clin Oncol, 2004. 22: p. 4500-6 5. Kalapurakal JA, Nan B, Norkool P, et al: Treatment outcomes in adults with favorable histologic type Wilms tumor-an update from the National Wilms Tumor Study Group. Int J Radiat Oncol Biol Phys, 2004. 60: p. 1379-84 6. Huszno J, Starzyczny-Słota D, Jaworska M, Nowara E. Adult Wilms’ tumor – diagnosis and current therapy. Cent European J Urol, 2013. 66: p. 39-44 7. Segers H, van den Heuvel-Eibrink MM, Pritchard-Jones K, Coppes MJ, Aitchison M, Bergeron C, de Camargo B, Dome JS, Grundy P, Gatta G, Graf N, Grundy P, Kalapurakal JA, de Kraker J, Perlman EJ, Reinhard H, Spreafico F, Vujanic G, Warwick AB; SIOP-RTSG and the COG-Renal Tumour Committee. Management of adults with Wilms’ tumor: recommendations based on international consensus. Expert Rev Anticancer Ther, 2011. 11(7): p. 1105-13

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16 Surgical Guidelines General recommendations for unilateral nephrectomy, metastectomy, nephron sparing surgery (NSS) and laparoscopic surgery (minimal invasive surgery (MIS)) are included. In addition, specific guidelines for WTs stage I-III, WTs stage IV, bilateral WTs, relapsed WTs and non-WTs are presented.

16.1 General surgical guidelines 16.1.1 Nephrectomy Access: the long transverse abdominal incision is the preferred option. The thoraco-abdominal approach may be useful in huge masses located high in the abdomen, preventing the rise in the rate of tumour rupture and other complications [1, 2]. Whatever the incision, lymph nodes sampling must be done. Inspection of the abdominal cavity The abdominal cavity should always be inspected prior to tumour removal. Metastases in the liver, lymph nodes and peritoneum should be searched for. Every lesion should be excised (if resectable) or biopsied (if unresectable) and its position marked. Excised material must be sent to the pathologist in a separate container and its origin clearly indicated. Thorough inspection of the opposite retroperitoneal space is obligatory only if pre-operative imaging (MRI is the preferred option) is unclear or indicates bilateral localisation of the tumour. In such cases intraoperative ultrasound may be useful to localise the lesion. Unequivocal stage V cases will be treated following ‘Stage V treatment guidance’. The procedure [1-3] The goal of the procedure is to perform a radical nephro-ureterectomy outside of the Gerota’s fascia, including perirenal fat in mono-bloc resection. The colon should be mobilized to expose the retroperitoneal structures. In left sided tumours, the spleen may also be mobilized. On the right side, Kocher’s maneuver is helpful in exposing the inferior vena cava and renal vein. The renal artery and vein should be identified and controlled. The artery should be ligated first to avoid venous congestion and possible tumour rupture. In very large or infiltrating tumours, primary ligation of the renal vessels may be difficult or risky. In such situations, the tumour can be dissected from surrounding structures first, and the vessels ligated when possible. The tumour should be removed together with its adipose capsule and Gerota’s fascia, and if possible with all invaded surrounding structures. Very extensive and mutilating resections (e.g. pancreatectomy) however are not recommended, as these tumours are sensitive both to chemotherapy and radiotherapy. The ureter should be ligated and divided as low as possible in the pelvis. The tumour bed could be marked with titanium clips to guide future radiotherapy if required. Tumour thrombus in the renal vein and inferior vena cava Preoperative evaluation, by MRI, CT or ultrasound scan, should state the patency of the renal veins and inferior vena cava (IVC). Although intravascular extension of the tumour is usually apparent on the preoperative imaging, a careful examination of both renal vein and inferior vena cava is required during the operation. The classification of venous thrombus is linked to the surgical procedure needed to remove it entirely: 1. A short thrombus in the renal vein may be resected together with the vein.

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2. A sub-hepatic thrombus (extending to the infra-hepatic vena cava but below the hepatic veins) should be removed through a vena cavotomy, after controlling the contralateral renal vein and vena cava above and below the thrombus. 3. Cardiopulmonary by-pass will be required in the case of intra-atrial thrombus. It may also be very useful in case of a longer thrombus, extending to or above the level of the hepatic veins. 4. In cases with very extensive infiltration of the vena cava wall, the risks and benefits of surgery should be reconsidered. Even with extensive vascular surgery it may be impossible to achieve complete excision and radiotherapy may be a better option as shown in SIOP9.

Adrenal gland [8] The adrenal gland should be left in-situ if a safe resection margin between the tumour and the gland can be guaranteed. In tumours originating from the upper part of the kidney, adrenal preservation should not be attempted unless clear margins had been identified on preoperative images, or unless there is a clear plane at operation.

Lymph nodes [9-12] Sampling and histological examination of lymph nodes (LN) is imperative for accurate staging and subsequent treatment. This includes hilar LNs if not included in the radical nephrectomy specimen, and inter-aorto-caval LNs below the level of the renal pedicle even if not suspicious. It is useful to note that inter-aorto-caval LN normally belong to the right sided tumours as the anatomical border between left and right is the aorta and not the midline of the vertebral bodies but should be also accessed for left- sided tumours. Suspicious LNs at the aortic bifurcation, ipsilateral iliac axis, origin of the celiac trunk and superior mesenteric artery should also be sampled. It is recommended that at least 7 nodes are sampled and preferably not ruptured including the LNs sampled together with the specimen [9–12]. They must be carefully labelled and sent to the pathologist separately with an accurate description of their position and character. Radical LN dissection does not enhance survival and therefore is not part of the surgical therapy.

Tumour rupture In case of a tumour rupture the anatomical site and potential spread within the operational field should be documented. Infiltrations into adjacent tissue, affected LNs, macroscopic residues and macroscopic tumour ruptures should be described in detail.

16.1.2 Nephron sparing surgery (NSS) in unilateral cases [20-29] Unilateral cases may also benefit from NSS, but the nephrologic advantages and risk of recurrence have to be precisely evaluated for each individual case. Contra-lateral urological and nephrological disorders and genetic syndromes of an increased risk of WT rather than a risk of hyperperfusion nephropathy in the remaining kidney are important criteria when this option is considered. NSS is acceptable in cases of unilateral non-syndromatic WT provided the following criteria are met: 1. Tumour restricted to one pole of kidney or peripheral at mid-kidney

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2. Volume < 300 ml at diagnosis (the risk of positive LNs approx. 2 % only in small tumours) 3. No preoperative rupture (The imaging criteria for rupture: retroperitoneal peritumoral effusion/hemorrhage, peritoneal hemorrhage or nodules, spontaneously or by open biopsy and no intraoperative rupture) 4. No intraluminal tumour on preoperative imaging in renal pelvis 5. No invasion of surrounding organs 6. No thrombus in the renal vein or vena cava 7. No multifocal tumour 8. Excision can be performed with oncological safe margin 9. Kidney remnant is expected to show remaining function 10. At least 66% of renal tissue should be spared after the tumour resection with a margin of healthy tissue, to give any worthwhile protection against hyper perfusion. If this is in doubt preoperative DMSA may be able to define expected post-operative function. In case of visceral metastasis, NSS should not be systematically ruled out but considered carefully. In all possible cases of NSS a reference surgical opinion is mandatory also stating if this patient should be referred for NSS to an experienced centre. Remarks A significant volume reduction after preoperative chemotherapy suggests a better chance of successful NSS but is not mandatory as some tumours have radiological modifications suggesting necrotic transformation without decreasing in size. Functional imaging of the kidneys should be considered prior to surgery. Resection must be performed with margins of healthy renal tissue. Enucleation is not adequate local treatment. In case of microscopically incomplete resection, further local treatment depends on a number of factors and should be discussed with the multidisciplinary team. In unfavourable subtypes of renal tumours, however, complete nephrectomy seems necessary. Positive LNs at pathology after NSS indicate radiotherapy but NOT necessarily a completion nephrectomy. Intraoperative ultrasound scanning can be useful in defining the intrarenal tumour extent. Following NSS, the remaining kidney should be carefully followed up in short and long term: Doppler sonography two days after surgery. The contribution of spared renal tissue in the total urinary excretion should be assessed 6 months later with scintigraphy (DMSA). Creatinine clearance, hypertension and indicators of renal failure should be looked for/assessed every 3 months during the 2 first years, every 6 months for the next 3 years, then every year. This is important to understand the potential benefits or otherwise on long term renal function of NSS. All members of the multidisciplinary team should take the decision for NSS together. The surgeon confirms the final feasibility during the operation. Nephroblastomatosis in the renal parenchyma of the NSS specimen may give rise to metachronous nephroblastoma in the residual kidney. These patients should be followed very carefully.

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Classification of nephron sparing surgery (NSS) A classification for NSS has been developed by Audry et al. [29] and approved by the panel of surgeons as follows: 1) SURGICAL TECHNIQUE - NSS (A) = Partial Nephrectomy = Resection of tumour with a rim of normal renal parenchyma - NSS (B) = Enucleation = Resection of tumour without a rim of normal renal parenchyma

2) SURGICAL RESECTION MARGIN (SRM) - Intact pseudo capsule = (0) - Doubt = (1) - Tumour breach = (2)

3) PATHOLOGICAL RESECTION MARGIN (PRM) - Safe rim of renal parenchyma on resection margin, except nephroblastomatosis = (0) - Intact pseudo-capsule along the resection margin = (1) - Tumour breach = (2)

4) REMAINING RENAL PARENCHYMA (RRP) A subjective evaluation is done by the surgeon of the percentage of renal parenchyma remaining on the operated kidney = (n %) For example, a polar nephrectomy usually corresponds to a RRP of 70%.

A classification for each case would be reported as follows: “NSS(X)-SRM(n)-PRM(n)-RRP(n%)”.

16.1.3 Laparoscopic nephroureterectomy [30-31] Although the classical open approach to renal tumours of childhood is recommended, laparoscopic or laparoscopic assisted total nephrectomy is acceptable in WTs provided the following criteria are met: 1. Resection must adhere to oncological principles and include lymph node sampling. 2. Small, central tumours with rim of “normal” renal tissue. 3. The extraction of the specimen in a bag, without morcellation, through an adequate abdominal wall incision, is mandatory, not only to control the risk of dissemination, but also to ensure adequate histopathological staging. 4. If a NSS is feasible, it should be preferred even if an open approach is needed.

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Contraindications for laparoscopic nephrectomy: 1. Tumour infiltrating extra renal structures or extended beyond the ipsilateral border of spinal column 2. Thrombus in the renal vein or vena cava 3. Peripheral location if NSS is not deemed feasible 4. Tumour without any response to chemotherapy due to the risk of tumour rupture 5. Little or no experience in laparoscopic nephrectomy (consider transfer to another unit or obtain more experienced help) In all possible cases of laparoscopic total nephrectomy a reference surgical opinion is mandatory also stating if this patient should be referred for laparoscopic surgery to an experienced centre.

16.1.4 Comments regarding pathology specimens All suspicious structures should be biopsied or resected, marked, described precisely and sent to the pathologist in separate containers, either at initial diagnosis or at relapse. The intact surgical specimen should be delivered fresh (not fixed in formalin) to the pathologist without being opened by the surgeon. Please leave sutures on the ureter, renal vein and artery so that the pathologist is able to find them easily for histological examination. For relapse, the specimen needs to be orientated and suspicious areas should be clearly marked. 16.1.5 The SIOP form The surgeon must adequately fill in the SIOP surgical form for the data to be recorded with the most accuracy whatever the type of tumour and surgery. At nephrectomy, areas of dubious complete excision should be marked and described precisely on both surgery and pathology forms. A copy of the complete surgical report should accompany the SIOP surgery form. Forms for every surgical procedure need to be signed if applicable according to local regulations and should include special situations of relevance for the pathologist.

16.2 Surgical guidelines Wilms Tumour 16.2.1 Wilms Tumour Stage I-III See general surgical guidelines above.

16.2.2 Wilms Tumour Stage IV [13–15] Lungs Metastasectomy for lung metastasis should not be a primary intervention. Response under preoperative chemotherapy needs to be assessed (See Chapter 15.3). The timing of lung surgery depends on the response to chemotherapy and the treatment protocol. Metastasectomy can be performed either as a single stage approach together with the nephrectomy or preferably as delayed surgery after one or two courses of post-operative chemotherapy, if metastasis are still present after preoperative chemotherapy. If a complete remission can be achieved by surgery after preoperative

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chemotherapy. Otherwise timing of lung surgery is recommended as detailed in the stage IV- section (REFER TO CHAPTER STAGE IV TREATMENT 15.3 ff.). A thoracotomy approach is recommended in case of one-sided operable nodules. Bilateral resectable lung metastases should be excised either via two lateral thoracotomies, median-sternotomy or a bilateral anterior thoracotomy (clamshell approach) depending on surgical choice and anatomy. Size and location of metastases should allow for complete removal of all the lesions with limited extension of resections. A safety margin is recommended for any kind of metastasectomy if possible. Wedge resections can frequently be the best approach. If wedge resection will not achieve complete excision, then segmentectomy or lobectomy is acceptable. Pneumonectomy is not justified. Peripheral nodules located sub-pleurally may be amenable to a thoracoscopic approach. All excised lung lesions should be labelled with highest anatomical accuracy in order to i) correlate with radiological imaging and ii) relate in case of pulmonary relapses whether the relapse occurred at a previously resected site or not. Liver For isolated liver metastases a wedge resection is appropriate after neoadjuvant chemotherapy. Extensive and potentially mutilating resections are not recommended before the possibility of further chemotherapy is explored. Other sites Metastases outside lung or liver should be excised completely if the operation can be done without mutilation, or loss of vital organs. Complete excision of metastases where reasonably possible is strongly recommended. Further local and systemic treatment can depend on it and its histologic finding (Compare Chapter 15.3). Furthermore, it possibly reduces the risk for local relapse. It is not recommended to operate on metastases that have progressed under pre-operative chemotherapy, as complete excision is rarely successful in such circumstances. Alternative chemotherapy and/or radiotherapy should be explored first.

16.2.3 Bilateral Wilms Tumours (Stage V) [16 – 19] Bilateral cases should be treated individually. The main surgical aim is to preserve as much functional renal tissue as possible. Due to the small numbers of such cases, it is recommended that a team experienced in the care of such patients is involved. Surgical intervention requires an experienced team and consideration should be given to centralising such cases to few centres. Thus:  Each patient should be discussed in the context of a surgical national meeting to take decision with the advice of national experts.  Surgery will be performed only at centres specialized in the treatment of this disease. Surgery is planned after tumour response to chemotherapy, by either decreased size or radiological changes in favour of necrosis. If there is no tumour reduction under chemotherapy, tumours might be of stromal subtype where surgical resection of the tumour needs to be considered before intensifying chemotherapy. Chemotherapy for longer than 12 weeks rarely brings further response prior to surgery and therefore it is not recommended. Surgical resections can be multiple for one kidney. The abiding principle is to achieve bilateral nephron sparing resections either in a single stage approach or in two separate operations performed not more than two post-operative courses apart. Biopsy of surgical margins to assess the complete resection of the tumour should be performed if not harmful for

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the remaining kidney. The less involved kidney should be operated on first. Complete nephrectomy on one side with NSS on the opposite side is acceptable providing enough functional renal tissue can be preserved [17]. If in spite of favourable appearances on imaging, the tumours appear unresectable at surgery, the tumours could be biopsied, preferably with the Tru-cut needle, and the patient treated with further chemotherapy. However, in rare cases bilateral complete nephrectomy may be the only surgical option. The options for radiotherapy as local treatment are limited after NSS but there are examples from the SIOP 9 study indicating that low dose radiotherapy (10Gy) and chemotherapy may result in long-term remission even after incomplete excision without any alteration of the renal function. This possibility should be taken into account in patients for whom bilateral nephrectomy would be the only means to achieve complete excision, or in patients with Denys-Drash syndrome with a life threatening blood pressure not responding to conservative treatment. If bilateral nephrectomy is performed, vascular access for dialysis (Permcath) should be inserted at the time of the second nephrectomy. Peritoneal dialysis may also be possible, although not usually in the immediate post-operative period. Transplantation should be planned provided there is no recurrent or residual disease, and preferably after 2 years of disease free survival. When bilateral tumours are diagnosed accidently, during operation in a previously untreated patient, NSS, if possible, is the preferred option. But if NSS is not possible open or retroperitoneal US guided Tru- cut biopsy and/or primary closure of the abdomen and upfront chemotherapy as described above needs to be balanced.

16.2.4 Relapsed Wilms Tumour First relapses, whether metastatic or local, are curable in a significant proportion of patients. Treatment should therefore be conducted with the intention of cure. A detailed imaging of the sites of relapse should be obtained before any surgical decisions. General considerations regarding surgery as described above do apply for relapses as well. The following paragraphs provide further statements for surgery in relapsed patients. The first treatment is second line chemotherapy. Exceptions are late (> 2 years) solitary lung metastasis. The nature of such lung lesions appearing a long time after the treatment for Wilms’ tumour may not be clear until histological examination. The CNS metastasis may be a surgical emergency. In the remaining cases, surgical resection should be undertaken after a response to chemotherapy is apparent and when all persisting sites of disease are amenable to complete excision. The goal of surgery should aim for clear resection margins. The tumour bed and any suspicious residual disease should be clearly described to provide detailed information for radiotherapy targeting this site. It is not recommended to operate on metastases that have progressed under chemotherapy, as complete excision is rarely successful in such circumstances. Alternative chemotherapy and/or radiotherapy should be explored first. Surgical excision of liver or lung nodules should be reserved for small numbers of operable metastases, after chemotherapy and only in patients with stable disease or partial response. The indication needs to be done together with surgeons, radiotherapists and oncologist. If the relapse occurs in the field of radiotherapy, all possible efforts should be undertaken to perform a complete resection. Local relapse and lung or liver metastases are frequently resectable. Lymph node relapse, especially if in a previously irradiated field is a very difficult problem. Even radical para-aortic

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lymphadenectomy may bring no benefit to the patient as the lymph node invasion frequently continues into the mediastinum. For contralateral kidney recurrence (metachronous WT), surgical intervention requires an experienced team. Surgery is planned after tumour reduction with chemotherapy also in these patients.

16.3 Surgical guidelines for non-Wilms Tumours 16.3.1 Clear cell Sarcoma of the Kidney (CCSK) Use surgical guidelines as described for Wilms tumours 16.3.2 Renal Cell Carcinoma (RCC) [32-41] Complete Surgical Tumour Resection (R0) is the mainstay of cure in pediatric RCC [32, 33]. The standard recommended surgical procedure is radical nephrectomy (RN). We recommend being cautious to consider partial nephrectomy in children. The EORTC led a prospective randomized clinical trial (RCT), comparing radical nephrectomy with partial nephrectomy in solitary T1-T2 N0M0 renal tumour < 5 cm with normal contralateral kidney function and WHO performance status of 0-2. At 9.3 years survival follow-up, 198 patients (72.5%) were alive after radical nephrectomy and 173 (64.4%) after NSS. Local recurrence occurred in one patient in the nephrectomy group and in six in the NSS group [34]. Indications to NSS are increasing for adult patients: it is now considered in cases of small tumours (i.e. < 4 cm [ up to < 7cm?] diameter, possibly all T1-T2 RCC) and – if technically feasible - in all cases with reduced renal function or/and renal malformation and in all cases with estimated higher risk of metachronic secondary RCC or secondary renal function deterioration such as especially RCC as part of a syndrome (Tuberous Sclerosis, VHL, others), RCC after chemotherapy or as second malignancy, RCC or renal failure in the family [35]. We need to remind that in children, still the differentiation between RCC and WT is not possible by imaging studies, even in the adolescence group, and WT remains the most frequent renal tumour type. However, we believe that any case of small renal neoplasm in children > 10 years should be regarded as a potential RCC, and consequently be discussed with the reference surgeons, with the aim of considering NSS procedures in selected cases. In addition it is difficult to extrapolate the tumour dimension criteria from adulthood experience, since the kidney dimensions in children are different. NSS is advised in such cases only after extensive discussion with the SIOP surgical panel [34, 35]. The standard indications for adult NSS in RCC according to the European Association of Urology guidelines [36] are divided into the following categories: 1. absolute (anatomic or functional solitary kidney), 2. relative (functioning opposite kidney that is affected by a condition that might impair renal function in the future), and 3. elective (localized unilateral RCC with a healthy contralateral kidney). Relative indications also include patients with hereditary forms of RCC who are at high risk of developing a tumour in the contralateral kidney in the future [36]. During the last decade, NSS has become the gold standard for the treatment of T1a tumours (< 4 cm) in adult patients with a normal contralateral kidney and – when performed in carefully selected patients in specialized centers - NSS can be safely applied in patients with larger renal tumours [37]. In adult patients several studies showed equivalence of NSS and RN in oncological outcome in localized RCC at least in T1RCC [37, 38]. Data for NSS in pediatric RCC are very limited. However no difference exist between NSS and RN concerning the oncological outcome [32, 39]. Nevertheless, before performing NSS in a child, we

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recommend to discuss the indication with the surgical board of SIOP-RTSG in all cases, similar to what is recommended in all other pediatric renal tumours.

General surgical principles in RCC:  The completeness of surgical resection is of prognostic importance [40]  Early stage disease has a better prognosis than later disease stages [41, 42].  NSS might have a role in low-volume localised cancer in carefully selected patients [41, 42].  Expert’s opinions are strongly recommended in case of partial nephrectomy and regional lymphadenectomy as these still need to be determined.

Regional lymph node positive RCC (stage T1-4, N1-2, M0) Most children with RCC will be diagnosed after initial WT treatment preoperatively. Therefore, the role of regional LN re-dissection in cases that were suspected of WT and had LN sampling only, is still under debate in pediatric patients with RCC. Pediatric patients with RCC and positive LNs seem to have a relatively more favorable outcome than adults [43, 44]. In case the diagnosis of RCC is known at the time of surgery, total regional lymphadenectomy needs to be done whenever feasible without significant surgical morbidity. In an Italian study of 16 pediatric RCC patients with local LN involvement, the survival rate in patients with extended retroperitoneal LN dissection (RLND) was markedly better than in cases with a more limited LN resection (8 of 9 with RLND alive and disease free vs. 1 of 7 without RLND) [45].

Distant metastatic RCC (stage T1-4, N0-2, M1) In case of metastatic RCC complete renal tumour resection with negative surgical margins as described above is strongly advised together with retroperitoneal LN sampling. After a medical treatment approach (recommendations see below) for response evaluation, surgical resection of all metastases should be attempted as completely as possible.

16.3.3 Malignant rhabdoid tumour of the kidney (MRTK) In most patients the diagnosis of MRTK is unknown before surgery. Therefore the general guidelines for surgery of pediatric kidney tumours as described above need to be adhered. Total resection of MRTK is significantly correlated with increased relapse free survival, as advanced stage has a significantly negative impact on survival. Van den Heuvel-Eibrink and colleagues reported 19% for stage III compared to 50% for stage I EFS after 5 years [46]. Tomlinson compared stage I&II with stage III, IV and V resulting in 41% OS compared to 19% OS [47]. Delayed surgery in non-completely resectable MRTK is preferred, instead of delaying treatment. MRTK usually shrinks on anthracycline containing treatment [48], and intensive treatment is often providing a setting in which a complete resection becomes possible. It is encouraged not to delay surgery too long, as the biology of the disease tends to give rise to early progression. Due to the aggressive nature of MRTK a NSS cannot be recommended. Tumournephrectomy is thus the surgical approach of choice.

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16.3.4 Congenital mesoblastic nephroma (CMN) Complete nephrectomy is the treatment of choice in localized disease and should be done according to the general surgical guidelines provided above. The perirenal fat needs to be removed always, as CMN tends to infiltrate in the surrounding tissue. Re-resection should be performed in case of incomplete tumour resection or incomplete removal of the perirenal fat. Metastectomy is advised in exceptional cases with solitary metastasis.

16.4 Chair and Members of the Surgical Panel

Name Profession Country Email

Chair Jan Godzinski Surgeon Poland [email protected]

Daniel C. Aronson Surgeon UK [email protected]

Georges Audry France [email protected] Surgeon

Torbjörn Backman Surgeon Sweden [email protected]

Davide Biasoni Surgeon Italy [email protected]

Gabriella Guillén Surgeon Spain [email protected] Burrieza

João Luís Castro Surgeon Portugal [email protected]

Giovanni Cechetto Surgeon Italy [email protected]

Tiago Henriques Surgeon Portugal [email protected] Members Coelho

Henrique Sa Couto Surgeon Portugal [email protected]

Virginie Fouguet Surgeon France [email protected]

Jörg Fuchs Surgeon Germany [email protected]

Frederic Gauthier Surgeon France [email protected]

Jan Godzinski Surgeon Poland [email protected]

Hugo Heij Surgeon The [email protected] Netherlands

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Sabine Irtan Surgeon France [email protected]

Lars Johansen Surgeon Denmark [email protected]

Rosa Cabello Surgeon Spain [email protected] Laureano

Marc-David Leclair Surgeon France [email protected]

Bruce Okoye Surgeon UK [email protected]

Mark Powis Surgeon UK [email protected]

Guido Seitz Surgeon Germany [email protected]

Kees van de Ven The [email protected] Surgeon Netherlands

Dietrich von Germany [email protected] Surgeon Schweinitz

Maximilian Stehr Surgeon Germany [email protected]

Raimund Stein Surgeon Germany [email protected]

Steven Warmann Surgeon Germany [email protected]

Jim Wilde The [email protected] Surgeon Netherlands

16.5 References

1. Fuchs J, Kienecker K, Furtwängler R, Warmann SW, Burger D, Thurhoff JW, et al. Surgical aspects in the treatment of patients with unilateral wilms tumor: a report from the SIOP 93-01/German Society of Pediatric Oncology and Hematology. Annals of surgery. 2009;249(4):666-71. 2. Godzinski J, Tournade MF, deKraker J, Lemerle J, Voute PA, Weirich A, et al. Rarity of surgical complications after postchemotherapy nephrectomy for nephroblastoma. Experience of the International Society of Paediatric Oncology-Trial and Study "SIOP-9". International Society of Paediatric Oncology Nephroblastoma Trial and Study Committee. Eur J Pediatr Surg 1998;8(2):83-6. 3. Powis M, Messahel B, Hobson R, Gornall P, Walker J, Pritchard-Jones K. Surgical complications after immediate nephrectomy versus preoperative chemotherapy in non-metastatic Wilms' tumour: findings from the 1991-2001 United Kingdom Children's Cancer Study Group UKW3 Trial. J Pediatr Surg. 2013;48(11):2181-6. 4. Szavay P, Luithle T, Semler O, Graf N, Fuchs J. Surgery of cavoatrial tumor thrombus in nephroblastoma: a report of the SIOP/GPOH study. Pediatric blood & cancer. 2004;43(1):40-5.

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5. Graf N, Tournade MF, de Kraker J. The role of preoperative chemotherapy in the management of Wilms’ tumor. The SIOP studies. Urol Clin North Am, 2000;27(3):443-54. 6. Thompson WR, Newman K, Seibel N. et al. A strategy for resection of Wilms’ tumour with vena cava or atrial extension. J of Pediatr Surg, 1992;27(7):912-915, 1992 7. Daum R, Roth H, Zachariou Z. Tumor infiltration of the vena cava in nephroblastoma. Eur J Pediatr Surg 1994;4:16-20. 8. Kieran K, Anderson JR, Dome JS, Ehrlich PF, Ritchey ML, Shamberger RC, et al. Is adrenalectomy necessary during unilateral nephrectomy for Wilms Tumor? A report from the Children's Oncology Group. J Pediatr Surg. 2013;48(7):1598-603. 9. Godzinski J, van Tinteren H, de Kraker J, Graf N, Bergeron C, Heij H, et al. Nephroblastoma: does the decrease in tumor volume under preoperative chemotherapy predict the lymph nodes status at surgery? Pediatr Blood & Cancer. 2011;57(7):1266-9. 10. Godzinski J, de Kraker J, Graf N, et al. Is the number of lymph nodes samples at Wilms Tumour nephrectomy predictive for detection of the regional extention of the disease? Pediatr Blood & Cancer 2004;43:329. Abstract SIOP Oslo: O.010 11. Shamberger RC, Guthrie KA, Ritchey ML, Haase GM, Takashima J, Beckwith JB, et al. Surgery-related factors and local recurrence of Wilms tumor in National Wilms Tumor Study 4. Annals of surgery. 1999;229(2):292-7. 12. Kieran K, Anderson JR, Dome JS, Ehrlich PF, Ritchey ML, Shamberger RC, et al. Lymph node involvement in Wilms tumor: results from National Wilms Tumor Studies 4 and 5. Journal of pediatric surgery. 2012;47(4):700-6. 13. De Kraker J, Lemerle J, Voute PA et al. Wilms’ tumour with pulmonary metastases at diagnosis: the significance of primary chemotherapy. J Clin Oncol 1990;8:1187-90. 14. DeKraker J, Tournade M.-F, Weirich A et al. Wilms tumour stage IV. A report from the SIOP-9 study. Med. Pediatr Oncol, 1997;29(5): 370. 15. Godzinski J. Tournade M.-F, deKraker J et al. Stage IV nephroblastoma with extra pulmonary metastatic involvement in the SIOP 6 and 9 Study. Med. Pediatr Oncol 1991;19:371. 16. Kieran K, Williams MA, Dome JS, McGregor LM, Krasin MJ, Davidoff AM. Margin status and tumor recurrence after nephron-sparing surgery for bilateral Wilms tumor. J Pediatr Surg. 2013;48(7):1481- 5. 17. Ritchey ML Coppes MJ. The management of synchronous bilateral Wilms’ tumor. Hematol/Oncol Clin North Am 1995;9:1303-15. 18. Herrera JM, Gauthier F, Tournade MF et al. Bilateral synchronous Wilms’ tumour (WT): is it a good model of conservative surgery for unilateral WT? Med Pediatr Oncol, 1996;27(4): 219. 19. Godzinski J, Tournade MF, Weirich A et al. Prognosis for the bilateral Wilms’ tumour patients after non-radical surgery: the SIOP-9 experience. Med Pediatr Oncol 1998;31(4):241. 20. Haecker FM, von Schweinitz D, Harms D, Buerger D, Graf N. Partial nephrectomy for unilateral Wilms tumor: results of study SIOP 93-01/GPOH. J Urology. 2003;170(3):939-42; discussion 43-4. 21. Wilde JC, Aronson DC, Sznajder B, Van Tinteren H, Powis M, Okoye B, et al. Nephron sparing surgery (NSS) for unilateral wilms tumor (UWT): The SIOP 2001 experience. Pediatr Blood & Cancer. 2014.61(12):2175-9 22. Moorman-Voestermans CG, Aronson DC, Staalman CR, Delemarre JF, de Kraker J. Is partial nephrectomy appropriate treatment for unilateral Wilms' tumor? J Pediatr Surgery. 1998;33(2):165- 70. 23. Gentil Martins A, Espana M. Partial nephrectomy for nephroblastoma – a plea for less radical surgery. Med. Pediatr Oncol 1989;17:320. 24. Green DM, Coppes MJ. Future directions in clinical research in Wilms’ tumour. Hematol/Oncol Clin North Am 1995;9:1329-39.

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25. Moorman-Voestermans CGM, Staalman CR, Delamarre JFM. Partial nephrectomy in unilateral Wilms’ tumour is feasible without local recurrence. Med. Pediatr Oncol 1994;23:218. 26. Cozzi DA, Schiavetti A, Morini F, Castello MA, Cozzi F. Nephron sparing surgery for unilateral primary renal tumor in children. J Pediatr Surg, 2001;36(2): 362-365. 27. Hoellwarth ME, Urban C, Linni K, Lackner H. Partial nephrectomy in patients with unilateral Wilms tumor. 3rd International Congress of Paediatric Surgery, Brussels, 6-8.05.1999, abstract book page: 038. 28. Guglielmi M, Cecchetto G, Dall’Igna P, Tchaprassian Z, d’Amore ESG, Carli M. Wilms tumor: does tumorectomy leave neoplastic tissue residual? Med. Pediatr Oncol. 2000;34(6): 429-431. 29. Godzinski J, Graf N, Audry G: Current concepts in surgery for Wilms tumor--the risk and function- adapted strategy. Eur J Pediatr Surg 2014;24:457-60. 30. Varlet F, Petit T, Leclair MD, Lardy H, Geiss S, Becmeur F, et al. Laparoscopic treatment of renal cancer in children: a multicentric study and review of oncologic and surgical complications. J Pediatr Urology. 2014;10(3):500-5. 31. Varlet F, Stephan JL, Guye E, Allary R, Berger C, Lopez M. Laparoscopic radical nephrectomy for unilateral renal cancer in children. Surgical laparoscopy, endoscopy & percutaneous techniques. 2009;19(2):148-52. 32. Selle B, Furtwängler R, Graf N, et al. Population-based study of renal cell carcinoma in children in Germany, 1980-2005. Cancer 2006;107:2906-14 33. Sausville JE, et al. Pediatric renal cell carcinoma. J Pediatr Urology 2009;5:308-314 34. Van Poppel H, Da Pozzo L, Albrecht W, Matveev V et al. A prospective, randomised EORTC intergroup phase 3 study comparing the oncologic outcome of elective nephron-sparing surgery and radical nephrectomy for low-stage renal cell carcinoma. Eur Urol. 2011 Apr;59(4):543-52 35. Spreafico F, Collini P, Terenziani M et al. Renal cell carcinoma in children and adolescents. Expert Rev Anticancer Ther. 2010;10(12):1967-78. 36. Ljungberg B, Hanbury DC, Kuczyk MA et al. Renal cell carcinoma guideline. Eur Urol. 2007;51(6):1502-10 37. Van Poppel H, Becker F, Cadeddu JA et al. et al. Treatment of localized renal cell carcinoma. Eur Urol 2011;60:662-672 38. Maehana T, Tanaka T, Kitamura H et al. Short-term functional and oncological outcomes of partial nephrectomy for renal cell carcinoma in patients with an anatomically or functionally solitary kidney: single-center experience. Int J Clin Oncol 2013;18:1049-53 39. Cook A, Lorenzo AJ, Salle JL et al. Pediatric renal cell carcinoma: single institution 25-year case series and initial experience with partial nephrectomy. J Urol;2006;175:1456-1460 40. Aronson DC, Medary I, Finlay JL et al. Renal cell carcinoma in childhood and adolescence: A retrospective survey for prognostic factors in 22 cases. J Pediatr Surg 1996;31:183-186 41. Hashim Uddin Ahmed, Manit Arya, Gill Levitt et al. Part I: Primary malignant non-Wilms’ renal tumours in children. Oncol 2007;8:730-37 42. Hashim Uddin Ahmed, Manit Arya, Gill Levitt et al. Part II: Treatment of primary malignant non- Wilms’ renal tumours in children. Lancet Oncol 2007;8:842-48 43. Ying Zhuge, M.D., Michael C. Cheung, M.D., Relin Yang et al. Pediatric Non-Wilms Renal Tumors: Subtypes, Survival, and Prognostic Indicators. J Surg Res 2010;163:257-263 44. Geller JL, Dome JS: Local lymph node involvement does not predict poor outcome in pediatric renal cell carcinoma. Cancer 2004;101:1575-83 45. Indolfi P, Bisogno G, Cecchetto G et al. Local lymph node involvement in pediatric renal cell carcinoma: a report from the Italian TREP project. Pediatr Blood & Cancer 2008;51:475-8 46. van den Heuvel-Eibrink MM, van Tinteren H, Rehorst H et al., Malignant rhabdoid tumours of the kidney (MRTKs), registered on recent SIOP protocols from 1993 to 2005: a report of the SIOP renal

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tumour study group. Pediatric blood & cancer, 2011. 56(5): p. 733-737. 47. Tomlinson GE, Breslow NE, Dome J et al., Rhabdoid tumor of the kidney in the National Wilms' Tumor Study: Age at diagnosis as a prognostic factor. Journal of Clinical Oncology, 2005;23(30):7641-7645. 48. Furtwängler R, Nourkami-Tutdibi N, Leuschner I et al. Malignant rhabdoid tumor of the kidney: significantly improved response to pre-operative treatment intensified with doxorubicin. Cancer Genet 2014;207:434-6

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17 Radiotherapeutic guidelines Depending on tumour stage, histology, chemotherapy response and resection status, in about 15 % of the children with nephroblastoma radiotherapy (RT) still plays an important role in curative treatment concepts. In addition, palliative RT may contribute to the treatment options in cases with no curative intent. In general, the indications and treatment strategies for RT in the SIOP– 2001 – protocol have been adequate and successful, so not many changes in the context of RT had to be implemented in the new protocol. Some important changes, which are described in detail later in this chapter, have been made: - No boost irradiation to the lymph nodes in stage III with initially positive lymph nodes and complete resection. - The dose of whole lung RT has been reduced to 12 Gy in intermediate risk histology cases. - Whole lung RT is indicated in the case of intermediate risk histology with complete resection but histological residual disease. - In cases of recurrent disease with lung metastases without prior lung irradiation during first line treatment whole lung RT should be performed in all histology- and remission types. - New irradiation techniques like IMRT and IGRT may be used as long as they contribute to a dose reduction in normal tissue at risk (e.g. liver, heart), include the target volume as recommended and avoid substantial dose scattering to non involved areas in a larger volume.

17.1 RADIATION THERAPY TREATMENT OF LOCAL ABDOMINAL DISEASE 17.1.1 Indications for post-operative local or flank RT: - Intermediate risk, stage III (lymph nodes positive N+, residual disease left after surgery, tumour rupture); (In adults also stage II, for exception see chapter 15.7) - High risk, stage II (except blastemal subtype5) - High risk, stage III (all histological subtypes) - Stage V according to local stage

17.1.2 Indications for post-operative whole abdominal RT: Whole abdominal RT is indicated for diffuse intra-abdominal tumour spread or gross preoperative or intraoperative “major rupture”.

17.1.3 Start of RT Abdominal/flank RT will start as soon as possible within 2 – 4 weeks after abdominal surgery.

5 Since the poor prognosis of blastemal subtype is caused by the occurrence of metastases and not by increased local recurrence unlike in other high risk tumours there is no necessity for a local radiation in stage II

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If there is an expectation for an additional RT for lung metastases, the abdominal RT will be postponed and starts after lung surgery. It is ideal to give lung- and flank RT in one field together. In cases with a high risk of local recurrence in the flank after surgery, mainly in cases with diffuse anaplasia, flank irradiation should not be delayed and can be delivered separately from whole lung RT. Age related dose reduction Total dose is dependent on pathology. The daily fraction dose to ICRU prescription points is conditioned by the age of the child (younger children <2 years and >2 years) and the volume (e.g. whole lung or abdomen) encompassed. Dose per fraction may be reduced in children <2 years of age to 1.5 Gy/fraction (flank irradiation) or 1.25 Gy/fraction (whole abdominal irradiation). The dose per fraction will be decided by the treating radiation oncologist and will depend upon the age of the child and the volume encompassed. One fraction per day should be treated daily, five days per week. In general a national RT reference institution should be contacted in these rare cases.

17.2 LOCAL FLANK RADIOTHERAPY - dose and fractionation - 17.2.1 Intermediate risk histology – local flank RT - Stage III based on positive lymph nodes or microscopic residual disease: 14.4 Gy in 8 fractions (1.8 Gy per fraction) to the involved flank or pre-operative tumour region - Boost to the macroscopic residual abdominal disease after surgery: 10.8 Gy boost in 6 fractions on macroscopic residual tumour (leading to a total dose of 25.2 Gy) - No boost on the lymph node area is needed, if no macroscopic residuals are detectable in this area.

17.2.2 High risk histology – local flank RT General Indication on Stage II and III (except blastemal type in stage II): Diffuse anaplastic type: Stage II and III: - 25.2 Gy in 14 fractions (1.8 Gy per fraction) to the involved flank. - Boost to macroscopic residual disease after surgery with 10.8 Gy to a total dose of 36 Gy. Blastemal type: Stage III: - 25.2 Gy in 14 fractions (1.8 Gy per fraction) to the involved flank. - Boost to macroscopic residual disease after surgery with 10.8 Gy to a total dose of 36 Gy.

For Rhabdoid tumour and CCSK see chapter 17.17 and 17.18.

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17.3 WHOLE ABDOMEN RADIOTHERAPY – dose and fractionation - Stage III based on spillage/rupture: - Whole abdomen with 15.0 Gy (IR) or 19.5 Gy (HR) in 10 or 13 fractions (1.5 Gy per fraction). - Consider reduction of the total dose in children younger than 2 years to 12.0 Gy. - Dose per fraction may be lowered to 1.25 Gy in case of toxicity and very young children (< 2 years). Stage III based on macroscopic peritoneal deposits (IR and HR): - Whole abdomen with 19.5 Gy in 13 fractions of 1.5 Gy. - Consider a boost to a limited area if necessary up to 25.2 Gy (IR) or 36 Gy (HR) for macroscopic remnants. - Reduction of the total dose in children younger than 2 years to 12 Gy. - Dose per fraction may be lowered to 1.25 Gy in case of toxicity and very young children (< 2 years).

17.4 Summary recommendation of radiation therapy treatment of abdominal disease

Stage II Stage III (except Stage III (major rupture) major rupture) Intermediate Risk no indication 14.4 Gy in 8 fractions, Whole abdomen +/- 10.8 Gy boost 15.0 Gy in 10 fractions +/- 10.8 Gy boost High risk 25.2 Gy in 14 fractions 25.2 Gy in 14 fractions Whole abdomen Diffuse anaplasia +/- 10.8 Gy boost +/- 10.8 Gy boost 19.5 Gy in 13 fractions +/- 10.8 Gy boost High Risk no indication 25.2Gy in 14 fractions Whole Abdomen Blastemal type +/- 10.8 Gy boost 19.5 Gy in 13 fractions +/- 10.8 Gy boost

17.5 Dose reduction of chemotherapy

Cytotoxic drugs (especially actinomycin D and doxorubicin) should be reduced to 2/3 of the normal dose during radiotherapy and during the first cycle after radiotherapy.

17.6 RADIATION THERAPY TREATMENT OF STAGE V (BILATERAL WILMS TUMOUR) Management of bilateral WT is constantly evolving and still stands as a therapeutic challenge. Patients receive postoperative therapy according to histology and to the highest assigned risk for either kidney. Local RT is indicated after nephrectomy according to the guidelines for unilateral disease. Furthermore RT may be adequate to prevent local recurrence in case of positive surgical margins (stage III) as part of nephron sparing surgery (enucleation, partial nephrectomy). A national radiotherapy reference institution should be contacted in these cases.

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17.6.1 Indications for postoperative local radiotherapy Bilateral Nephroblastoma (Stage V) with complete resection of all gross tumours - according to local stage - Intermediate risk histology, stage III (nodes positive N+, positive margins, residual disease left after surgery, tumour rupture) - High risk histology, stage II (except blastemal type) and stage III (all high risk histologies)

17.6.2 Radiation therapy treatment after nephron sparing surgery - dose and fractionation - In case of irradiating a remaining kidney after nephron sparing surgery the dose to the whole kidney should not exceed 10-12 Gy (12 Gy maximum dose), even if there is high risk histology. Boost irradiation focussed to areas at risk (surgical margin) is difficult to perform because of small target size and target movement; nevertheless it may be taken into account if the technical preconditions make a proper dose distribution reliable. (Brachytherapy in selected cases). In general a radiotherapy reference institution should be contacted in these cases. Stage II: In cases of high risk histology except blastemal type the area at risk (surgical margin) or the whole kidney should be irradiated to a total dose of 10.8-12 Gy in 1.5 – 1.8 Gy per fraction. No indication for radiotherapy to cases with intermediate risk histology. Stage III: In patients with positive margins (local stage III), any histologically type should be irradiated to the area at risk (surgical margin) or the whole kidney to a total dose of 10.8-12 Gy in daily fractions of 1.5-1.8 Gy. In patients with stage III because of lymph node involvement and local stages I or II (intermediate risk) or stage I (high risk) only the para-aortic lymph node area should be irradiated with 14.4 Gy (intermediate risk) or 25.2 Gy (high risk), 1.8 Gy per fraction. In patients with local stage III (intermediate risk) or local stages II and III (high risk) and lymph node involvement, a flank irradiation should be performed; the target volume has to be reduced after 12 Gy maximum dose to the remaining kidney using a shrinking field to the lymph node area.

17.7 RADIATION THERAPY TREATMENT OF METASTATIC SITES 17.7.1 Indications for pulmonary radiotherapy (primary treatment) Low Risk Histology: Indication of RT in LR tumours should be discussed with national PI, since it is usually not indicated.

Intermediate risk histology: - No complete remission of the lung metastases after chemotherapy (week 10 of postoperative chemotherapy) and/or surgery in computed tomography.

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- Histological viable tumour after chemotherapy and surgery in resected metastases. - RT should not be given if a CR is achieved in computed tomography at week 10 of postoperative chemotherapy. High risk histology: - All cases of primary lung metastases regardless of the response to chemotherapy or surgical treatment. Dose and fractionation - The total dose is 12 Gy (intermediate risk) and 15 Gy (high risk) for both lungs. - The dose per fraction is 1.5 Gy (with homogeneity correction), delivered within 8-10 fractions. A boost of 10-13 Gy (intermediate risk histology) and 15-20 Gy (high risk histology) should be considered for areas of macroscopic residual disease after surgery using highly conformal radiation techniques (SBRT), if possible and normal tissue dose constraints can be respected. 17.7.2 Timing of pulmonary radiotherapy In case of abdominal RT with pulmonary RT it is recommended to treat the chest and the abdomen simultaneously in one planning procedure (resp. in one field) in order to avoid any gap or overlap of both fields (cardiotoxicity). In case of resected abdominal stage III disease with a high probability of local recurrence such as cases with diffuse anaplasia histology, abdominal RT may be applied after surgery and pulmonary radiotherapy may given later in a separate field. 17.7.3 Indications for hepatic radiotherapy Liver metastases which do not respond completely to chemotherapy and which cannot be completely resected with negative margins should be irradiated either with total liver irradiation or partial liver irradiation depending on the number and the distribution of the metastases. In principle, a similar approach as for lung metastases may be used. Dose and fractionation A total dose of 14.4 Gy for intermediate risk and 19.8 Gy for high risk histology should be given to the whole liver; unresected residual metastases after chemotherapy or the area of incomplete resection of metastases may be boosted up to 25.2 (IR) or 36.0 Gy (HR); a simultaneous integrated boost (SIB) technique or stereotactic RT may be used. Dose per fraction is 1.5-1.8Gy. The total dose to the whole liver should not exceed 20 Gy.

17.7.4 Indications for radiotherapy to other metastatic sites Metastatic disease to other organs than lung or liver is a rare event in nephroblastoma; therefore the treating radiotherapist should discuss in such cases the radiation therapy options with panel radiotherapists.

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17.7.4.1 Brain and bone metastases Patients with haematogenous metastases to the brain and/or bone metastases at diagnosis should be treated with the appropriate RT fields regardless of response to chemotherapy.

17.7.4.2 Treatment dose and fractionation Brain: The whole brain is treated to a dose of 15.0 Gy for intermediate risk and 25.2 Gy for high-risk histology (1.8 Gy per fraction). A boost of 10.8 Gy may be given to a total of 25.2 Gy (IR) resp. 36 Gy (HR). A simultaneous integrated boost (SIB) technique may be used. Bone: Bone metastases may be treated with a dose of 30 or 30.6 Gy. Dose per fraction is 1.8-3 Gy (individual decision by performance status, prognosis, localisation of the metastases and individual palliative situation).

17.8 Summary recommendations of radiation therapy treatment of metastatic sites

Metastatic Site Lung Liver Brain Bone (incomplete resection) Intermediate Risk Whole lung Whole liver/ Whole brain Local histology 12.0 Gy in 8 local 15.0 Gy in 10 30.6 Gy in 17 fractions 14.4 Gy in 8 fractions fractions fractions (boost +/- 10.5 Gy boost or 30 Gy in 10 10.8 Gy) fractions High Risk histology Whole lung Whole liver/ Whole brain Local 15.0 Gy in 10 local 25.2 Gy in 14 30.6 Gy in 17 fractions 20-25.2 Gy in 11 fractions fractions fractions (boost +/- 10.5 Gy boost or 30 Gy in 10 16.2 Gy) fractions

17.9 RADIATION THERAPY TREATMENT OF RECURRENT DISEASE 17.9.1 RADIATION TREATMENT OF LOCAL RELAPSES IN THE ABDOMEN 1. Recurrent local tumour in abdomen/flank without previous radiotherapy Radiotherapy according to histologically type and chemotherapy response; the same procedure as in the first line treatment (see chapter: “Radiotherapy of local abdominal disease”)

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2. Recurrent local tumour in abdomen/flank after a previous radiotherapy All cases receive radiotherapy according to the localisation and the dose and volume of the first line RT. Because of many different situations it is impossible to develop standard recommendations. Nevertheless in many cases a re-irradiation should be possible because of the low doses necessary. New techniques like IMRT should be taken into account as they may contribute to a reduction of dose in normal tissues such as small bowel or liver. The treating radiotherapist should discuss the radiation therapy options with panel radiotherapists.

17.9.2 RADIATION TREATMENT OF RELAPSES IN THE LUNG 1. No lung irradiation in the first line treatment: Whole lung RT is always indicated in cases of lung metastases as a recurrent disease and no prior lung irradiation for patients with intermediate as well as high risk histology. Target volume and doses see chapter: “Radiotherapy treatment of metastatic sites, pulmonary RT” RT should take place after chemotherapy and surgery (if performed).

2. Lung irradiation in the first line treatment: In this case, no further whole lung RT can be performed. In case of residual disease small volume RT (stereotactic body radiotherapy (SBRT)) should be considered, according to the histological type. The treating radiotherapist should discuss the radiation therapy options with panel radiotherapists.

17.10 INTERRUPTIONS and BREAKS Breaks must be kept to an absolute minimum. Interruptions due to treatment machine service and public holidays must be avoided unless absolute necessary. Both breaks and interruptions should be compensated if longer than 2 days. Interruptions for myelotoxicity: - RT should be interrupted if the neutrophil count falls below 0,5x109/l and should not be resumed until the count is at least 1,0x109/l. - RT should be interrupted if the platelet count falls below 25x109/l and should not be resumed until the count is at least 50x109/l. - The haemoglobin level should be maintained at a minimum of 10 g/dl during RT with correction by transfusion if necessary. - GCSF may be used in the case of the neutrophil count falling below 0,5, and continued until it is greater than 1,0.

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17.11 EQUIPMENT AND TREATMENT TECHNIQUE (SIMULATION/TREATMENT PERFORMANCE) Megavoltage equipment (photons form a modern linear accelerator energy usually 4-6 MV, radiation technique AP/PA opposing fields, 3D conformal or intensity modulated radiotherapy (IMRT)-technique, IGRT techniques should be available. All patients will undergo a simulation procedure with conventional simulator or preferred a virtual CT- simulator (especially to optimize critical organs sparing (heart, liver, contralateral kidney etc.). The CT based radiation treatment planning is the standard of care. All Patients will generally be treated in supine position.

17.12 TARGET VOLUME DEFINITION Target volumes are defined according to ICRU guidelines (ICRU 50 and 62). 17.12.1 Dose uniformity and Reference Points (ICRU) Target dose is calculated and reported according to the ICRU criteria. The reference point is in a central part of the target volume. The dose variation within the target volume should not exceed ±5% until ±7-10% of the prescribed dose. CT based computed dose have to follow the same rules for target dose specification. Margins for PTV will be influenced by individual departmental policy (availability of 4D-CT scan radiation treatment planning and IGRT, individual IGRT-frequency). In general the margins that will be applied will be as follows:

17.13 TARGET VOLUMES 17.13.1 Localisation of primary tumour and kidney for flank/abdominal RT For RT planning the preoperative tumour extent should be localised according to the preoperative contrast-enhanced CT and/or preoperative T2-weighted MRI scan. A preoperative CT or MRI scan is the optimal imaging modality and should be performed to optimize target volume delineation as a standard of care. The boundaries of tumour residuals during surgery should be marked with clips, particularly in the case of areas suspicious of incompletely resected disease. In addition, important information may be available in the surgical and histopathological reports. For volume-reduced IMRT-techniques a supplemental protocol will be available. These techniques should only be undertaken in centres with extensive IMRT and IGRT experience. In the case of pre-operative or intra-operative rupture the anatomic location and the intra-abdominal space (intra/retroperitoneal) should be clearly indicated in the surgical note and drawing. Infiltration of the peri-renal fat, diaphragm, involved lymph nodes, macroscopic incomplete resection, microscopic or macroscopic ruptures have to be stated clearly.

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17.14 CLINICAL TARGET VOLUME (CTV) AND PLANNING TARGET VOLUME (PTV) 17.14.1 Flank-Radiotherapy The clinical target volume (CTV) is defined as the preoperative gross tumour volume (GTV)+ 1 cm margin. The CTV may occasionally be adapted or modified in order to spare nearby organs at risk. The internal margin for breathing movements and interfractional movements to PTV is defined as CTV plus 1 cm. The PTV should extend across the midline to achieve homogeneous irradiation of the full width of the vertebral bodies to prevent a scoliosis. The contralateral kidney should be spared as good as possible. The radiation field should not be extended into the dome of the diaphragm unless there is tumour extension. If possible do not irradiate major parts of the heart (left sided tumours). In case of positive lymph nodes (stage III) that have been removed, the entire length of the para-aortic chain of lymph nodes will be included. Lymph node groups that were involved at presentation should be included. Nodal areas will be treated in continuity with the primary tumour area. The cranial field border should be at the thoracic vertebra TH10-/11 level while almost 50% of the celiac axis arises from the aorta at the level of the pedicle of the 12th vertebral body.

Boost volume for residual macroscopic disease: Extent of residual macroscopic disease at surgery (postoperative MRI/CT scan is necessary) with a 1-2 cm safety margin.

17.14.2 Whole abdominal RT CTV/PTV: This includes the entire abdominal contents and peritoneum extending from the dome of the diaphragm to the pelvic floor (lower border of obturator foramen).

17.14.3 Pulmonary RT CTV/PTV: This encompasses both lungs including the apices and costo-diaphragmatic recessus. If abdominal radiotherapy also is to be given, both fields may be matched and irradiated simultaneously in order to avoid any gap or overlap.

17.14.4 Liver RT CTV/PTV: This includes the whole liver and as a boost the extent of incompletely resected or residual tumour with margin of 0.5 - 1cm. Margins for PTV depends on the internal target motion and will be influenced by individual departmental policy (availability of 4D-CT scan radiation treatment planning and IGRT). A simultaneous integrated boost technique (SIB) may be used.

17.14.5 RT for brain metastases CTV/PTV: The whole brain is treated; a boost to single metastases may be performed. A simultaneous integrated boost technique (SIB) may be used.

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17.14.6 RT for haematogenous metastases to bone CTV/PTV: For bone metastases it is not necessary to treat the entire bone. The field include the obvious disease visible on imaging examination, with an appropriate margin depending on the skeletal area involved.

17.15 NORMAL TISSUE SPARING Dose in organs at risk is calculated and reported for each organ separately. It is recommended to add the estimated volume of the organ irradiated to the reported dose. Typical organs at risk in nephroblastoma treatment are the vertebral column, lung, iliac bone, contralateral kidney, small bowel, soft tissue of irradiated flank, liver, ovaries, testes, thyroid gland, mammary gland and heart.

17.15.1 Critical organ dose Kidney: The dose to the remaining kidney should not exceed more than 10 – 12 Gy (V10 < 25-30%). Liver: The dose to the whole liver should not exceed 20 Gy. A dose exceeding 20 Gy should not be received by more than half of the liver (V20 < 50 %, D mean < 20 Gy). Lung: The whole lung dose should not be more than 15 Gy in 15 fractions (HR-patients) with correction for inhomogeneity. A dose exceeding 15 Gy should not be received by more than 25% of the lung volume. (V15 < 25%).

17.15.2 Shielding: Joints: For pulmonary RT the shoulder joints should be shielded. For whole abdominal RT the hips should be shielded.

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17.16 EXAMPLES FOR TYPICAL TARGET VOLUMES AND RADIATION PORTALS (Related to anatomical landmarks)

17.16.1 Stage II high risk, stage III (Fig. 1a-c) Cranial border: - Right sided tumours: if feasible 1-2 cm below the dome of the diaphragm (sparing of the liver) - Left sided tumours: 1-2 cm above the macroscopic tumour (e.g. dome of diaphragm), if possible do not irradiate the heart.

Caudal border: 1-2 cm below the macroscopic tumour e.g. within the iliac fossa often including the iliac crest (Position of the ovaries (homo-/contralateral) is important.

Lateral border: Including der abdominal wall

Medial border: Depending on tumour extension: including the vertebral bodies and shield the contralateral kidney.

Boost volume only for macroscopic residual disease after surgery with 1-2 cm margin (Fig. 1b, c, 2b)

Fig. 1a: Right-sided tumor with stage III (microscopic residual disease, minor rupture). Radiation portal covering the tumor region including the vertebral column, the iliac crest and major parts of the right liver.

The same type of radiation portal would apply for a nephroblastoma stage II, high grade.

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Fig. 1b: Extensive left-sided tumor from the dome of the diaphragm to the fossa iliaca with macroscopic

residual disease at the splenic hilus (Stage III). Radiation portal including the major part of the left hemiabdomen with the vertebral column; boost portal including the left upper abdomen without the

vertebral column.

Fig. 1c: Right-sided tumor with paraaortic lymphnode metastases infiltrating the vena cava inferior up to the diaphragm and tumor thrombus up to the right atrium (Stage III): lymph nodes and tumor thrombus

could not be completely removed macroscopically by surgery. Radiation portal encompassing the tumor region, the paraaortic lymphnode chain, and the vena cava inferior including part of the right atrium. Boost portals covering the area of the macroscopic residual disease: paraaortic lymphnode chain, vena cava inferior, and the part of the right atrium.

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17.16.2 Stage III intermediate and high risk (Fig. 2a, b) (if lymphnode involvement under the level of the renal artery)

Target volume encompasses the whole para-aortic lymphnode chain including the homo-lateral pararenal lymphnodes and the macroscopic tumour extends at surgery + 1-2 cm safety margin. The safety margin may not be feasible towards the contra-lateral kidney and to large volumes of the liver.

Examples for typical Target Volumes and Radiation Portals:

Cranial border: Right sided tumours: if feasible 1-2 cm below the dome of the diaphragm (sparing of the liver)

Left sided tumours: 1-2 cm above the macroscopic tumour (e.g. dome of diaphragm), if possible do not irradiate the heart.

Caudal border: 1-2 cm below the macroscopic tumour e.g. within the iliac fossa often including the iliac crest (Position of the ovaries (homo-/contralateral) is important!

Lymphnode chain: lower plate of L IV but for preventing inhomogeneous dose to the bone, the border is often the elongation of the caudal border.

Lateral border: including the abdominal wall

Medial border: including the transverse process of the vertebral column.

Boost volume only for macroscopic residual disease in the lymphnodes after surgery

Fig. 2a: Right-sided tumor with one homolateral para-renal lymphnode involved and removed (stage III).

Radiation portal covering the tumor region (including the right dome of diaphragm and the iliac crest) and the whole para-aortic chain.

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Fig. 2b: Right-sided tumor with several para-aortic lymphnode involved (stage III) and suspicious macroscopic residual disease in the lymphnode chain at surgery (stage III macroscopic residual disease).

Radiation portal covering the tumor region and the whole para-aortic lymphnode region. Boost volume in

case of macroscopic residual disease refined to the lymphnode chain including the homo-lateral renal hilus.

17.16.3 Stage III (all histologies): major intra-/retroperitoneal rupture

Examples for typical Target Volumes and Radiation Portals:

1) Major intraperitoneal rupture (Fig. 3)

The target volume encompasses the whole intraperitoneal cavity

Cranial border: including both domes of the diaphragm

Caudal border: upper part of the symphysis

Caudal and lateral border: line along the inguinal ligament (sparing the epiphyses of the femoral head).

Lateral border: including abdominal wall. Cave on remaining kidney dose and dose at the testes.

Boost volume only for macroscopic residual disease after surgery with a 1-2 cm safety margin.

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Fig. 3: Massive intraperitoneal rupture during surgery as right-sided tumor broke into many pieces and spread around the intraperitoneal cavity (stage III major rupture). Radiation portal covering the whole

intraperitoneal cavity. No boost indicated as no detectable macroscopic residual disease was seen at surgery.

2) Major retroperitoneal rupture (Fig. 4)

Cranial border: including both domes of the diaphragm

Caudal border: upper part of the symphysis

Caudal and lateral border: line along the inguinal ligament (sparing the epiphyses of the femoral head).

Homo-lateral border: including abdominal wall. Cave on remaining kidney dose and dose at the testes.

Contra-lateral border: including the vertebral bodies, line from edge of LV to symphysis (cave contralateral ovary dose and dose at the testes!)

Boost volume only for macroscopic residual disease after surgery with a 1-2 cm safety margin.

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Fig. 4: Extensive retroperitoneal rupture in a huge tumor without contamination of the intraperitoneal

cavity (stage III major retroperitoneal rupture). Target volume encompasses the whole homo-lateral

retroperitoneal space (right) including the prevertebral space. Boost is indicated if there is macroscopic disease left in the retroperitoneal space during surgery.

17.16.4 PULMONARY RADIOTHERAPY (Stage IV)

Examples for typical target volumes and radiation portals: stage IV (lung)

If local abdominal radiotherapy has to be performed, pulmonary and abdominal targets are defined on the same portal imaging. If the targets overlap, a decision has to be taken related to target matching of two adjoining fields or using intensity modulated radiotherapy techniques.

Cranial border: including the top of the lung (some cm above the clavicle)

Caudal and lateral border: including the lung, shielding the shoulder region

Caudal border: including the bottom of the costodiaphragmatic recesses: e.g. 2-4 cm below the radiobiologically visible diaphragm, depending much on the phase of respiration which is to be seen at lateral recesses or on a 4D-CT-planning CT scan (if object the planning tool)

Lateral border: including the thoracic wall

Boost volume (e.g. stereotactic radiotherapy Boost with 10-15 Gy to metastases remnants visible after surgery or chemotherapy at the start of radiotherapy). In very young children, protect as much lung tissue as possible.

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Pulmonary metastases at diagnosis (stage IV lung) with residual inoperable disease in the left and central right lung after preoperative chemotherapy. Indication for pulmonary radiotherapy if there is disease after post-operative aggressive chemotherapy (all histologies) or in case of a histological high risk primary tumor, regardless of metastatic response. Radiation portal including both lungs with its recesses. Remember air-correction when calculating dose. Lateral view needed to calculate dimension of lung tissue.

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17.17 GENERAL GUIDELINES FOR RADIATION THERAPY FOR PATIENTS WITH RHABDOID TUMOURS OF THE KIDNEY (RTK)

Children with a rhabdoid tumour of the kidney should be registered at the European Rhabdoid Registry and be treated according to this protocol (EU-Rhab, extracerebral disease). Contact the European Rhabdoid Registry protocol (EU-Rhab) and their reference center: Pediatric oncology: [email protected] and /or [email protected] Radiation oncology: [email protected]

Indications for postoperative RT to the flank: Stage I-III RTK (19.8 Gy for children > 1 year, 10.8 Gy for children < 1 year), boost for gross residual tumour after surgery at a total dose of 10.8 Gy. Indications for the whole abdominal RT: - 19.5 Gy (1.5 Gy) > 1 year; < 1 year reduce at 10.5 Gy (1.5 Gy) - Stage III- ascites positive for rhabdoid cells - Preoperative tumour rupture - Diffuse operative spill - Peritoneal seeding Indication for RT to the lung: Lung metastases (15 Gy, in 10 fractions) (not in children below three years of age, may be reduced the dose to 10.5 Gy in 7 fractions, boost dose of up to 7.5 Gy (persisting localized lung foci after whole lung irradiation) Indications for RT to the liver: Liver metastases, diffusely involved, 19.8 Gy, single dose fraction 1.8 Gy (in infants may be reduced the dose to 15 Gy in 10 fractions. Additional boost irradiation dose of 5.4-10.8 Gy may be administered to limited volumes. Indications for whole brain RT: Brain metastases (21.6 Gy) plus boost of 10.6 Gy Indications for bone metastases RT: Bone metastases (25.2 Gy)

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17.18 Clear Cell Sarcoma of the Kidney (CCSK)

The radiation dose necessary to prevent local recurrence of CCSK is discussed controversial. As most of the children with CCSK are very young it was agreed to lower the dose from 25.2 Gy to 10.8 Gy according to the data presented by the COG. Close follow up and a stopping rule are defined to rule out an increase of local recurrences. We propose a mechanism to stop the study early in the event that the estimated relapse rate becomes unacceptably high. The rule described in the following is based on an a priori guess for the relapse rate of 0.02 and an unacceptable relapse rate of 0.05. The approach is Bayesian. We postulate a Beta(1,49) prior for the relapse rate which is the least informative prior that is consistent with a prior expectation of a relapse rate of 0.02. The prior belief is then that there is a probability of 0.08 that the relapse rate will exceed 0.05. (This does not necessarily mean that there is a 0.08 probability that the stopping rule will be trickered since both type I and type II errors can occur: however, there is a 0.08 probability that the stopping rule ought to be trickered). The posterior mean of the relapse rate will then be exactly 0.05 if we observe 2 relapses in ten patients, 3 relapses in 30 patient, 4 relapses in 50 patients etc.

Bayes on the shape of the EFS curve for stage I in Furtwangler et al. in which the survival curve appears linear in the first two years and flattens thereafter, a patient will count as relapse free after a two years of follow up but will count relapse free with a weight m/24 after m months of follow up when m<24. The steering committee will take other factors into considerations, so the 0.05 cut-off is not set in stone. But the posterior mean will be calculated before each steering committee meeting so that it can be used for making decisions about protocol amendments or, possibly, to stop the recommendation of reduced RT early. General guidelines and target delineation as nephroblastoma (see there).

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Local stage II-III: - 10.8 Gy to the flank; in 6 fractions, 5 fractions a week. - 10.8 Gy boost to macroscopic residual

Metastatic disease: - Whole lung: 12 Gy in 8 fractions, five fractions a week - Whole brain: 15 Gy in 10 fractions, 5 fractions a week, optional boost of 10.5 Gy - Liver: focal as well as diffuse involvement: 19.8 Gy (infants <13 months 10.8 Gy and no boost, patients with residual tumour: optional boost 5-10.8 Gy) - Lymphnode involvement: resected: 10.8 Gy, unresected: boost with 10.8 Gy to suspect areas

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17.19 ORGANS AT RISK 17.19.1 Bone and soft tissue It is not clear to what degree a radiation dose of 15 Gy in young children will impair bone and soft tissue growth, which takes place years after radiotherapy. The amount of impairment is dependent on radiation dose, irradiated volume, and age of the child and reveals as kyphoscoliosis, hypoplasia and osteochondroma. The impairment is expected to be smaller after a lower dose of radiotherapy (<15 Gy). It can be assumed that, if there is impairment, this will only be small and without significant clinical relevance. The amount of impairment is certainly larger after radiation dose of 30 Gy. The whole vertebral column should be included within radiation portal area in order to avoid inhomogeneity, which is known to produce scoliosis. Nevertheless, the radiation portal should not include major parts of the contralateral kidney. The iliac crest contains the apophysis from which the growth of the iliac bone mainly takes place. In order to avoid asymmetric iliac bone growth radiation dose at this apophyseal line should not be more than 15 Gy. The epiphyseal lines of the acetabulum cannot be shielded, if the whole intraperitoneal cavity is to be adequately irradiated (“abdominal bath”). The femoral head should not be included in the treatment volume as it does not belong to the target volume and epiphyseal slipping is a possible consequence after radiotherapy in young children. The shoulder is not to be included within the treatment volume if pulmonary radiotherapy is indicated. Reduction of lung volume and dynamic compliance can develop to some degree after radiotherapy to both lungs, more so in young children, because of insufficient growth of the rib–cage.

17.19.2 Liver Radiation tolerance of the liver depends on total dose and volume irradiated. A radiation dose of 15 to 20 Gy to the whole liver does not by itself produce severe side effects and is indicated in whole abdominal irradiation and may be advisable in some extensive right sided tumours and whole liver irradiation because of metastases. If the boost volume is indicated in the upper right abdomen at least one quarter of the liver should be shielded after 20 Gy. If less than half of the liver is within the treatment volume no special shielding is necessary. If veno-occlusive disease (VOD) occurred during chemotherapy, the radiation tolerance of the liver might be reduced. Special attention should be paid to further liver shielding.

17.19.3 Gastrointestinal tract Because of the radiosensitivity of the rapidly proliferating mucosa sparing from irradiation volume is advisable but only possible by adequately tailoring the treatment portal. Side effects like diarrhoea and vomiting may be observed during abdominal radiotherapy in particular if large volumes are treated. Symptomatic treatment for vomiting and for diarrhoea is necessary including intravenous fluids are required. A diet free of lactose and saccharose and with low fat content is recommended for treatment of acute and late radiation enteritis.

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17.19.4 Kidney Dose of the remaining kidney should not exceed 12 Gy (maximum dose). Irradiation of the remaining kidney up to 12 Gy is indicated in total abdominal radiotherapy and in some cases of stage V tumours. Radiation dose to the contralateral kidney in radiotherapy of the prevertrebral space due to the penumbra at the field margin and scattered radiation usually does not exceed 10-20 % of the radiations dose at the reference point. It may be somewhat higher in medial parts in the remaining kidney lying close to the vertebral column.

17.19.5 Ovary Ovarian insufficiency induced by irradiation doses of about 15 Gy, if the true pelvis had to be included into the irradiated volume. At least one ovary should not receive a radiation dose of more than 10-15% of the dose at the reference point (15 Gy). As the necessary distance between the field margin and the location of the ovary to achieve this dose can be estimated before treatment performance, much attention should be paid to adequate localization in this regard. If the target dose is 30 Gy the ovarian dose should not exceed 5- 10% of the reference dose. Only in total abdominal radiotherapy both ovaries are irradiated to more than 15 Gy. Nevertheless, little is known about the ovarian tolerance doses in young girls. Hormonal function and fertility can probably be preserved if the ovarian dose can be kept below 2-3 Gy.

17.19.6 Testes Impairment of spermatogenesis may occur even after scattered radiations dose above 50 to 100 cGy to the testes. Leydig cell function is much less radiosensitive and not influenced by such low scatter radiation dose. Radiation dose to the testes from scattered radiation should be clearly below 5% of the radiation dose at the reference point (15 Gy). Special attention is necessary in total abdominal radiotherapy because of the close relationship between the caudal border and the position of the testes, particularly in small boys.

17.19.7 Mammary gland/areola The mammary gland bud is known to be very radiosensitive even in low dose radiotherapy; hypoplasia of the mammary gland may occur after a dose as low as 1-3 Gy in young girls. Therefore it should be spared from radiotherapy whenever possible. Special attention has to be paid when target volume includes the upper abdomen and the dome of the diaphragm. In radiotherapy of both lungs some sparing of the mammary gland bud may only be achieved by the build-up-effect in high megavoltage beams.

17.19.8 Cardiac toxicity Cardiomyopathy in case of pulmonary irradiation, previous treatment with doxorubicin or radiotherapy followed by this drug may increase the chance of this complication. In radiotherapy of both lungs the

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heart should be spared with new techniques like IMRT whenever possible. Echocardiography should be done at regular intervals to detect early toxicity

17.20 QUALITY ASSURANCE DOCUMENTATION – Case Report Form (CRF) The quality assurance information should be sent on request to the national clinical trials office or to the SIOP Secretariat along with copies of the radiotherapy trial forms (Radiotherapy Data reporting forms (Online- Radiotherapy-eCRF; standardized questionnaire to document the data of radiotherapy).

17.21 Chair and Members of the Radiotherapy Panel

Name Profession Country Email

Chair Christian Rübe Radiotherapist Germany [email protected]

Lorenza Gandola Radiotherapist Italy [email protected]

Aymeri Huchet Radiotherapist France [email protected]

The Geert Janssens Radiotherapist [email protected]

Netherlands

The [email protected] Foppe Oldenburger Radiotherapist Netherlands

Members Daniel Saunders Radiotherapist UK [email protected]

Stéphane Supiot Radiotherapist France [email protected]

Patrick Melchior Radiotherapist Germany [email protected]

Farida Alam Radiotherapist UK [email protected]

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18 Non-Wilms tumours of the kidney 18.1 Clear Cell Sarcoma 18.1.1 Introduction Clear Cell Sarcoma of the Kidney (CCSK) is an uncommon renal tumour that comprises 3-5% of all primary renal tumours in children [1]. This tumour is observed most often in children between 2 and 4 years of age and is characterized by a highly malignant potential. CCSK represents the second most common paediatric renal tumour after Wilms tumour. In the past, CCSK was considered an unfavourable histology Wilms tumour variant. However, in 1970 it was recognized as a separate clinico-pathologic entity by Kidd [2]. Marsden et al. subsequently called the tumour ‘bone metastasizing tumour of the kidney’ and Beckwith and Palmer referred to it as ‘clear cell sarcoma of the kidney’ [3, 4]. Histologically CCSK shows a remarkable morphologic diversity, which renders it sometimes difficult to distinguish this tumour from other renal tumours, such as Wilms tumour, malignant rhabdoid tumour of the kidney and congenital mesoblastic nephroma [5]. In earlier studies, up to 50% of all CCSKs have initially been classified as an entity different from CCSK by local pathologists [6]. Gene expression profiling studies have reported up-regulation of neural markers and apparent expression of members of the Sonic hedgehog signalling pathway and the Akt cell proliferation pathway (including strong activation of EGFR) in CCSK [7, 8, 12, 37]. A cytogenetic marker, which has been consistently identified in CCSK patients is a clonal balanced translocation involving t(10;17)(q22;p13) [8 - 10]. O’Meara et al. identified that the breakpoints of this translocation involve the genes NUTM2B/E (chromosome 10) and YWHAE (chromosome 17), and identified the YWHAE-NUTM2B/E fusion transcript in 6 of 50 studied CCSK cases [11]. Very recently, recurrent internal tandem duplications (ITD) of the X- linked BCL-6 co-repressor (BCOR) gene have been described in CCSK [39]. This BCOR ITD and t(10;17)(q22;p13) seem to be mutually exclusive; all reported CCSK patients without t(10;17) harbour the BCOR ITD, while cases with t(10;17) do not harbour BCOR ITDs [41]. A large scale screening of 159 cases confirmed mutual exclusivity of the duplications that are found in most cases and the rare translocations, but also identified a subset of 12 % of CCSK that lacked both alterations [43]. These may form a novel subentity of CCSK that may behave differently. With current intensive treatment schedules, including radiotherapy and multi-agent chemotherapy regimens, outcome has significantly improved (5-year event-free survival ranging from 65% - 85%, 5- year overall survival ranging from 75% - 90%) [5, 13]. The current intensive therapy schedule aims to maintain and even improve survival, but also to limit serious late and direct toxicity, such as cardiotoxicity, infertility, metabolic syndrome, obesity and second malignancies [14 - 16] by using safe cumulative dosages of anthracyclines, combining ifosfamide and cyclophosphamide use, and reducing radiotherapy dosages. Ongoing biological studies offer the chance to identify molecular targets for more targeted future therapy strategies.

18.1.2 Aims and endpoints Aims Primary aim:  To provide the best available care for children with CCSK, including a harmonised diagnostic and treatment guideline.

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Secondary aims:  To determine the prevalence, clinical characteristics and outcome of paediatric CCSK patients based on a prospective registration of complete national cohorts of paediatric CCSK patients, in order to identify risk categories, which may benefit from either reduced or alternative/ intensified therapy.  To identify specific radiological characteristics that may discriminate CCSK from other renal tumours at presentation.  To study specific biological and genetic characteristics in paediatric CCSK: o To determine the prognostic value of the YWHAE-FAM22 fusion transcript in CCSK patients o To determine the prognostic value of BCOR ITDs in CCSK patients o To characterize CCSK that lack both, the duplication and translocation events o To identify significant biomarkers, genetic and molecular alterations in CCSKs by a genomics approach and functionally validate these genetic alterations Endpoints Primary endpoint:  Outcome of paediatric CCSK patients Secondary endpoints:  The prevalence of paediatric CCSK based on the registry of renal tumours (SIOP-RTSG).  The characterization of CCSK by molecular genetics o The prognostic value of the YWHAE-FAM22 fusion transcript in CCSK o The prognostic value of BCOR ITDs in CCSK o Additional genetic causes of CCSK o The clinical relevance of novel significant genetic and molecular aberrations in CCSK

18.1.3 Background information Epidemiology and clinical features CCSK comprises 3-5% of all primary renal tumours in children and is observed most often in children under 3 years of age [5, 13]. CCSK is extremely rare in the first 6 months of life and in adults, where it has been the subject of only isolated case reports [17 - 19]. A male predominance has been noted in all large CCSK reports (average male to female ratio of about 2:1) [5, 13]. Some studies suggested a predilection for involvement of the right kidney [13, 20, 21]. Most patients present with stage I, II and III disease, and only 6-7% of the patients present with stage IV disease [5, 6]. The most frequent sites of metastases at diagnosis are bone, lung and liver [34]. Unlike nephroblastoma, CCSK is never bilateral. Relapses occur in about 15% of the cases [34]. CCSK has historically been associated with late recurrences, occurring up to 8 years after initial diagnosis (median time to relapse 17-20 months) [5, 6, 22]. Recent reports from the International Society for Pediatric Oncology (SIOP) and the National Wilms Tumour Study Group (NWTSG) have indicated that, following intensified upfront treatment, the pattern of relapses is changing with brain metastases now more common than the classical site of bone metastases in its original description as the ‘bone metastasising renal tumour of childhood’ [23, 24]. This indicates that the brain might be a sanctuary for cells that are protected from intensive chemotherapy that patients currently receive.

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Histology CCSK tumours have a deceptively bland appearance and many histological patterns. The most common pattern is the classic pattern, with features present at least focally in over 90% of tumours [5]. The classical subtype is characterized by a uniform appearance of a diffuse growth of relatively small cells with normochromatic nuclei, inconspicuous nucleoli, pale staining cytoplasm and ill-defined cell membrane [5, 25]. In only 20% of the cases the tumour cells do have clear cytoplasm. The most characteristic feature is a peculiar vascular pattern consisting of arborizing blood vessels that create an alveolar or trabecular pattern. The classical pattern of CCSK is relatively simple to diagnose, but others including the myxoid, sclerosing, cellular, epithelioid, palisading, spindle cell, storiform and anaplastic pattern can cause problems in reaching the diagnosis [5]. In the differential diagnosis blastemal nephroblastoma, mesoblastic nephroma, PNET and rhabdoid tumour must be considered. The histogenesis of CCSK is uncertain. Recent studies have shown that tumour cells are positive for Cyclin D1 and NGFR, so these two markers should be used in diagnostically challenging cases. Other markers including cytokeratin, factor VIII associated antigen, epithelial membrane antigen, desmin and S100 protein are negative in CCSK [5, 25].

Genetics and Biology One of the recurring molecular markers which has been identified in patients with CCSK is a balanced translocation involving t(10;17)(q22;p13) [8 - 10]. O’Meara et al. identified that the breakpoints of this translocation involve the genes NUTM2B/E (chromosome 10) and YWHAE (chromosome 17); the YWHAE-NUTM2B/E transcript was detected in 6 of 50 studied CCSK cases [11]. In addition, CCSKs have been shown to reveal a strong immunoreactivity for the EGF-receptor tyrosine kinase; also, EGFR gene amplification and an EGFR mutation have been reported in 2 cases [12]. Gene-expression profiling CCSK studies have reported up-regulation of genes known to act within the Sonic hedgehog signalling pathway and the phosphoinositide-3-kinase/Akt cell proliferation pathway [7]. Very recently, recurrent internal tandem duplications (ITDs) of the X-linked BCL6 co-repressor (BCOR) gene have been described in CCSK [39, 40]. This BCOR ITD and the t(10;17)(q22;p13) seem to be mutually exclusive [41, 43]. CCSK tumours harbouring BCOR ITDs exhibit high expression of BCOR mutant transcripts and protein [39, 41]. Consistent with possible disruption of the PRC1.1/BCOR complex in CCSKs, transcriptome profiling reveals widespread upregulation of PRC2 targets in these tumours, suggesting disruption of polycomb regulation [41]. This may explain the, by Gooskens et al identified hypermethylation and consequent down-regulation of the gene TCF21 in all CCSKs (n = 17) except for the samples harbouring the 10;17 translocation [42]. However, additional studies to analyse this connection are needed. Furthermore, the absence of both genetic alterations affecting BCOR and NUTM2B/E-YWHAE in 12 % of cases provides clear evidence for additional molecular subclasses present [43]. In contrast to WT, CCSK does not appear to be associated with genetic predisposition syndromes, and familial CCSK cases have not been reported so far.

Previous data on treatment and outcome of paediatric CCSK and background for the current protocol Results from the first three National Wilms’ Tumour Study Group (NWTS) protocols suggested that the addition of doxorubicin to the combination of vincristine and actinomycin-D improved the 6-year relapse-free survival (RFS) for patients with CCSK from 25% to 63% [22]. The addition of cyclophosphamide to the regimen of the NWTS-3 trial did not improve the 6-year EFS [22]. However, in the most recent NWTS-5 clinical trial, patients diagnosed with CCSK irrespective of stage were treated with a radical nephrectomy followed by treatment with vincristine, doxorubicin and cyclophosphamide,

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alternating with cyclophosphamide and etoposide for 24 weeks, and postoperative radiotherapy (10.8 Gy) [24]. Five-year EFS and 5-year OS for CCSK patients treated on NWTS-5 were 79% and 89%, respectively. Only one of the recurrences was in the tumour bed and two in the abdomen, indicating that local control achieved after radiological treatment with 10.8 Gy was sufficient. Stage was found to be highly predictive of outcome; 5-year EFS rates for stage I, II, III and IV on NWTS- 5 were 100%, 87%, 74% and 36%, respectively [24]. Current treatment according to the Children’s Oncology Group (COG) consists of surgery of resectable tumours followed by vincristine, cyclophosphamide, doxorubicin and etoposide for stage I-III disease. Stage IV patients are treated on an intensified regimen with additional carboplatin. Stage II-III patients receive local radiotherapy (10.8 Gy).

Report Study Treatment EFS OS Chemotherapy Radiotherapy Green 1994 [22] NWTS 1-2 AMD/VCR (8 pt) 0 - 37.8 Gy 25% (6y) 25% (6y) AMD/VCR/DOX (58 pt) 63.5% 71.9% (6y) (6y) Green 1994 [22] NWTS 3 AMD/VCR/DOX (43 pt) 0 - 37.8 Gy 64.4% 71.3% AMD/VCR/DOX/CPM (30 pt) (6y) (6y) 58.2% 60.8% (6y) (6y) Seibel 2004 [24] NWTS 4 6m CT (AMD/VCR/DOX) (23 pt) 10.8 Gy 65.2% 95.5% 15m CT (AMD/VCR/DOX) (17 pt) (5y) (5y) 87.8% 87.5% (5y) (5y) Seibel 2006 [38] NWTS 5 VCR/DOX/CPM/VP-16 (110 pt) Stage II/III 79% (5y) 89% (5y) 10.8 Gy

Current COG AREN0321 VCR/DOX/CPM/VP-16 Stage II/III - - protocol 10.8 Gy Tournade 2001 SIOP 09 AMD/VCR/DOX/IFO (10 pt) Stage II/III 30 75% (2y) 88% (5y) [35] Gy

Furtwängler 2005 SIOP 93-01 / Stage I: AMD/VCR/DOX (27 pt) Stage II/III and 86% 91% [23] GPOH Stage II-IV: DOX/IFO/CAR/VP-16 III 30 Gy (5.9y) (5.9y) (26 pt) Furtwängler 2013 SIOP 93-01 St I-IV: VP-16/CARBO/IFO/EPI Stage II/III 78% (5y) 86% (5y) [6] SIOP 2001 St I: AMD/VCR/DOX 25.2 Gy St II-IV: VP-16/CARBO/CPM/DOX Mitchell 2000 [36] UKWT2 AMD/VCR/DOX (16 pt) ≥ Stage III 30 82% (4y) 88% (4y) Gy Current UK SIOP 2001 St I-III: VCR/AMD/DOX Stage II/III - - protocol St IV: VP-16/CARBO/CPM/DOX 25.2 Gy Current AIEOP AIEOP-TW- St I-IV: ADM/VCR/DOX; ADM/IFO; Stage I-III 84% (5y) 91% (5y) protocol [33] 2003 VP-16/CARBO 19.8 Gy

Abbreviations: NWTS: National Wilms’ Tumor Study Group, JWiTS: Japanese Wilms Tumor Study Group, SIOP: International Society of Pediatric Oncology, GPOH: German Society of Pediatric Oncology and Hematology, UKWT: United Kingdom Wilms Tumour Study Group, AMD: actinomycin-D, VCR: vincristine, DOX: doxorubicin, CPM: cyclophosphamide, VP-16: etoposide, IFO: ifosfamide, CAR: carboplatin, pt: patients, RFS: relapse free survival, OS: overall survival, y: year

Table 18.1.1: CSSK treatment and outcome according to different treatment regimens

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Treatment in the United Kingdom Wilms’ Tumour Study Group 2 trial (UKWT2) consisted of three chemotherapeutic agents (vincristine, dactinomycin and doxorubicin) administered for 12 months after initial surgery [26]. Only stage III and IV patients received irradiation on the original side of the tumour (30 Gy). Results of the UK-WT-2 study demonstrated a 4-year EFS of 82% and a 4-year OS of 88%. This regimen revealed a high local relapse rate in stage II patients, which urged to include local irradiation in stage II patients in the current CCSK protocol, in order to avoid local recurrence [27]. SIOP trials include pre-operative treatment with vincristine and dactinomycin for 4 weeks for localized disease, and treatment with vincristine, actinomycin-D and an anthracycline (doxorubicin or epirubicine) for 6 weeks for stage IV patients [6]. Patients registered on the SIOP 93-01 protocol were treated postoperatively with etoposide, carboplatin, ifosfamide and doxorubicin (GPOH patients) or epirubicin (all other patients). Patients registered on the SIOP 2001 protocol were treated post-operatively with actinomycin-D, vincristine and doxorubicin for stage I patients and with ifosfamide, etoposide, carboplatin and doxorubicin for patients with stage II-IV disease. Both SIOP protocols included additional irradiation of the flank (25.2 Gy) for stage II and III patients. The 5-year EFS of all 191 CCSK patients treated according to SIOP 93-01 and SIOP 2001 was 78% and the 5-year OS was 86% [6]. Stage I patients treated according to the SIOP 93-01 protocol (treated with 4 drugs: etoposide, carboplatin, ifosfamide, doxorubicin) had better 5-year EFS and OS rates (82.6% and 90.1%, respectively) compared to stage I patients treated according to SIOP 2001 (treated with 3 drugs: actinomcyin, vincristine, doxorubicin) (5-year EFS 71.5%, 5-year OS 79.6%). In addition treatment of stage I CCSK patients according to current COG protocol (AREN0321): includes also 4 drugs (etoposide, vincristine, cyclophosphamide, doxorubicin). AREN0321 is the first protocol in COG in which stage I CCSK patients are treated without radiotherapy. Stage IV disease and young age were significant adverse prognostic factors for EFS. Patients with stage I disease had 5-year EFS and OS rates of 79% and 87% respectively, not as excellent as survival rates described in previous studies (Kalapurakal et al 5-year OS 100%, Argani et al 5-year OS 98%) [5, 6, 28]. Five-year EFS and OS rates of patients treated post-operatively with alkylating agents (i.e. ifosfamide, cyclophosphamide) (n = 146) were respectively 83% and 88% versus 67% and 78% for patients treated without alkylating agents (n = 28). Five-year EFS and OS rates of patients treated with ifosfamide (n = 80) were respectively 89% and 94%, and 5-year EFS and OS rates of patients treated with cyclophosphamide (n = 66) were respectively 74% and 78% [6]. The current SIOP-2016 CCSK guideline aims to include chemotherapy regimens that have been proven to be of value for CCSK patients, in order to maintain excellent survival in localized stage CCSK and further improve survival where possible. It also takes into account that survival is already reasonable for some groups of children, thus intensive therapy may be selectively modified to avoid serious short and long term toxicity. For those purposes, the challenge is to limit the total cumulative dose of doxorubicin to 250 mg/m2 to avoid cardiotoxicity [29, 30], to combine cyclophosphamide and ifosfamide in an alternating setting to avoid serious renal toxicity, and reduce the risk of fertility problems [31, 32], to include chemotherapeutic agents that guarantee CNS penetration, such as ifosfamide and carboplatin, and to limit the dose of abdominal radiotherapy to 10.8 Gy according to the current COG strategy [24]. For CCSK patients with stage I disease, treatment will be commenced according to SIOP 93-01 (but with alternating ifosfamide and cyclophosphamide), as 5-year EFS and OS rates of stage I CCSK patients treated according to SIOP 2001 (treated with 3 drugs) were inferior to SIOP 93-01 (treated with 4 drugs) [6].

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18.1.4 Treatment recommendations Treatment recommendations are given in table 18.1.2.

Stage Pre-operative Post-operative chemotherapy Radiotherapy chemotherapy

I AV VP16, CARBO, IFO/CYC, DOX No II AV VP16, CARBO, IFO/CYC, DOX 10.8 Gy III AV VP16, CARBO, IFO/CYC, DOX 10.8 Gy IV AVD VP16, CARBO, IFO/CYC, DOX 10.8 Gy + metastasis in case of incompleteness/impossibility of resection

Table 18.1.2: Recommendations for the treatment of CSSK Detailed descriptions of the chemotherapy treatment schedules are given here: VP-16 = etoposide = 150 mg/m2/d in 1 hour i.v. CARBO = carboplatin = 200 mg/m2/d in 1 hour i.v. CYCLO = cyclophosphamide = 450 mg/m2/d in 1 hour i.v. IFO = ifosfamide = 2 g/m2/d in 1 hour i.v. DOX = doxorubicin = 50 mg/m2/ in 4-6 hours i.v. , just before the first cyclophosphamide administration (5 courses, omit DOX in week 31)

VP16 150 mg/m2   CARBO 200 mg/m2   CYCLO 450 mg/m2   IFO 2 g/m2  DOX 50 mg/m2  *    RTo              Weeks  1-----2------3-----4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 

 = Echocardiography: at start of treatment, before week 19, 31 and at end of treatment * = no doxorubicin in week 31; maximum cumulative dose of doxorubicin should not exceed 250 mg/m2 (in case of stage IV: 300 mg/m2; replace DOX with VCR (1.5 mg/m2, max dose 2 mg/m2) after 300 mg/m2)  = GFR (measure at every third course, or more frequently if there is evidence of renal dysfunction RTo= Abdominal RT in local stage II and III disease

Dose reductions (see table in section 14.5.3)

Surgery: Recommendations are given in chapter 16.

Radiotherapy: Abdominal RT in local stage II and III. Dose: 10.8 Gy. Postpone DOX during radiotherapy (switch with IFO/CARBO). In case of stage IV irradiation of metastasis is needed. In case of pulmonary irradiation: Pneumocystic carinii pneumonia prophylaxis with cotrimoxazol is indicated. There is a stopping rule for the reduced radiation dosage implemented. For more details go to section 17.18.

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18.1.5 Follow-up guideline for surveillance Follow-up care is directed towards identification of early (and late) relapses as well as identifying treatment related toxicity. In a recent survey by the SIOP RTSG, it turned out that of 37 relapses nearly all relapses were metastatic (n = 35). Most common site of relapse was the brain (n = 13). Relapses also involved lungs (n = 7) and bone (n = 5). The median time to relapse was 17 months (range 5.5 months – 6.6 years). All pulmonary relapses occurred within 38 months after initial diagnosis, indicating that pulmonary screening after 4 years can be based on clinical symptoms. Younger patients (<12 months) all relapsed within 17 months after initial diagnosis (n = 12) The follow-up surveillance should be done according to section 11.6.5.

18.1.6 Recommendations treatment relapsed CCSK SIOP recently found that consolidation of a second complete remission seems to be a challenge in relapsed CCSK [34]. Intensive treatment, including chemotherapy as well as achieving local control by complete surgery (where possible) and/or radiotherapy seems to be of benefit to enhance survival. In addition, high-dose chemotherapy followed by autologous bone marrow transplantation seems to consolidate second complete remission. Possible high-dose chemotherapy drug combinations are:  CET: carboplatin, etoposide, thiotepa  CEM: carboplatin, etoposide, melphalan  Carboplatin and etoposide  Carboplatin and thiotepa

18.1.7 References 1. Ahmed HU, Arya M, Levitt G, Duffy PG, Mushtaq I, Sebire NJ. Part I: Primary malignant non-Wilms' renal tumours in children. Lancet Oncol, 2007. 8: p. 730-737 2. JM K. Exclusion of certain renal neoplasms from the category of Wilms tumours. Am J Pathol, 1970. Abstract 16A 3. Marsden HB LW. Bone-metastasizing renal tumour of childhood. Br J Cancer, 1978. 38: p. 437-441 4. Beckwith JB, Palmer NF. Histopathology and prognosis of Wilms tumors: results from the First National Wilms' Tumor Study. Cancer 1978. 41: p. 1937-1948 5. Argani P, Perlman EJ, Breslow NE, et al. Clear cell sarcoma of the kidney: a review of 351 cases from the National Wilms Tumor Study Group Pathology Center. Am J Surg Pathol, 2000. 24: p. 4-18 6. Furtwangler R GS, van Tinteren H, de Kraker J, Schleiermacher G, Bergeron C, de Camargo B, Acha T, Godzinski J, Sandstedt B, Leuschner I, Vujanic GM, Pieters R, Graf N, van den Heuvel-Eibrink MM. Clear Cell Sarcomas of the Kidney (CCSK) registered on SIOP 93-01 and SIOP 2001 protocols: A report of the SIOP Renal Tumour Study Group. Eur J Cancer, 2013. 49(16): p. 3497-506 7. Cutcliffe C, Kersey D, Huang CC, et al. Clear cell sarcoma of the kidney: up-regulation of neural markers with activation of the sonic hedgehog and Akt pathways. Clin Cancer Res, 2005. 11: p. 7986- 7994

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8. Punnett HH, Halligan GE, Zaeri N, Karmazin N. Translocation 10;17 in clear cell sarcoma of the kidney. A first report. Cancer Genet Cytogenet, 1989. 41: p. 123-128 9. Brownlee NA, Perkins LA, Stewart W, et al. Recurring translocation (10;17) and deletion (14q) in clear cell sarcoma of the kidney. Arch Pathol Lab Med, 2007. 131: p. 446-451 10. Rakheja D, Weinberg AG, Tomlinson GE, Partridge K, Schneider NR. Translocation (10;17)(q22;p13): a recurring translocation in clear cell sarcoma of kidney. Cancer Genet Cytogenet, 2004. 154: p. 175- 179 11. O'Meara E, Stack D, Lee CH, et al. Characterization of the chromosomal translocation t(10;17)(q22;p13) in clear cell sarcoma of kidney. J Pathol, 2012. 227: p. 72-80 12. Little SE, Bax DA, Rodriguez-Pinilla M, et al. Multifaceted dysregulation of the epidermal growth factor receptor pathway in clear cell sarcoma of the kidney. Clin Cancer Res, 2007. 13: p. 4360-4364 13. Gooskens SL, Furtwangler R, Vujanic GM, Dome JS, Graf N, van den Heuvel-Eibrink MM. Clear cell sarcoma of the kidney: a review. Eur J Cancer. 2012, 48: p. 2219-2226 14. Scully RE, Lipshultz SE. Anthracycline cardiotoxicity in long-term survivors of childhood cancer. Cardiovasc Toxicol, 2007. 7: p. 122-128 15. van Waas M, Neggers SJ, Raat H, van Rij CM, Pieters R, van den Heuvel-Eibrink MM. Abdominal radiotherapy: a major determinant of metabolic syndrome in nephroblastoma and neuroblastoma survivors. PLoS One, 2012. 7: e52237 16. Meadows AT, Friedman DL, Neglia JP, et al. Second neoplasms in survivors of childhood cancer: findings from the Childhood Cancer Survivor Study cohort. J Clin Oncol, 2009. 27: p. 2356-2362 17. van den Heuvel-Eibrink MM, Grundy P, Graf N, et al. Characteristics and survival of 750 children diagnosed with a renal tumor in the first seven months of life: A collaborative study by the SIOP/GPOH/SFOP, NWTSG, and UKCCSG Wilms tumor study groups. Pediatr Blood Cancer, 2008. 50: p. 1130-1134 18. Hung NA. Congenital "clear cell sarcoma of the kidney. Virchows Arch, 2005. 446: p. 566-568. 19. Amin MB, de Peralta-Venturina MN, Ro JY, et al. Clear cell sarcoma of kidney in an adolescent and in young adults: a report of four cases with ultrastructural, immunohistochemical, and DNA flow cytometric analysis. Am J Surg Pathol, 1999. 23: p. 1455-1463 20. Sotelo-Avila C, Gonzalez-Crussi F, Sadowinski S, Gooch WM, 3rd, Pena R. Clear cell sarcoma of the kidney: a clinicopathologic study of 21 patients with long-term follow-up evaluation. Hum Pathol, 1985. 16: p. 1219-1230 21. Sandstedt BE, Delemarre JF, Harms D, Tournade MF. Sarcomatous Wilms' tumour with clear cells and hyalinization. A study of 38 tumours in children from the SIOP nephroblastoma file. Histopathology. 1987;11: 273-285 22. Green DM, Breslow NE, Beckwith JB, Moksness J, Finklestein JZ, D'Angio GJ. Treatment of children with clear-cell sarcoma of the kidney: a report from the National Wilms' Tumor Study Group. J Clin Oncol, 1994. 12: p. 2132-2137 23. Furtwangler R RH, Beier R. Clear-cell sarcoma (CCSK) of the kidney - results of the SIOP 93-01/GPOH trial. Pediatr Blood Cancer, 2005. 50: p. 1

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24. Seibel N SJ, Li S, Breslow JB, et al. Effect of duration of treatment on treatment outcome for patients with clear-cell sarcoma of the kidney: a report from the National Wilms' Tumor Study Group. J Clin Oncol, 2004. 22(3): p. 468-73 25. Balarezo FS, Joshi VV. Clear cell sarcoma of the pediatric kidney: detailed description and analysis of variant histologic patterns of a tumor with many faces. Adv Anat Pathol, 2001. 8: p. 98-108 26. Mitchell C, Pritchard-Jones K, Shannon R, et al. Immediate nephrectomy versus preoperative chemotherapy in the management of non-metastatic Wilms' tumour: results of a randomised trial (UKW3) by the UK Children's Cancer Study Group. Eur J Cancer, 2006. 42: p. 2554-2562 27. Stoneham S KM, Moroz V, et al. Clear Cell Sarcoma of the Kidney (CCSK) - Combined 20 year experience of therapeutic outcomes from United Kingdom (UK) and France. Pediatr Blood Cancer, 2009. 0.145, p. 753; SIOP XXXXI Congress Meeting Abstracts; DOI: 10.1002/pbc.22234 28. Kalapurakal JA, Perlman EJ, Seibel NL, Ritchey M, Dome JS, Grundy PE. Outcomes of patients with revised stage I clear cell sarcoma of kidney treated in National Wilms Tumor Studies 1-5. Int J Radiat Oncol Biol Phys, 2013. 85: p. 428-431 29. Kremer LC, Caron HN. Anthracycline cardiotoxicity in children. N Engl J Med, 2004. 351: p. 120-121 30. van Dalen EC, Michiels EM, Caron HN, Kremer LC. Different anthracycline derivates for reducing cardiotoxicity in cancer patients. Cochrane Database Syst Rev, 2006. 2(5):CD005006. doi: 10.1002/14651858.CD005006.pub4 31. Green DM, Kawashima T, Stovall M, et al. Fertility of female survivors of childhood cancer: a report from the childhood cancer survivor study. J Clin Oncol, 2009. 27: p. 2677-2685 32. Green DM, Kawashima T, Stovall M, et al. Fertility of male survivors of childhood cancer: a report from the Childhood Cancer Survivor Study. J Clin Oncol, 2010. 28: p. 332-339 33. Spreafico F, Gandola L, Melchionda F. Stage I clear cell sarcoma of the kidney: is it the time for a less intensive adjuvant treatment? Translational Pediatrics, 2014. 3: p.1-3 34. Gooskens SL, Furtwangler R, Spreafico F, et al. Treatment and outcome of patients with relapsed Clear Cell Sarcoma of the Kidney (CCSK): a combined SIOP and AIEOP study. Br J Cancer, 2014. 111: p. 227-33 35. Tournade MF, Com-Nougué C, de Kraker J, et al. Optimal duration of preoperative therapy in unilateral and nonmetastatic Wilms' tumor in children older than 6 months: results of the Ninth International Society of Pediatric Oncology Wilms' Tumor Trial and Study. J Clin Oncol, 2001. 19(2): p. 488-500 36. Mitchell C, Jones PM, Kelsey A, et al. The treatment of Wilms' tumour: results of the United Kingdom Children's cancer study group (UKCCSG) second Wilms' tumour study. Br J Cancer, 2000. 83(5): p. 602-8 37. Jet AwS, Hong KUIK C, Hwee Yong M et al. Novel Karyotypes and Cyclin D1 Immunoreactivity in Clear cell sarcoma of the kidney. Pediatr Dev Pathol, 2015. 18(4): p. 297-304 38. Seibel NL, Sun J, Anderson JR, et al. Outcome of clear cell sarcoma of the kidney (CCSK) treated on the National Wilms Tumor Study-5 (NWTS). J Clin Oncol, 2006. 24(18S): p. 9000 39. Ueno-Yokohata H, Okita H, Nakasato K, et al.: Consistent in-frame internal tandem duplications of BCOR characterize clear cell sarcoma of the kidney. Nat Genet, 2015. 47(8): p. 861-3

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40. Astolfi A, Melchionda F, Perotti D, et al.: Whole transcriptome sequencing identifies BCOR internal tandem duplication as a common feature of clear cell sarcoma of the kidney. Oncotarget, 2015. 6(38): p. 40934-9 41. Karlsson J, Valind A and Gisselsson D. BCOR internal tandem duplication and YWHAE-NUTM2B/E fusion are mutually exclusive events in clear cell sarcoma of the kidney. Genes Chromosomes Cancer, 2015. doi: 10.1002/gcc.22316. [Epub ahead of print] 42. Gooskens SL, Gadd S, Guidry Auvil JM, et al: TCF21 hypermethylation in genetically quiescent clear cell sarcoma of the kidney. Oncotarget, 2015. 6(18):15828-41 43. Kenny C, Bausenwein S, Lazaro A, et al. Mutually exclusive BCOR internal tandem duplications and YWHAE-NUTM2 fusions in clear cellsarcoma of kidney: not the full story. J Pathol, 2016. 238(5): p. 617-20

18.1.8 Chairs and members of the CCSK Panel

Name Profession Country Email

Chair Marry van den Oncologist The m.m.vandenheuvel- Heuvel-Eibrink Netherlands [email protected]

Co-Chair Rhoikos Furtwängler Oncologist Germany [email protected]

Tomás Acha Oncologist Spain [email protected]

Christophe Bergeron Oncologist France [email protected]

Beatriz de Camargo Oncologist Brazil [email protected]

Oncologist The Saskia Gooskens [email protected] Netherlands

Norbert Graf Oncologist Germany [email protected]

Kathy Pritchard-Jones Oncologist UK [email protected] Members Sara Stoneham Oncologist UK [email protected]

The Harm van Tinteren Statistician [email protected] Netherlands

Fraia Melchionda Oncologist Italy [email protected]

Hélène Sudour- Oncologist France [email protected] Bonnange

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Estelle Thébaud Oncologist France [email protected]

Gordan Vujanic Pathologist UK [email protected]

Molecular Manfred Gessler Germany [email protected] Geneticist

Ivo Leuschner Pathologist Germany [email protected]

NN Surgeon

NN Radiologist

NN Radiotherapist

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18.2 Renal Cell Carcinoma (RCC) 18.2.1 Introduction Renal cell carcinomas (RCC) in children are rare, accounting for only 2 to 6 % of paediatric malignant renal tumours [1-3]. Compared with the adult counterpart, the majority of paediatric RCCs show a different histologic subtype, special morphological features and unique genetic abnormalities [4]. Paediatric RCC are predominantly of the translocation type and of the papillary subtype, whereas the clear cell RCC with von Hippel-Lindau (VHL) gene abnormalities at 3p25-26 dominates in adults [5-7]. Morphologically, translocation type RCC (20-70% of the paediatric RCC) typically show a distinctive voluminous pale or clear cytoplasm with focal papillary/pseudo-papillary architecture and with psammona bodies [5]. Nevertheless, they also show a considerable morphological overlap with clear cell RCC and papillary RCC [1]. Translocation type RCCs are characterized by specific chromosome translocations involving the transcription factor gene TFE3 located on Xp11.2 or – rarely - TFEB located on 6p21 [8-10]. Both TFE3 and TFEB belong to the microphthalmia transcription factor (MiTF)/TFE family. The various chromosome translocations in paediatric RCC result in gene fusions, most frequently between the TFE3 gene and the PRCC (papillary renal cell carcinoma) gene located on 1q21.2 or the ASPL (alveolar soft part sarcoma locus) gene located on 17q25, respectively [10]. Translocations involving the TFE3 and TFEB genes induce an aberrant overexpression of these proteins and can be specifically identified by immunohistochemistry or FISH [10]. Microarray analyses revealed a very different gene expression profile of translocation type RCC compared to other RCC types, reflecting a distinct biological behaviour [11, 12]. In adult clear cell RCCs, the inactivation of the VHL gene results in an up-regulation of the VEGF-and PDGF signalling pathway successfully targeting with VEGF – and PDGF- receptor tyrosine kinases (RTK) inhibitors [13]. In contrast, in translocation RCC the MET-RTK has been shown to be the most up-regulated RTK [12]. Corresponding to the distinct morphological and genetic characteristics, children and adolescents suffering from RCC show particular clinical features and a different course of disease in comparison to adults. Paediatric RCCs occur with equal frequency in girls and boys with a mean age of over 10 years [2]. A significant proportion of paediatric RCC patients suffer from a special underlying disorder such as tuberous sclerosis, chronic renal insufficiency, urogenital malformations or have been previously treated with chemotherapy [1, 2, 14]. Furthermore, a special subgroup of paediatric RCC exists as a second malignancy after neuroblastoma [15]. The most significant difference to adult RCC, is the better outcome of paediatric RCC with regional lymph node metastases without distant metastases with survival rates of over 70% without adjuvant therapy based on retrospective studies (in adults below 30%) [7, 16]. The majority of paediatric RCC are localized diseases, which can be cured with surgical tumour resection alone (survival rate over 90%) [2]. The relatively small subgroup of children suffering from a distant metastatic RCC has a poor prognosis, as in adults [2, 17, 18]. Nephron-sparing surgery is established in adult RCC smaller than 4 cm [19]. In small cohorts of paediatric RCC patients no relapses were observed after nephron sparing surgery [20, 21]. Because of an increased risk of metachronic disease in the other kidney in paediatric RCC especially after underlying disorders, the nephron sparing surgery would be desirable if possible. Currently there are no published prospective study data of an unselected paediatric RCC cohort and their treatment. Therefore, we do not know which adjuvant therapy could improve the outcome of distant metastatic paediatric RCC patients and – possibly – of a subgroup of particular advanced regional lymph node metastatic RCC.

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Former small studies and single case reports showed objective responses in paediatric metastatic RCC patients after INFa/IL2 therapy at a comparable rate as in adult RCC [22-24]. Recently a retrospective analysis of various targeted therapies in translocation RCCs showed a significant objective response rate with the RTK inhibitor Sunitinib (PR in 3 of 3 patients) in comparison with Sorafenib (only SD as best response) and Temsirolimus/Everolimus (1 PR, 6 SD) [23]. In a preclinical study, translocation-type RCC cells with ASPL-TFE3-gene fusion responded to MET receptor tyrosine kinase inhibitor administration with decreased cell growth [12]. The objective of a SIOP paediatric RCC study is to obtain new knowledge about RCC in children and adolescents including their biological behaviour and clinical course in a large unselected prospective cohort. This will help to improve treatment in the different subtypes of RCC. The most significant challenges of treatment are the development of an effective adjuvant therapy in advanced paediatric RCC patients (distant metastatic RCC, possibly advanced local lymph node metastatic RCC) and nephron- sparing surgery in selected RCC patients.

18.2.2 Aims and endpoints Aims Primary Aim:  To provide the best available care including a diagnostic and treatment guideline for RCC in children Secondary Aims:  To get insight in the prevalence, clinical characteristics, and outcome of paediatric RCC (including all subgroups) based on a prospective registration of a full cohort of paediatric RCCs registered in SIOP-RTSG  To study specific biological and genetic characteristics in paediatric RCC  To develop a risk scoring system (INDEX) for children with RCC Endpoints Primary endpoint:  Outcome of paediatric RCC patients Secondary endpoint:  The prevalence of paediatric RCC based on a European cohort registry of renal tumours (SIOP- RTSG)  The molecular characteristics of RCC

18.2.3 Background information Epidemiology The SEER program reports an age-adjusted incidence of RCC of 0.5 cases per million people under the age of 19 years [25]. A German population-based study of childhood RCC (including children < 16 yrs) showed a median age at diagnosis of 10.6 years, with a male: female ratio of 1 to 1.1 [2].

Clinical Features

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The most common symptoms at diagnosis are pain (30-40%), gross hematuria (30-40%), and abdominal mass (20-25%). Non-specific constitutional symptoms such as fever, weight loss, and lethargy are seen in 15-40% of children. [17, 26] Imaging Imaging is done according to the guidelines given in chapter 11.3 and Appendix 4.

Pathology The histology of paediatric RCC is distinct from that of adult RCC. In literature before 1990, many cases of paediatric RCC were described as having clear cells with a papillary pattern [27, 28]. Renshaw described tumours with distinctive voluminous clear cytoplasm, which he proposed were a newly recognized type of RCC involving translocations of Xp11 [29]. In 2004, the World Health Organization officially recognized translocation RCC, which is associated with a family of translocations involving the TFE3 or TFEB genes, as a distinct class of RCC [30]. It is now estimated that translocation RCC accounts for 20-50% of paediatric and young adult RCC [6, 31-33]. Other histologic types of RCC described in children include papillary RCC (about 30%), chromophobe RCC, sarcomatoid RCC, collecting duct carcinoma, RCC arising from Wilms tumour, renal medullary carcinoma, RCC after neuroblastoma, and RCC not otherwise specified [6, 31, 33, 34]. The clear cell (conventional) subtype, by far the most common type of RCC in adults, is uncommonly observed in children. A careful morphological and molecular analysis by Bruder included 6 paediatric patients with the histologic appearance of clear cell RCC [33]. However, none of these cases had LOH at chromosome 3p, the site of the VHL gene, or mutations of VHL, indicating that the clear cell RCC in children is distinct from adult clear cell RCC.

Paediatric RCC [32]: Translocation Translocation RCC involving RCC involving t(X;17) t(X;1)

Translocation Papillary RCC RCC involving t(6;11)

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Renal Oncocytic RCC medullary following carcinoma neuroblastoma

Paediatric RCC can be classified according to The WHO 2016 classification system of RCC which –beside the adult type RCC- also considers RCC types with predilection for young age groups, such as translocation-RCC and post Neuroblastoma-RCC [33, 35, 36]. Papillary RCC: paediatric papillary RCC has the classic papillary architecture as described in the WHO classification, comprising 20-50% of all RCC. Clear cell RCC also can be classified according the WHO classification.

Table 1. Staging RCC

Primary Tumour (T)a aReprinted with permission from AJCC: Kidney. In: Edge SB, Byrd DR, Compton CC, et al., eds.: AJCC Cancer Staging Manual. 7th ed. New York, NY: Springer, 2010, pp 479-89.

TX Primary tumour cannot be assessed.

T0 No evidence of primary tumour.

T1 Tumour ≤7 cm in greatest dimension, limited to the kidney.

T1a Tumour ≤4 cm in greatest dimension, limited to the kidney.

T1b Tumour >4 cm but not >7 cm in greatest dimension, limited to the kidney.

T2 Tumour >7 cm in greatest dimension, limited to the kidney.

T2a Tumour >7 cm but ≤10 cm in greatest dimension, limited to the kidney.

T2b Tumour >10 cm, limited to the kidney.

T3 Tumour extends into major veins or perinephric tissues but not into the ipsilateral adrenal gland and not beyond Gerota fascia.

T3a Tumour grossly extends into the renal vein or its segmental (muscle containing) branches, or tumour invades perirenal and/or renal sinus fat but not beyond Gerota fascia.

T3b Tumour grossly extends into the vena cava below the diaphragm.

T3c Tumour grossly extends into the vena cava above the diaphragm or invades the wall of the vena cava.

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T4 Tumour invades beyond Gerota fascia (including contiguous extension into the ipsilateral adrenal gland).

Regional Lymph Nodes (N)a aReprinted with permission from AJCC: Kidney. In: Edge SB, Byrd DR, Compton CC, et al., eds.: AJCC Cancer Staging Manual. 7th ed. New York, NY: Springer, 2010, pp 479-89.

NX Regional lymph nodes cannot be assessed.

N0 No regional lymph node metastasis.

N1 Metastases in regional lymph node(s).

Distant Metastasis (M)a aReprinted with permission from AJCC: Kidney. In: Edge SB, Byrd DR, Compton CC, et al., eds.: AJCC Cancer Staging Manual. 7th ed. New York, NY: Springer, 2010, pp 479-89.

M0 No distant metastasis.

M1 Distant metastasis.

Anatomic Stage/Prognostic Groupsa aReprinted with permission from AJCC: Kidney. In: Edge SB, Byrd DR, Compton CC, et al., eds.: AJCC Cancer Staging Manual. 7th ed. New York, NY: Springer, 2010, pp 479-89.

Stage T N M

I T1 N0 M0

II T2 N0 M0

III T1 or T2 N1 M0

T3 N0 or N1 M0

IV T4 Any N M0

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Any T Any N M1

18.2.4 Genetics and Biology Germline mutations/syndromes Several genetic syndromes are associated with predisposition to RCC [37]. The best described is von Hippel Lindau (VHL) Syndrome, caused by mutations in the VHL gene at chromosome 3p25. VHL encodes a protein that regulates the level of the hypoxia inducible factor (HIF) family of transcription factors. VHL is a component of a protein complex that promotes the ubiquitin-mediated degradation of HIF, which binds to promoters of genes involved in angiogenesis, erythropoiesis, energy metabolism, iron metabolism, cell proliferation, apoptosis, and other processes that are dysregulated in human cancer [37]. Tuberous sclerosis is caused by mutations in the TSC1 and TSC2 genes, which encode the hamartin and tuberin proteins, central regulators of the mammalian target of rapamycin (mTOR) pathway. The most common renal tumour in individuals with tuberous sclerosis is angiomyolipoma, but affected individuals are also susceptible to RCC with clear cell morphology [38]. Hereditary papillary RCC is caused by mutations in the MET oncogene, which encodes the hepatocyte growth factor receptor, which signals through the phosphatidylinositol 3-kinase (PI3K) pathway [39]. The Birt-Hogg-Dubé Syndrome is caused by mutations in FLCN, which encodes the protein folliculin, which interacts with the mTOR pathway [40]. Individuals with germline mutations of two tricarboxylic acid (Krebs) cycle genes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), are also susceptible to RCC. FH is the gene responsible for hereditary leiomyomatosis and papillary RCC, a cancer predisposition syndrome in which patients develop uterine and cutaneous leiomyomas as well as papillary RCC subtype 2 [41]. Germline mutations of the SDHB, SDHC, and SDHD genes are responsible for familial paraganglioma and pheochromocytoma. Individuals with SDHB mutations are also at risk for early onset RCC [42, 43]. Mutations of the FH and SDH genes impair progression through the tricarboxylic acid cycle, thereby diminishing oxidative phosphorylation and leading cells to rely on glycolysis for energy metabolism even in normoxic conditions [37].

Tumour-specific genetics Translocation RCC is associated with translocations involving genes that encode members of the microophthalmia (MiTF) family of transcription factors. The most commonly involved gene is TFE3 on chromosome Xp11, which can fuse to several partners including ASPL (17q25), PRCC (1q21), PSF (1p34), NonO (Xq12), and CLTC (17q23) [44]. The TFE3-ASPL translocation is the same translocation seen in alveolar soft part sarcoma [45]. A recent gene expression study has identified several novel genes that are differentially expressed between the Xp11 translocation carcinomas and conventional renal carcinomas. This has shown that Xp11 translocation carcinomas may be more similar to alveolar soft part sarcoma than to conventional renal carcinomas [12]. Additionally, gene expression profiling has identified potential therapeutic targets in the Xp11 translocation RCC. For example, the ASPL-TFE3 fusion protein transactivates the promoter of the MET receptor tyrosine kinase, leading to MET protein overexpression. Inhibition of the MET receptor tyrosine kinase may therefore be a potential avenue of targeted therapy for these RCC [12]. Translocation RCC also express high levels of phosphorylated S6, a measure of mTOR pathway activation, which suggests that mTOR inhibition may be effective in this

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tumour type [11]. A less common type of translocation RCC involves a fusion of the untranslated alpha gene (11q12) to the TFEB gene (6p21) [9, 46]. Interestingly, 15% of translocation RCC occur in individuals who were previously treated with chemotherapy for a variety of pediatric malignancies and non- malignant conditions [14].

Gene (chromosome) Major clinical manifestations von Hippel-Lindau VHL (3p25-26) Clear-cell renal cell carcinoma; CNS haemangioblastomas; pheochromocytoma; retinal angiomas; pancreatic endocrine tumours; paragangliomas; cystadenoma of broad ligamnt or epididymis Hereditary papillary C-Met proto-oncogene (7q31- Type 1 papillary renal cell carcinoma renal carcinoma 34) Hereditary Fumarata hydratase (1q42-43) Type 2 papillary renal cell carcinoma; leiomyomatosis renal leiomyomas of skin or uterus; uterine cell carcinoma leiomyosarcomas Birt-Hogg-Dubé BHD1 (17p11) Chromophobe renal cell carcinoma; oncocytoma (benign); transitional tumours; cutaneous fibrofolliculomas; lung cysts or pneumothorax Tuberous sclerosis TSC1 (9q34) or TSC2 (16p13) Multiple renal angiomyolipomas; renal cell carcinoma; renal cysts/ploycystic kidney disease; cardiac rhabdomyomas; neurological disorders or seizures; multiple skin findings, including angiofibromas, fibromas, and nevi

Possibly related underlying conditions in children (Selle 2006 Cancer 107; 2906-14): preexisting renal cysts, urogenital malformation (horse shoe kidney, cryptorchism), renal failure/post renal transplant (RCC in non removed own kidney), genetic syndromes/malformation (tuberous sclerosis, Saetre-Chotsen syndrome, XYY syndrome, supernummerous nipple), previous neuroblastoma, coccygeal immature teratoma, angiomatous formation, family history of urogenital tumour and kidney malformations [2].

18.2.5 Inclusion criteria prospective RCC registry and guideline  Patients from birth up to 18 years of age  Registered in SIOP RTSG  Histological diagnosis will be confirmed by SIOP RTSG pathology panel, including an expert on adult RCC

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18.2.6 Treatment Background information Tumour resection is the mainstay of therapy for pediatric RCC. It is important to underline that most children and adolescents with a renal mass are presumed to have Wilms tumour and usually undergo radical nephrectomy and lymph node sampling/resection (after neo-adjuvant chemotherapy) according to Wilms tumour surgical guidelines. Hence, despite we are aware about the increasing indication of conservative surgery, like nephron sparing surgery, it is likely that in most cases the nephrectomy has been already done when the diagnosis of RCC has been made. In cases which are highly suggestive for RCC, it should be discussed pre-operatively if to prefer NSS – if technically applicable- or complete nephrectomy. The role of radical lymph node dissection remains to be determined [47]. There is no evidence for efficacy of radical lymph node dissection in childhood RCC. Lymph node sampling is recommended. Many patients with localized disease have fared well without adjuvant therapy. Among adults and children with translocation RCC, age ≥ 25 years, lymph node involvement, high Fuhrman grade, and presence of distant metastatic disease were associated with poor survival [16]. In pure pediatric series, however, local lymph node involvement was not associated with unfavorable outcome, even among patients who did not receive adjuvant therapy [31]. The Children’s Oncology Group AREN0321 study is prospectively registering cases of RCC in children and adolescents, and collecting data concerning which adjuvant treatment was applied in metastatic cases. Children with metastatic RCC have a poor outcome. Although successes with high-dose interleukin-2 have been reported [48], it is recognized in that non-clear cell renal cell carcinomas do not typically respond well to immunotherapy [23, 24]. Emerging data on translocation RCC suggest that some tumours respond to vascular endothelial growth factor receptor (VEGF)-targeted therapy (sunitinib, sorafenib, ramucirumab) [23, 49]. Among the agents reported, sunitinib seems to be most active. In one series, 7 of 14 patients (50%) treated with sunitinib as either first or second-line therapy for translocation RCC had partial or complete response [23]. Seven of 7 patients who had progressive disease on VEGF-directed therapy and switched to mTOR inhibitors showed at least transient disease stabilization, including one with a partial response. Responses to gemcitabine/doxorubicin alternating with gemcitabine/oxaliplatin have also been observed [31]. Prospective studies to evaluate therapies for metastatic and recurrent childhood RCC are warranted. Renal medullary carcinoma (RMC) is a renal epithelial neoplasm that has been described as the “7th sickle cell nephropathy” [50]. It is an aggressive cancer that occurs in adolescent and young adult patients with sickle cell trait or hemoglobin SC disease [50]. The mean age of presentation is 19 years, with a reported range from 5 to 40 years. There is a male predominance, with a male to female ratio of 2 to 1[51]. There is no single pathognomonic genetic abnormality seen in RMC, but BCR-ABL translocations or ABL gene amplification have been described in rare cases, as have ALK gene rearrangements [51-53]. Absence of SMARCB1 (INI1/hSNF5) protein staining by immunohistochemistry has been observed in RMC, suggesting that rhabdoid tumour of the kidney and RMC may have common biological, as well as clinical, features [54]. Both are characterized by an aggressive metastatic pattern and relative chemotherapy resistance. Patients with RMC almost always present with metastatic disease and have fatal outcomes [50, 51]. Transient responses have been observed after treatment with methotrexate / vinblastine / doxorubicin

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/ cisplatin (MVAC) or platinum / gemcitabine / taxane [51, 55-58]. A patient with RMC was shown to have a complete tumour response after treatment with the proteosome inhibitor bortezomib [59]. Until now, the optimal treatment of the different subtypes of pediatric RCCs is widely unclear because of the rarity of RCC in children and the complete lack of results from prospective studies with sufficient patient numbers [32]. On the other hand there is a huge number of published studies concerning adult RCCs and adequate treatment strategies for them. But because of the significant biological differences and differences in prevalence of the subtypes, between adult and pediatric RCCs, namely the great majority of adult RCC displays the clear cell histology and not the translocation one, applicability to pediatric RCC management is restricted.

18.2.7 Treatment recommendations In general, complete surgery is the most adequate approach in all children with RCC. In patients with metastasis an adjuvant medical treatment is necessary.

Surgical recommendations: For surgical recommendations, see chapter 16.

Localized RCC (Stage T1-4, N0M0): Complete Surgical Tumour Resection (R0) is the mainstay of cure in pediatric RCC [2, 60]. The standard recommended surgical procedure is radical nephrectomy (RN = removal of the tumour-bearing kidney). We recommend to be cautious to consider partial nephrectomy in children. The EORTC led a prospective randomised controlled trial (RCT), comparing radical nephrectomy with partial nephrectomy in solitary T1-2 N0M0 renal tumour < 5cm with normal contralateral kidney function and WHO PS 0-2. At 9.3 years survival follow-up, 198 patients (72.5%) were alive after radical nephrectomy and 173 (64.4%) after NSS. Local recurrence occurred in one patient in the nephrectomy group and in six in the NSS group [61]. Though prospective data are absent, partial nephrectomy is now considered in cases of small tumours (i.e. < 4cm-≤7cm) diameter, possibly all T1-T2 RCC) and –if technically feasible- in all cases with reduced renal function or/and renal malformation and in all cases with estimated high risk of metachronic secondary RCC or secondary renal function deterioration such as RC as part of a syndrome (Tuberous Sclerosis, VHL, others), RCC after chemotherapy or as second, RCC or renal failure in the family [1]. Indications to partial nephrectomy (PN= nephron-sparing surgery[NSS]) are increasing for adult patients: it is now considered in cases of small tumours (i.e. < 4cm [-< 7cm?] diameter, possibly all T1-T2 RCC) and – if technically feasible - in all cases with reduced renal function or/and renal malformation and in all cases with estimated higher risk of metachronic secondary RCC or secondary renal function deterioration such as especially RCC as part of a syndrome (Tuberous Sclerosis, VHL, others), RCC after chemotherapy or as second malignancy, RCC or renal failure in the family [1]. We need to be reminded that in children, still the differentiation between RCC and Wilms tumour is not possible by imaging studies, even in the adolescence group, and Wilms tumour remains the most frequent renal tumour type. However, we believe that any case of small renal neoplasm in children > 10 years should be regarded as a potential RCC, and consequently be discussed with the reference surgeons, with the aim

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of considering NSS procedures as well, though only in selected cases. In addition it is difficult to extrapolate the tumour dimension criteria from adulthood experience, since the kidney dimensions in children are very different, and partial nephrectomy is advised in such cases only after extensive discussion with the SIOP surgical panel [1]. The standard indications for adult NSS in RCC according to the European Association of Urology guidelines are divided into the following categories: (1) absolute (anatomic or functional solitary kidney), (2) relative (functioning opposite kidney that is affected by a condition that might impair renal function in the future), and (3) elective (localized unilateral RCC with a healthy contralateral kidney). Relative indications also include patients with hereditary forms of RCC who are at high risk of developing a tumour in the contralateral kidney in the future [62, 63]. During the last decade, NSS has become the gold standard for the treatment of T1a tumours (<4 cm) in adult patients with a normal contralateral kidney” and – when performed in carefully selected patients in specialized centers - PN can be safely applied in patients with larger renal tumours [61]. In adult patients several studies showed equivalence of PN and RN in oncological outcome in localized RCC at least in T1RCC [61, 64]. Data for PN in pediatric RCC are very limited, however revealed no difference between PN and RN concerning the oncological outcome [2, 20]. Therefore, before performing NSS in a child, we recommend this to be discussed with the surgical board of SIOP-RTSG in all cases, similar to what is recommended in all other pediatric renal tumours.

General surgical principles in RCC:  The completeness of surgical resection is of prognostic importance [65].  Early stage disease has a better prognosis than later stage disease stages [66].  Partial nephrectomy might have a role in low-volume localised cancer in carefully selected patients [66].  Experts opinions are strongly recommended in case of partial nephrectomy and regional lymphadenectomy as these still need to be determined.

Regional Lymph Node Positive RCC (stage T1-4, N1-2, M0) First it is advised to perform a complete renal tumour (if necessary RE-) resection with negative surgical margins as described in section1). In addition, in case the diagnosis of RCC is known at time of surgery, total regional lymphadenectomy is recommended at the time of renal tumour resection whenever feasible without significant surgical morbidity, as the fact that the surgical tumour resection seems to be the crucial mainstay of cure among the treatment modalities in pediatric RCC [2]. Apart from a more correct tumour staging, that procedure may improve the outcome. In an Italian study of 16 pediatric RCC patients with local lymph node involvement, the survival rate in patients with extended retroperitoneal lymph node dissection (RLND= on the left; excision of hilar, periaortic, and ipsilateral common iliac nodes; on the right, hilar, interaortocaval, retro-paracaval, and common iliac nodes) was markedly better than in cases with a more limited lymph node resection (8 of 9 with RLND alive and disease free vs. 1 of 7 without RLND [17]. Most children with RCC will be diagnosed after initial WT treatment preoperatively. Therefore, the role of regional lymph node re-dissection in cases that were suspected of WT and had lymph node sampling

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only, is still under debate in pediatric RCC’s, especially as lymph node positive pediatric RCCs seem to have a relatively more favorable outcome than adult RCC patients [18]. There is no clear evidence (from pediatric nor adult literature) that, in completely resected, more advanced regional lymph node involvement (i.e. >6 LN RCC+?) an adjuvant therapy is of any value.

Distant Metastatic RCC (stage T1-4, N0-2, M1) Surgical treatment: complete renal tumour resection with negative surgical margins as described above is strongly advised, and retroperitoneal lymph node sampling (not dissection) is recommended in case of enlarged lymph nodes. Adult data have shown that an initial reduction of tumour burden in RCC through nephrectomy, i.e. cytoreductive nephrectomy may improve the treatment results after adjuvant therapy with interferon- alpha [67]. In cases of initial unresectable renal tumours a neo-adjuvant use of drug treatment (see below) may be considered, in order to perform surgery after tumour shrinkage. As a secondary surgical measure, after a medical treatment approach for (6-)12(-18) weeks (recommendations see below) for response evaluation reasons, surgical resection of all metastases should be attempted as completely as possible. The value of radiotherapy to metastatic disease is limited [68].

18.2.8 Medical treatment: Multi targeted tyrosine kinase inhibitor: Sunitinib We recommend to use Sunitinib in metastatic pediatric RCC as first-line adjuvant drug. Adult guidelines now recommend Sunitinib, Pazobanib and Bevacizumab + interferon-alpha, though the latter is not well accepted because of its intravenous route. Sunitinib is a multitargeted tyrosine kinase inhibitor (TKI) that acts mainly on the vascular endothelial growth-factor receptor (VEGFR) and platelet-derived growth- factor receptor (PDGFR), which play a role in both tumour angiogenesis and tumour cell proliferation. In addition, Sunitinib inhibits receptor tyrosine kinases (RTK) RET and c-KIT. Sunitinib significantly improved the progression-free survival (PFS) in metastatic adult clear cell RCC as compared to interferon following its accelerated FDA approval for the treatment of metastatic adult RCC. However, we have underlined that there is lack of comprehensive evidence for Sunitinib in non- clear cell RCC, and only very limited outcome data with Sunitinib in pediatric RCC are available. Prospective data of an unselected pediatric RCC cohort for judgment of the most appropriate adjuvant treatment in metastatic pediatric RCC are completely lacking. A few case reports have shown objective responses with Sunitinib in metastatic translocation-type RCCs in adults. In 2012 a first case report documented a persistent response during 24 months in a pediatric metastatic translocation RCC patient [69-72]. In a retrospective analysis of various targeted therapies in advanced Xp11 translocation type RCCs (mostly confirmed only by TFE3/TFEB immunostaining without genetic analysis) the best objective response rate was found with the RTK inhibitor Sunitinib (PR/CR in 7 of 14 patients) in comparison with Sorafenib (only SD as best response) and Temsirolimus (1 PR, SD), including 5 patients under 18 years

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(2-16) with one CR for 16 months and one PR for 15 months and one SD for 8 months after Sunitinib [23]. In a retrospective review of 14 metastatic RCCs in children and adolescents from Italy (1973-2010) 2 patients received RTK inhibitors. In both cases objective responses were noticed: in one case with Sunitinib during 3 months and in one case with Sorafenib for 6 months [17].

Dosages and schedule Sunitinib: A phase I study including pharmacokinetic analyses in pediatric patients with refractory solid tumours showed that the clearance of Sunitinib was similar between children and adults but the maximum tolerated dose was only 15 mg/m2/d in this intensive pre-treated patient population [73]. Other Sunitib studies performed in pediatric patients showed safe dosing of 20-25 mg/m2/day [74, 75]. In adults the approved Sunitinib dose is 50 mg/d [76]. Therefore, in the first course, a dose of 20-25 mg/m2/d (not higher than 25 mg/d absolute daily dose in patients < 12 yrs for the first dose) for 28 days and 14 days break is recommended. Higher doses can be considered in later courses in case of no relevant (WHO grade 3 or 4) toxicity, on expert advice. In patients > 16 years and/or > 50 kg body weight, adult dosages could be considered from the beginning. Close clinical control of possible toxicity and a monitoring of blood counts, liver transaminases, bilirubin, renal function, hypothyroidism and cardiac function are obligatory. Tumour response assessment will be documented after every treatment course at least in the first six months using the RECIST criteria (www.recist.com).

Drug treatment duration: In case of objective response, drug treatment should be administered for at least 1 year, possibly a longer duration for 2 (-3) years –depending on therapy tolerance and toxicity- may improve the outcome. Resection of residual metastases should be done after (8) – 12 – (16) weeks whenever possible. If residual - unchanged - tumour lesions are existent longer than 3 (-6) months, an alternative drug treatment should be considered. In case of tumour progression or relapse a second-line treatment is recommended. mTOR inhibitor therapy (evirolimus, temsirolimus) Temsirolimus and everolimus are inhibitors of the mammalian target of rapamycin (mTOR), a serine- threonine kinase, that regulates cell growth and survival. In adults, mTOR inhibitors are recommended in poor risk metastatic RCC or/and as second-line therapy. Very few cases with Temsirolimus use in translocation RCC and temporary responses have been reported [77]. Experiences with mTOR inhibitor therapy in pediatric RCC are extremely limited. Everolimus is currently approved for subependymal giant cell astrocytoma (SEGA) treatment in children with tuberous sclerosis, a genetic disorder with a predisposition to RCC. In a currently published case report on a child with tuberous sclerosis, the authors reported that Temsirolimus treatment had led to substantial shrinkage of multifocal RCC tumours assessed as PR [78].

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MET tyrosine kinase inhibitor (TKI) therapy Aberrant transcriptional up-regulation of MET by TFE3 fusion proteins in translocation-associated RCCs and preclinical evidence of anti-proliferative effect of MET TKI exposition have raised hopes for the therapeutic potential of MET inhibitors in these RCCs [12]. First data of a phase II study with Tivantinib, a selective inhibitor of MET, however, were disappointing with no objective responses in 6 translocation- type RCC [79]. Thus, in our opinion, MET TKI therapy in pediatric RCC should currently not be recommended.

Alternative and second line drug treatment: Immunotherapy. Several years ago, before the targeted drug era had evolved, immunotherapy was considered the standard treatment of metastatic adult RCC, producing single-agent objective response rates below 20% but with a curative potential in a small subset of patients (especially patients with good-risk features as good performance status and normal organ function, low LDH, normal BC and BSG, according to the prognostic index in adult RCC). In pediatric RCC, where such prognostic profiles are not identified as yet, a very few small studies and retrospective analyses documented objective responses and a survival benefit following interferon-a (IFN-a)–and/or interleukin-2 (IL-2)–based therapy in a small percentage/number of patients comparable with adult experiences [2, 22, 80]. Combined IFN-a plus bevacizumab and high dose IL-2 therapy are optional and recommended immunotherapy protocols in metastatic adult RCCs. Following IFN-a/IL-2/capecitabine/13-cis-retinoic acid combination with maintenance immunotherapy for three years in adult metastatic RCC patients, an objective response rate of 54%, a median PFS of 14.7 months and a 2-year survival rate of 52 % were observed, suggesting a possible improvement after a combined version with prolonged immunotherapy [81]. We observed one child with metastatic translocation-type RCC surviving disease-free > 10 years after combined IFN-a/IL-2/capecitabine/13-cis- retinoic acid according to the German DGCIN protocol published by Atzpodien et al [82].

Allogeneic hematopoietic stem cell transplantation (HSCT) In case of refractory/relapsed metastatic RCC, HSCT has been considered as a rescue possibility if there is no evidence for a better alternative. So far, no evidence exists that this is of benefit. A clear graft versus tumour (GVT) effect by allogeneic reduced intensity stem cell transplantation in metastatic RCC has been shown and the rescue potential of HSCT in adult cytokine-refractory RCC has been reported. The use of HSCT in adults was hardly compromised because of a high treatment-related mortality rate. In children a higher HSCT tolerance could be expected. In a recently published case the curative potential of reduced intensity HSCT in a pediatric metastatic RCC patient was demonstrated with a 5.7 year-progression-free survival after the initial HSCT [83].

Table 2: Treatment approaches for paediatric RCC Disease stage Treatment approach Localized disease (T1-4M0N0) - Complete nephrectomy - Nephron sparing surgery only if feasible and always after consultation of the SIOP surgical panel

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Regional lymph node metastases - Complete tumour resection including lymph node sampling (T1-4M0N1-2) Distant metastases (T1-4M1N0-2) 1. Surgery: Complete radical tumour nephrectomy + lymph node sampling. Surgery of distant metastases only after adjuvant therapy (& response judgment). 2. Adjuvant therapy: best available suggestion: Sunitinib. If non-response: mTOR inhibitor (evirolimus, temsirolimus), TKI (tivantinib) or lastly immunotherapy can be considered. 18.2.9 Radiotherapy The role of radiotherapy is limited in RCC. For details see chapter 17.

18.2.10 Follow-up guideline for surveillance The follow-up surveillance should be done according to section 11.6.5.

18.2.11 Second line treatment after relapse of RCC For patients with relapsed RCC the National Coordinator and the chairs of the RCC Panel should be approached to get the most approbriate treatment recommendation.

18.2.12 Side effects of medical treatment

Sunitinib General The most common side effects included fatigue, asthenia, fever, diarrhea, nausea, mucositis/stomatitis, vomiting, dyspepsia, abdominal pain, constipation, hypertension, peripheral edema, rash, hand and foot syndrome, skin discoloration, dry skin, hair color changes, altered taste, headache, back pain, arthralgia, extremity pain, cough, dyspnea, anorexia, and bleeding. The most serious adverse reactions included hepatotoxicity, renal failure, heart failure, pulmonary embolism, gastrointestinal perforation, and hemorrhages Gastrointestinal - Very common (10% or more): Diarrhea (up to 66%), nausea (up to 58%), mucositis/stomatitis (up to 49%), abdominal pain (up to 44%), vomiting (up to 39%), dyspepsia (up to 34%), constipation (up to 28%), dry mouth (up to 13%), flatulence (up to 15%), oral pain (up to 14%), GERD/reflux esophagitis (up to 12%), glossodynia (up to 11%), laboratory abnormalities including lipase elevations (up to 56%), amylase elevations (up to 35%) - Common (1% to 10%): Hemorrhoids, dysphagia, esophagitis, abdominal discomfort, rectal hemorrhage, gingival bleeding, mouth ulceration, proctalgia, cheilitis, oral discomfort, eructation - Uncommon (0.1% to 1%): Pancreatitis, intestinal perforation, anal fistula - Postmarketing reports: Esophagitis

Hematologic

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- Very common (10% or more): Neutropenia (18.3%), anemia (22%), thrombocytopenia (16.6%), bleeding events (18% to 37%), laboratory abnormalities including decreases in neutrophils (up to 77%), lymphocytes (up to 68%), leukocytes ( up to 78%), platelets (up to 68%), and hemoglobin (up to 79%) - Common (1% to 10%): Leukopenia, lymphopenia - Uncommon (0.1% to 1%): Pancytopenia - Postmarketing reports: Thrombotic microangiopathy Postmarketing bleeding events have included gastrointestinal, respiratory, tumour, urinary tract, and brain hemorrhages, some of which have been fatal. During clinical trials, bleeding events were reported in 37% (140 of 375) of patients with renal cell carcinoma (RCC) receiving sunitinib compared with 10% receiving interferon alpha. In the gastrointestinal stromal tumour (GIST) study, bleeding events were reported in 18% (37 of 202) of the sunitinib-treated patients compared with 17% (17 of 102) in placebo. Epistaxis was commonly reported; less frequent reports included rectal, gingival, upper gastrointestinal, genital, and wound bleeding.

Nervous system - Very common (10% or more): Altered taste (21% to 47%), headache (13% to 26%), dizziness (up to 13.6%) - Common (1% to 10%): Peripheral neuropathy, paresthesia, hypoesthesia, hyperesthesia - Uncommon (0.1% to 1%): Reversible posterior leukoencephalopathy syndrome, cerebrovascular accident, transient ischemic attack - Postmarketing reports: Seizures

Hepatic - Very common (10%): Laboratory abnormalities including elevations of ALT (up to 72%), AST (up to 61%), alkaline phosphatase (up to 63%), total bilirubin (up to 37%), and indirect bilirubin (up to 13%) - Uncommon (0.1% to 1%): Liver failure, cholecystitis, hepatitis, abnormal hepatic function - Postmarketing reports: Emphysematous cholecystitis, acalculous cholecystitis

Hypersensitivity - Uncommon (0.1% to 1%): Hypersensitivity - Postmarketing reports: Hypersensitivity reactions, including angioedema

Dermatologic - Very common (10% or more): Skin discoloration (up to 30%), rash (up to 30%), hand-foot syndrome (14% to 29%), hair color changes (up to 29%), dry skin (15% to 23%), alopecia (up to 14%), erythema (14%), pruritus (14%) - Common (1% to 10%): Skin exfoliation, skin reaction, eczema, blister, acne, pruritus, hyperkeratosis, dermatitis, nail disorder - Uncommon (0.1% to 1%): Stevens-Johnson syndrome, toxic epidermal necrolysis Frequency not reported: Erythema multiforme, necrotizing fasciitis - Postmarketing reports: Pyoderma gangrenosum

Cardiovascular

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- Very common (10% or more): Hypertension (up to 34%), peripheral edema (up to 24%), chest pain (13%), laboratory finding of decreased left ventricular ejection fraction (up to 16%) - Common (1% to 10%): Venous thromboembolic events including deep vein thrombosis and pulmonary embolism, hot flush/flushing - Uncommon (0.1% to 1%): Congestive cardiac failure, cardiac failure, cardiomyopathy, pericardial effusion, left ventricular failure Rare (less than 0.1%): Torsades de pointes - Postmarketing reports: Arterial thromboembolic events including cerebrovascular accident, transient ischemic attack, and cerebral infarction.

Musculoskeletal - Very common (10% or more): Myalgia/limb pain (11% to 40%), back pain (24% to 28%), arthralgia (19% to 23%), creatine kinase elevation (up to 49%) - Common (1% to 10%): Muscle spasms, muscular weakness - Uncommon (0.1% to 1%): Osteonecrosis of the jaw, fistula - Postmarketing reports: Fistula formation, myopathy and/or rhabdomyolysis

Respiratory - Very common (10% or more): Epistaxis (21%), cough (27%), dyspnea (16% to 26%), nasopharyngitis (14%), oropharyngeal pain (14%), upper respiratory infection (11%) - Common (1% to 10%): Pulmonary embolism, pleural effusion, hemoptysis, nasal congestion, nasal dryness - Uncommon (0.1% to 1%): Pulmonary hemorrhage, respiratory failure - Postmarketing reports: Pulmonary embolism Renal - Very common (10% or more): Increased serum creatinine (up to 70%), - Rare (less than 0.1%): Nephrotic syndrome - Frequency not reported: Proteinuria - Postmarketing reports: Renal impairment and/or failure

Metabolic - Very common (10% or more): Anorexia (up to 48%), weight loss (up to 16%), laboratory abnormalities including decreased potassium (up to 21% ), increased potassium (up to 18%), decreased calcium (up to 42%), increased calcium (13%), increased uric acid (up to 46%), increased glucose (up to 71%), decreased glucose (up to 22%), decreased sodium (up to 29%), increased sodium (up to 13%), decreased phosphorus (up to 36%), decreased magnesium (19% ), decreased albumin (up to 41%) - Common (1% to 10%): Dehydration - Uncommon (0.1% to 1%): Tumour lysis syndrome

Other - Very common (10% or more): Fatigue (33% to 76%), asthenia (16% to 34%), fever (up to 22%), chills (up to 14%) - Common (1% to 10%): Pain, influenza like symptoms - Uncommon (0.1% to 1%): Impaired wound healing

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Ocular - Common (1% to 10%): Periorbital edema, eyelid edema, increased lacrimation

Endocrine - Very common (10% or more): Hypothyroidism (16%) - Uncommon (0.1% to 1%): Hyperthyroidism

Psychiatric - Very common (10% or more): Insomnia (15% to 18%), depression (up to 11%)

Evirolimus General The most common side effects included stomatitis, infection, rash, fatigue, diarrhea, edema, peripheral edema, anemia, nausea, hyperlipidemia, headache, abdominal pain, fever, asthenia, cough, constipation, hypertension, urinary tract infection, leukopenia, and decreased appetite.

Metabolic - Very common (10% or more): Hypercholesterolemia (up to 85%), cholesterol increased (up to 77%), glucose increased (up to 75%), alkaline phosphatase increased (up to 74%), triglycerides increased (up to 73%), bicarbonate decreased (56%), hypertriglyceridemia (up to 52%), creatinine increased (up to 50%), hypophosphatemia (up to 49%), phosphate decreased (up to 40%), calcium decreased (37%), appetite decreased (up to 30%), potassium decreased (29%), weight decreased (up to 28%), anorexia (25%), hyperlipidemia (up to 21%), hyperkalemia (18%), sodium decreased (16%), dyslipidemia (15%), hyperglycemia (14%), hypomagnesemia (14%), hypokalemia (12%), diabetes mellitus (up to 10%) - Common (1% to 10%): Dehydration, blood urea increased, acidosis, gout, hypercalcemia, hyperuricemia, hypocalcemia, hypoglycemia, hyponatremia, iron deficiency, vitamin B12 deficiency, potassium increased

Hematologic - Very common (10% or more): Decreased hemoglobin (up to 92%), elevated partial thromboplastin time (72%), anemia (up to 61%), WBC decreased (up to 58%), lymphocytes decreased (up to 54%), platelets decreased (up to 54%), neutropenia (up to 46%), leukopenia (up to 37%), albumin decreased (up to 33%), neutrophils decreased (up to 31%), lymphopenia (up to 20%), thrombocytopenia (up to 19%) - Common (1% to 10%): Hemorrhage, leukocytosis, lymphadenopathy, pancytopenia - Uncommon (0.1% to 1%): Pure red cell aplasia

Gastrointestinal

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- Very common (10% or more): Stomatitis (up to 78%), diarrhea (Up to 50%), constipation (up to 38%), abdominal pain (up to 36%), nausea (up to 32%), vomiting (up to 29%), dry mouth (up to 11%), gastroenteritis (10%) - Common (1% to 10%): Abdominal distention, dyspepsia, dysphagia, epigastric discomfort, flatulence, gastritis, gastroesophageal reflux disease, gingival hypertrophy, hematemesis, hemorrhoids, ileus, mouth ulceration, oral candidiasis, oral pain, peritonitis

Other - Very common (10% or more): Fatigue (up to 45%), peripheral edema (up to 45%), edema (39%), asthenia (up to 33%), pyrexia (up to 31%), mucosal inflammation (19%), incision site pain (16%), procedural pain (15%) - Common (1% to 10%): Mucosal inflammation, irritability, blood lactate dehydrogenase increased, non-cardiac chest pain, chills, incisional hernia, edema

Dermatologic - Very common (10% or more): Rash (up to 59%), cellulitis (29%), nail disorders (22%), acne (up to 22%), pruritus (up to 21%), dry skin (13%), alopecia (10%) - Common (1% to 10%): Dermatitis acneiform, erythema, folliculitis, hand-foot syndrome, hirsutism, hyperhidrosis, hypertrichosis, night sweats, onychoclasis, onychomycosis, oral herpes skin exfoliation, skin lesion, tinea pedis - Uncommon (0.1% to 1%): Angioedema, Herpes zoster

Respiratory - Very common (10% or more): Respiratory tract infection (up to 31%), cough (up to 30%), dyspnea (up to 24%), epistaxis (up to 22%), pneumonitis (up to 19%), oropharyngeal pain (up to 11%), streptococcal pharyngitis (10%) - Common (1% to 10%): Nasopharyngitis, pharyngitis, pneumonia, pulmonary embolism, bronchitis, sinusitis, pleural effusion, rhinorrhea, atelectasis, nasal congestion, pulmonary edema, sinus congestion, wheezing - Uncommon (0.1% to 1%): Hemoptysis, acute respiratory distress syndrome

Genitourinary - Very common (10% or more): Amenorrhea (up to 17%), urinary tract infection (up to 16%), hematuria (12%), dysuria (11%), menorrhagia (up to 10%), menstrual irregularities (up to 10%) - Common (1% to 10%): Urethritis, bladder spasm, micturition urgency, pollakiuria, polyuria, pyuria, urinary retention, erectile dysfunction ovarian cyst, scrotal edema, blood luteinizing hormone increased, vaginal hemorrhage, blood follicle stimulating hormone increased, metrorrhagia, dysmenorrhea, delayed menstruation

Musculoskeletal

204

UMBRELLA Protocol SIOP 2016

- Very common (10% or more): Arthralgia (up to 20%), back pain (up to 15%), extremity pain (up to 14%), muscle spasms (up to 10%) - Common (1% to 10%): Osteomyelitis, jaw pain, joint swelling, muscular weakness, myalgia, osteonecrosis, osteopenia, osteoporosis, spondylitis

Hepatic - Very common (10% or more): AST increased (up to 69%), ALT increased (up to 51%), hepatitis C (up to 11%), bilirubin increased (up to 10%) - Common (1% to 10%): Transaminases increased

Nervous system - Very common (10% or more): Headache (up to 30%), dysgeusia (22%), dizziness (up to 12%) - Common (1% to 10%): Tremor, paresthesia, hemiparesis, hypoesthesia, lethargy, neuralgia, somnolence, syncope - Uncommon (0.1% to 1%): Ageusia

Psychiatric - Very common (10% or more): Behavioral disturbances (up to 21%), insomnia (up to 17%) - Common (1% to 10%): Depression, agitation, anxiety, hallucination

Cardiovascular - Very common (10% or more): Hypertension (up to 30%) - Common (1% to 10%): Angina pectoris, hot flush, atrial fibrillation, congestive cardiac failure, hypotension, palpitations, tachycardia, venous thromboembolism (including deep vein thrombosis)

Immunologic - Very common (10% or more): Infections (50%) - Common (1% to 10%): BK virus infection, bacteremia, candidiasis, influenza, otitis media, sepsis

Renal - Common (1% to 10%): Renal failure, proteinuria, pyelonephritis, hydronephrosis, interstitial nephritis, renal artery thrombosis

Ocular - Common (1% to 10%): Cataract, conjunctivitis, blurred vision, eyelid edema

Endocrine - Common (1% to 10%): Cushingoid, hyperparathyroidism

Hypersensitivity

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UMBRELLA Protocol SIOP 2016

- Common (1% to 10%): Hypersensitivity

Temsirolimus Hepatic - Very common (10% or more): Increased AST (up to 38%) - Common (1% to 10%): Increased total bilirubin, increased ALT

Immunologic - Very common (10% or more): Bacterial and viral infections (including abscess, bronchitis, cellulitis, herpes simplex, oral herpes, herpes zoster; up to 28.3%) - Common (1% to 10%): Sepsis, candidiasis, fungal infection, flu syndrome

Hypersensitivity - Common (1% to 10%): Allergic/hypersensitivity reactions

Respiratory - Very common (10% or more): Dyspnea (up to 28%), cough (up to 29%), epistaxis (up to 21.5%), pharyngitis (up to 12%), pneumonia (up to 10.9%), rhinitis (up to 10%) - Common (1% to 10%): Upper respiratory tract infection, pleural effusion, sinusitis, interstitial lung disease/pneumonitis (including fatalities) - Uncommon (0.1% to 1%): Laryngitis - Postmarketing reports: Pneumocystis jiroveci pneumonia

Renal - Very common (10% or more): Increased creatinine (up to 57%) - Common (1% to 10%): Renal failure

Hematologic - Very common (10% or more): Decreased hemoglobin (up to 94%), decreased lymphocytes (up to 53%), anemia (up to 41.1%), decreased platelets (up to 40%), decreased leukocytes (up to 32%), thrombocytopenia (up to 30.2%), decreased neutrophils (up to 19%), neutropenia (up to 14.3%) - Common (1% to 10%): Leukopenia, lymphopenia

Cardiovascular - Very common (10% or more): Chest pain (up to 16%) - Common (1% to 10%): Hypertension, venous thromboembolism (including deep vein thrombosis and pulmonary embolism, some fatal), thrombophlebitis, pericardial effusion

Dermatologic

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UMBRELLA Protocol SIOP 2016

- Very common (10% or more): Rash (including eczema, exfoliative dermatitis, maculopapular rash, pustular rash, vesiculobullous rash; up to 47%), pruritus (up to 21.5%), nail disorder (up to 14%), dry skin (up to 11%), acne (up to 10%) - Common (1% to 10%): Folliculitis, ecchymosis, petechiae - Postmarketing reports: Stevens-Johnson syndrome

Other - Very common (10% or more): Asthenia (up to 51%), fatigue (up to 41.4%), edema (including facial and peripheral; up to 38%), pyrexia (up to 28.3%), pain (up to 28%) - Common (1% to 10%): Wound infection/post-operative wound infection, impaired wound healing - Postmarketing reports: Angioneurotic edema-type reactions, extravasations Endocrine - Very common (10% or more): Increased glucose (up to 89%), hyperglycemia (up to 26%) - Common (1% to 10%): Diabetes mellitus

Gastrointestinal - Very common (10% or more): Mucositis (including stomatitis, glossitis, mouth ulceration; up to 41%), nausea (up to 37%), diarrhea (up to 34%), abdominal pain (up to 21%), constipation (up to 20%), vomiting (up to 19%) - Common (1% to 10%): Bowel perforation (including fatal cases), gastrointestinal hemorrhage, rectal hemorrhage, gastritis, dysphagia, abdominal distension, oral pain, gingivitis, mouth pain, oral moniliasis, hemorrhoidal hemorrhage - Uncommon (0.1% to 1%): Intestinal/duodenal perforation, lip hemorrhage, mouth hemorrhage

Genitourinary - Very common (10% or more): Unspecified urogenital adverse reaction (up to 30%), urinary tract infection (including cystitis, dysuria, hematuria, urinary frequency; up to 15%)

Metabolic - Very common (10% or more): Increased total cholesterol (up to 87%), increased triglycerides (up to 83%), increased alkaline phosphatase (up to 68%), decreased phosphorus (up to 49%), decreased calcium (up to 39%), decreased appetite (up to 33.3%), anorexia (up to 32%), hyperlipidemia (up to 27%), decreased potassium (up to 21%), weight loss (up to 19%), hypercholesterolemia (up to 18.8%), hypertriglyceridemia (up to 17.4%), hypokalemia (up to 13.7%), increased lactic dehydrogenase (up to 11%) - Common (1% to 10%): Dehydration, hypocalcemia, hypophosphatemia

Musculoskeletal - Very common (10% or more): Back pain (up to 20%), arthralgia (up to 18%), muscle cramp (up to 12%) - Common (1% to 10%): Myalgia - Postmarketing reports: Rhabdomyolysis

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Nervous system - Very common (10% or more): Dysgeusia (including taste loss, taste perversion; up to 20%), headache (up to 17.1%) - Common (1% to 10%): Dizziness, paresthesia, somnolence, convulsion - Uncommon (0.1% to 1%): Intracranial hemorrhage - Postmarketing reports: Complex regional pain syndrome/reflex sympathetic dystrophy

Ocular - Common (1% to 10%): Conjunctivitis (including lacrimation disorder) - Uncommon (0.1% to 1%): Eye hemorrhage Psychiatric - Very common (10% or more): Insomnia (up to 14%) - Common (1% to 10%): Depression, anxiety

Tivantinib [84]

The safety profile of tivantinib in advanced hepatocellular carcinoma was first explored in 21 patients treated with 360 mg twice-daily in a phase 1b study (NCT00802555); grade 3 or worse neutropenia was noted more frequently than was expected from previous non-hepatocellular carcinoma studies of the drug (38% in the phase 1b trial vs <5% elsewhere) but was generally well managed, and the overall safety profile of tivantinib in patients with hepatocellular carcinoma was regarded as acceptable (Santoro A, unpublished data). Adverse events noted in the tivantinib group of this study were much the same as for placebo, apart from the incidence of grade 3 or worse neutropenia with tivantinib 360 mg twice-daily, which occurred mainly in the first 45 days of treatment. Of particular concern were deaths related to neutropenia. These events prompted a protocol amendment to reduce the dose and institute a stricter dose-reduction scheme. At the amended dose of 240 mg twice-daily with proactive dose modifications, the incidence of grade 3 or worse neutropenia was reduced substantially, and efficacy was preserved. A population pharmacokinetics analysis, which included data from this study, has shown that tivantinib exposure is about three times higher in patients with hepatocellular carcinoma receiving 360 mg twice-daily than it is in patients with other solid tumours, and tivantinib exposure correlated with the incidence of grade 3 or worse neutropenia. This finding is consistent with evidence that tivantinib is extensively metabolised in the liver by CYP2C19 and CYP3A4.

18.2.13 References

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42. Tomlinson IP, Alam NA, Rowan AJ, Barclay E, Jaeger EE, Kelsell D, Leigh I, Gorman P, Lamlum H, Rahman S, Roylance RR, Olpin S, Bevan S, Barker K, Hearle N, Houlston RS, et al. Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet. 2002; 30(4):406-410. 43. Vanharanta S, Buchta M, McWhinney SR, Virta SK, Peczkowska M, Morrison CD, Lehtonen R, Januszewicz A, Jarvinen H, Juhola M, Mecklin JP, Pukkala E, Herva R, Kiuru M, Nupponen NN, Aaltonen LA, et al. Early-onset renal cell carcinoma as a novel extraparaganglial component of SDHB-associated heritable paraganglioma. Am J Hum Genet. 2004; 74(1):153-159. 44. Neumann HP, Pawlu C, Peczkowska M, Bausch B, McWhinney SR, Muresan M, Buchta M, Franke G, Klisch J, Bley TA, Hoegerle S, Boedeker CC, Opocher G, Schipper J, Januszewicz A and Eng C. Distinct clinical features of paraganglioma syndromes associated with SDHB and SDHD gene mutations. JAMA. 2004; 292(8):943-951. 45. Argani P and Ladanyi M. Translocation carcinomas of the kidney. ClinLab Med. 2005; 25(2):363-378. 46. Argani P, Antonescu CR, Illei PB, Lui MY, Timmons CF, Newbury R, Reuter VE, Garvin AJ, Perez- Atayde AR, Fletcher JA, Beckwith JB, Bridge JA and Ladanyi M. Primary renal neoplasms with the ASPL-TFE3 gene fusion of alveolar soft part sarcoma: a distinctive tumor entity previously included among renal cell carcinomas of children and adolescents. Am J Pathol. 2001; 159(1):179-192. 47. Davis IJ, His BL, Arroyo JD and et al. Cloning of a novel Alpha-TFEB fusion in renal tumors harboring the t(6;11)(p21;q12) chromosome translocation. Proc Natl Acad Sci U S A. 2003; (100):6051-6056. 48. Geller JI and Dome JS. Retroperitoneal lymph node dissection for pediatric renal cell carcinoma. Pediatr Blood Cancer. 2009; 52(3):430. 49. MacArthur CA, Isaacs H, Jr., Miller JH and Ozkaynak F. Pediatric renal cell carcinoma: a complete response to recombinant interleukin-2 in a child with metastatic disease at diagnosis. Medical and Pediatric Oncology. 1994; 23(4):365-371. 50. Choueiri TK, Lim ZD, Hirsch MS, Tamboli P, Jonasch E, McDermott DF, Dal Cin P, Corn P, Vaishampayan U, Heng DY and Tannir NM. Vascular endothelial growth factor-targeted therapy for the treatment of adult metastatic Xp11.2 translocation renal cell carcinoma. Cancer. 2010; 116(22):5219-5225. 51. Davis CJ, Jr., Mostofi FK and Sesterhenn IA. Renal medullary carcinoma. The seventh sickle cell nephropathy. Am J Surg Pathol. 1995; 19(1):1-11. 52. Simpson L, He X, Pins M, Huang X, Campbell SC, Yang XJ, Perlman EJ and Bergan RC. Renal medullary carcinoma and ABL gene amplification. J Urol. 2005; 173(6):1883-1888. 53. Stahlschmidt J, Cullinane C, Roberts P and Picton SV. Renal medullary carcinoma: prolonged remission with chemotherapy, immunohistochemical characterisation and evidence of bcr/abl rearrangement. Med Pediatr Oncol. 1999; 33(6):551-557. 54. Marino-Enriquez A, Ou WB, Weldon CB, Fletcher JA and Perez-Atayde AR. ALK rearrangement in sickle cell trait-associated renal medullary carcinoma. Genes Chromosomes Cancer. 2011; 50(3):146-153. 55. Cheng JX, Tretiakova M, Gong C, Mandal S, Krausz T and Taxy JB. Renal medullary carcinoma: rhabdoid features and the absence of INI1 expression as markers of aggressive behavior. Mod Pathol. 2008; 21(6):647-652. 56. Pirich LM, Chou P and Walterhouse DO. Prolonged survival of a patient with sickle cell trait and metastatic renal medullary carcinoma. J Pediatr Hematol Oncol. 1999; 21(1):67-69. 57. Strouse JJ, Spevak M, Mack AK, Arceci RJ, Small D and Loeb DM. Significant responses to platinum- based chemotherapy in renal medullary carcinoma. Pediatr Blood Cancer. 2005; 44(4):407-411. 58. Walsh A, Kelly DR, Vaid YN, Hilliard LM and Friedman GK. Complete response to carboplatin, gemcitabine, and paclitaxel in a patient with advanced metastatic renal medullary carcinoma. Pediatr Blood Cancer. 2010; 55(6):1217-1220.

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59. Bell MD. Response to paclitaxel, gemcitabine, and cisplatin in renal medullary carcinoma. Pediatr Blood Cancer. 2006; 47(2):228. 60. Ronnen EA, Kondagunta GV and Motzer RJ. Medullary renal cell carcinoma and response to therapy with bortezomib. J Clin Oncol. 2006; 24(9):e14. 61. Sausville JE, Hernandez DJ, Argani P and Gearhart JP. Pediatric renal cell carcinoma. J Pediatr Urol. 2009; 5(4):308-314. 62. Van Poppel H, Da Pozzo L, Albrecht W, Matveev V, Bono A, Borkowski A, Colombel M, Klotz L, Skinner E, Keane T, Marreaud S, Collette S and Sylvester R. A prospective, randomised EORTC intergroup phase 3 study comparing the oncologic outcome of elective nephron-sparing surgery and radical nephrectomy for low-stage renal cell carcinoma. Eur Urol. 2011; 59(4):543-552. 63. Ljungberg B, Hanbury DC, Kuczyk MA, Merseburger AS, Mulders PF, Patard JJ, Sinescu IC and European Association of Urology Guideline Group for renal cell c. Renal cell carcinoma guideline. Eur Urol. 2007; 51(6):1502-1510. 64. European Association of Urology Guideline Group for renal cell carcinoma. 65. Maehana T, Tanaka T, Kitamura H, Masumori N and Tsukamoto T. Short-term functional and oncological outcomes of partial nephrectomy for renal cell carcinoma in patients with an anatomically or functionally solitary kidney: single-center experience. Int J Clin Oncol. 2013; 18(6):1049-1053. 66. Aronson DC, Medary I, Finlay JL, Herr HW, Exelby PR and La Quaglia MP. Renal cell carcinoma in childhood and adolescence: a retrospective survey for prognostic factors in 22 cases. J Pediatr Surg. 1996; 31(1):183-186. 67. Ahmed HU, Arya M, Levitt G, Duffy PG, Mushtaq I and Sebire NJ. Part I: Primary malignant non- Wilms' renal tumours in children. Lancet Oncol. 2007; 8(8):730-737. 68. Flanigan RC, Mickisch G, Sylvester R, Tangen C, Van Poppel H and Crawford ED. Cytoreductive nephrectomy in patients with metastatic renal cancer: a combined analysis. J Urol. 2004; 171(3):1071-1076. 69. Mourad WF, Dutcher J and Ennis RD. State-of-the-art management of renal cell carcinoma. Am J Clin Oncol. 2014; 37(5):498-505. 70. Chowdhury T, Prichard-Jones K, Sebire NJ, Bier N, Cherian A, Sullivan MO, O'Meara A and Anderson J. Persistent complete response after single-agent sunitinib treatment in a case of TFE translocation positive relapsed metastatic pediatric renal cell carcinoma. J Pediatr Hematol Oncol. 2013; 35(1):e1- 3. 71. Choueiri TK, Mosquera JM and Hirsch MS. A case of adult metastatic Xp11 translocation renal cell carcinoma treated successfully with sunitinib. Clin Genitourin Cancer. 2009; 7(3):E93-94. 72. Pwint TP, Macaulay V, Roberts IS, Sullivan M and Protheroe A. An adult Xp11.2 translocation renal carcinoma showing response to treatment with sunitinib. Urol Oncol. 2011; 29(6):821-824. 73. Numakura K, Tsuchiya N, Yuasa T, Saito M, Obara T, Tsuruta H, Narita S, Horikawa Y, Satoh S and Habuchi T. A case study of metastatic Xp11.2 translocation renal cell carcinoma effectively treated with sunitinib. Int J Clin Oncol. 2011; 16(5):577-580. 74. Dubois SG, Shusterman S, Ingle AM, Ahern CH, Reid JM, Wu B, Baruchel S, Glade-Bender J, Ivy P, Grier HE, Adamson PC and Blaney SM. Phase I and pharmacokinetic study of sunitinib in pediatric patients with refractory solid tumors: a children's oncology group study. Clin Cancer Res. 2011; 17(15):5113-5122. 75. Janeway KA, Albritton KH, Van Den Abbeele AD, D'Amato GZ, Pedrazzoli P, Siena S, Picus J, Butrynski JE, Schlemmer M, Heinrich MC and Demetri GD. Sunitinib treatment in pediatric patients with advanced GIST following failure of imatinib. Pediatr Blood Cancer. 2009; 52(7):767-771. 76. Baker SD, Zimmerman EI, Wang YD, Orwick S, Zatechka DS, Buaboonnam J, Neale GA, Olsen SR, Enemark EJ, Shurtleff S, Rubnitz JE, Mullighan CG and Inaba H. Emergence of polyclonal FLT3

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tyrosine kinase domain mutations during sequential therapy with sorafenib and sunitinib in FLT3- ITD-positive acute myeloid leukemia. Clin Cancer Res. 2013; 19(20):5758-5768. 77. Molina AM, Lin X, Korytowsky B, Matczak E, Lechuga MJ, Wiltshire R and Motzer RJ. Sunitinib objective response in metastatic renal cell carcinoma: analysis of 1059 patients treated on clinical trials. Eur J Cancer. 2014; 50(2):351-358. 78. Parikh J, Coleman T, Messias N and Brown J. Temsirolimus in the treatment of renal cell carcinoma associated with Xp11.2 translocation/TFE gene fusion proteins: a case report and review of literature. Rare Tumors. 2009; 1(2):e53. 79. Pressey JG, Wright JM, Geller JI, Joseph DB, Pressey CS and Kelly DR. Sirolimus therapy for fibromatosis and multifocal renal cell carcinoma in a child with tuberous sclerosis complex. Pediatr Blood Cancer. 2010; 54(7):1035-1037. 80. Wagner AJ, Goldberg JM, Dubois SG, Choy E, Rosen L, Pappo A, Geller J, Judson I, Hogg D, Senzer N, Davis IJ, Chai F, Waghorne C, Schwartz B and Demetri GD. Tivantinib (ARQ 197), a selective inhibitor of MET, in patients with microphthalmia transcription factor-associated tumors: results of a multicenter phase 2 trial. Cancer. 2012; 118(23):5894-5902. 81. Bauer M, Reaman GH, Hank JA, Cairo MS, Anderson P, Blazar BR, Frierdich S and Sondel PM. A phase II trial of human recombinant interleukin-2 administered as a 4-day continuous infusion for children with refractory neuroblastoma, non-Hodgkin's lymphoma, sarcoma, renal cell carcinoma, and malignant melanoma. A Childrens Cancer Group study. Cancer. 1995; 75(12):2959-2965. 82. Recchia F, Saggio G, Amiconi G, Di Blasio A, Cesta A, Candeloro G, Necozione S, Fumagalli L and Rea S. Multicenter phase II study of chemo-immunotherapy in the treatment of metastatic renal cell carcinoma. J Immunother. 2007; 30(4):448-454. 83. Atzpodien J, Kirchner H, Jonas U, Bergmann L, Schott H, Heynemann H, Fornara P, Loening SA, Roigas J, Muller SC, Bodenstein H, Pomer S, Metzner B, Rebmann U, Oberneder R, Siebels M, et al. Interleukin-2- and interferon alfa-2a-based immunochemotherapy in advanced renal cell carcinoma: a Prospectively Randomized Trial of the German Cooperative Renal Carcinoma Chemoimmunotherapy Group (DGCIN). J Clin Oncol. 2004; 22(7):1188-1194. 84. Baek HJ, Han DK, Hwang TJ, Bae SH, Choi YD and Kook H. Long-term graft-versus-tumor effect following reduced intensity hematopoietic stem cell transplantation in a child with metastatic renal cell carcinoma. Pediatr Blood Cancer. 2012; 59(3):583-585. 85. Santoro A, Rimassa L, Borbath I et al. Tivantinib for second-line treatment of advanced hepatocellular carcinoma: a randomized, placebo-controlled phase 2 study. The Lancet Oncology 2013;14(1):55-63

18.2.14 Chairs and members of the RCC Panel

Name Profession Country Email

Lieve Tytgat / g.a.m.tytgat@@prinsesmaximacentru The Chair Oncologists m.nl / Marry van den Netherlands Heuvel-Eibrink m.m.vandenheuvel-

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[email protected]

Co-Chair Barbara Selle Oncologist Germany [email protected]

Tomás Acha Oncologist Spain [email protected]

Christophe Bergeron Oncologist France [email protected]

Beatriz de Camargo Oncologist Brazil [email protected]

Rhoikos Furtwängler Oncologist Germany [email protected]

Jan Godzinski Surgeon Poland [email protected]

Oncologist The Saskia Gooskens [email protected] Netherlands

Norbert Graf Oncologist Germany [email protected]

Ivo Leuschner Pathologist Germany [email protected]

Kathy Pritchard-Jones Oncologist UK [email protected] Members Sara Stoneham Oncologist UK [email protected]

The Harm van Tinteren Statistician [email protected] Netherlands

Gordan Vujanic Pathologist UK [email protected]

Filippo Spreafico Oncologist Italy [email protected]

Paola Collini Pathologist Italy [email protected]

Estelle Thebaud Oncologist France [email protected]

Christina Hulsbergen The Christina.Hulsbergen- Pathologist - van de Kaa Netherlands [email protected]

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18.3 Malignant Rhabdoid Tumour of the Kidney (MRTK)

18.3.1 Introduction Rhabdoid tumours represent a group of highly malignant childhood tumours characterized by common histology (1) and usually the loss of INI1 expression in immunostaining (2, 3). They can arise in the CNS, the soft tissues, the liver and the kidney. Though arising in different tissues, they usually share biallelic SMARCB1 or SMARCA4 inactivation (4, 5). This suggests a common genetic development of Rhabdoid tumours. Rhabdoid tumours can arise at different sites in a single patient if the patient suffers from a germline mutation (5, 6).

Malignant Rhabdoid Tumour of the Kidney (MRTK) shows an early onset with a median age at diagnosis of 10-18 months (7-9). 22-38% of MRTK patients have metastasis at diagnosis (7, 8). Metastatic disease often arises within the first two years of life, in contrast to nephroblastoma where stage IV patients younger than two years are an absolute rarity (10). Furthermore the number stage III and IV tumours, is high compared to other renal tumours. Their prognosis is still unsatisfactory with many relapses occuring early, often shortly after end of treatment or even during treatment.

Standard high risk renal tumour regimens as well as non-rhabdomyosarcoma regimen (EpSSG) have been unsatisfactory so far, resulting in 20-40% OS (7, 11). Mainly due to the rarity of the disease, no randomized trials investigating the efficacy of single agents or drug combinations in MRTK have commenced. So far in vitro testing, case reports and small series suggest sensitivity of MRTK to anthracyclins (12-14), alkylating agents, such as platinum derivates and oxazophosphorines (15), and radiation therapy (11, 16). A positive contribution of high dose treatment with stem cell rescue (HDSCT) has been reported in case series only (17). Despite multimodal treatment including these agents and treatment modalities OS remains poor (7, 18). This remains true for cases currently treated according to the ongoing UH1 protocol in the COG high-risk renal tumour studies whereas relevant toxicity causing repeated suspension of the latter protocols is a serious issue (Dome J., personal communication on preliminary results).

Gain of knowledge about response and outcome to a specific treatment is hampered by the rarity of MRTK and lately by several conflicting treatment protocols. Common international treatment guidelines specifically designed for MRTK will significantly accelerate gain of knowledge in this rare disease.

18.3.2 Aims Primary aim

 To unify and optimize the best available treatment for children with a MRTK in all SIOP collaboration centers by means of offering state of the art recommendations

Secondary aims

 To collect and analyze complete clinical information available concerning treatment and outcome of MRTK  To identify biomarkers and radiological characteristics that enhance early diagnosis in this rare disease in the future

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 To facilitate and maintain biology studies on MRTK refining future stratification and explore potential treatment targets  To serve as a platform for introducing high risk and relapsed MRTK patients into phase II trials where possible  To gain clinical and biological knowledge that will drive future randomized clinical trial(s)  To establish a blood and urine borne proteins and miRNA diagnostic tool to diagnose MRTK at presentation

18.3.3 Endpoints Primary endpoint:

 Outcome (Event free survival, overall survival) of paediatric MRTK patients treated according to a uniform recommendation.

Secondary endpoints:

 The prevalence and clinical characteristics of paediatric MRTK based on an International/SIOP cohort registry of renal tumours (SIOP-RTSG).  Characterization of blood and urine born circulating MRTK-biological markers including miRNA  Characterization of risk profils in expression analyses and specific gene mutations  Identification of MRTK subsets that are at risk of early relapse/progression

18.3.4 Inclusion criteria

Patients fulfilling the following criteria are eligible for inclusion:

 Histology proven RTK, for which central reference pathology review is mandatory  SMARCB1/INI1 staining  Obtained informed consent of the legal guardians to be treated according to the EU-RHAB guidelines and to transfer of data and/or tumour material and reference material

18.3.5 Exclusion criteria  Missing informed consent of the legal guardians  Histology other than MRTK

18.3.6 Background Information 18.3.6.1 Epidemiology MRTK accounts for about 1.4 to 2.4% of all childhood renal tumours in (7, 8, 11, 19, 20). The UK age standardized incidence between 1993 and 2010 was 0.6 per million children (9). In Germany 32 MRTK had been registered at the national childhood cancer registry over a time span of 16 years resulting in

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0.23 per million (8). A report from the Automated Childhood Cancer Information System Project including 45 MRTK reported an incidence of 0.1 per million for 0-14 years and 1.0 per million for the first year of life (19) presumably underestimating the real incidence. Median age at diagnosis is 10 - 18 months with 22-38% of patients presenting with metastasis already at a young age (7, 8, 11, 21). Thus, MRTK is the most frequent metastatic renal tumour in children younger than two years (10, 22). MRTK usually metastasizes to the lungs. In progressed cases bones, CNS and other tissues have been reported. Synchronous involvement of cerebral, liver and/or soft tissue Rhabdoid tumours are highly suspicious for Rhabdoid Tumour Predisposition Syndrome (RTPS) (5, 23) with a germline mutation in one of the genes involved in the SWI/SNF complex. The incidence of RTPS is not clear yet. RTPS might be associated with improved survival, as reported in seven cases with AT/RT showing long term survival (6). Of all childhood Rhabdoid tumours 45-48% arise in the kidneys, 14 – 18% in the head and neck and 36- 38% in the liver and soft tissue (8, 9). Soft tissue sites are more frequently seen in older children and adults where they account for 60-88% of all Rhabdoid tumours (9, 18). MRTK show a relatively equal gender distribution. 18.3.6.2 Biology Jaclyn Biegel and colleagues in 1989 reported the association of monosomy 22 to AT/RT (24) and later on could narrow down to biallelic inactivation of SMARCB1 on 22q11.2 in most cases (25). In roughly 15% of cases RTs have maintained SMARCB1 expression, suggesting other SWI/SNF complex inhibiting mutations (23). Recently Schneppenheim, Hasselblatt and colleagues identified both a germline and a somatic nonsense mutation of SMARCA4/BRG 1 in a RTPS family and a boy as reason for SWI/SNF complex disruption (3, 5). The proportion of MRTK patients with germline SWI/SNF complex disruption is not determined yet. In recent studies it was estimated with 15-35% (26). However since RTPS patients are probably overrepresented in the analyzed cohorts the true percentage still needs to be determined in a cross- sectional study irrespective of familial predisposition. Bourdeaut and colleagues found only one germline affected parent out of 21 parent pairs examined (27) suggesting a high rate of de novo germline mutations. Children suffering from germline mutations are significantly younger than the somatic MRTK children (27, 28). Various mutations including deletions, duplications, non-sense mutations, premature stop-codons, frameshift-mutations and others have been described for SMARCB1. Interestingly exons hit by a mutation seem to have an association for MRT, AT/RT or MRTK (26, 29-31). In all cases the loss of SMARCB1/INI expression leads to a loss of SWI/SNF chromatin-remodeling complex integrity. Similarly biallelic SMARCA4 inactivation causes the lack of BRG1, another crucial subunit of SWI/SNF complex. Both defects eventually cause a loss of function of SWI/SNF-complex in chromatin compaction. This presumably causes easier access of polymerases to chromosomes and thus a non-specific activation of many downstream pathways involving amongst others sonic hedgehog pathways, Wnt-pathway, aurora-kinase pathway and cell-cycle controls, f.e. cyclin D1, p16 and p14(32- 35). This is in contrast to the genetic stability of RTs, which harbor only very few mutations as compared to other tumours (36-38) and show no oncogenic canonical pathway mutations (39). EZH2 dependent polycomb repressor complex (PRC2) has been shown to work antagonistic to the SWI/SNF-complex in vitro (40). Thus targeting EZH2 might be another potential future treatment option.

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18.3.6.3 Treatment In EU-RHAB Consensus Therapy Recommendations are given for MRTK. All patients with a MRTK should be registered in EU-RHAB and treated according to these recommendations that are described in detail in Part II of the European Rhabdoid Registry V4 from 2015 (starting page 129). The protocol of the Multinational Registry for Rhabdoid Tumours of any anatomical site (EU-RHAB) can be requested from the Study centre in Augsburg/Germany (Frühwald M: European Rhabdoid Registry. A multinational registry for rhabdoid tumours of any anatomical site. V4, 2015):

[email protected] Klinik für Kinder und Jugendliche, Klinikum Augsburg, Germany Stenglinstr. 2, 86156 Augsburg, Phone: 0049 821 400 4342, Fax: 0049 821 400 17 4243

It is agreed that patients with an MRTK and registered in SIOP-RTSG need to be registered in EU-RHAB as well, if informed consent is given. The same is true for patients primarily registered in EU-RHAB to be registered in SIOP-RTSG, so that the incidence of Rhabdoid Tumuors in the kidney can be calculated. For that purpose data exchange will be done between SIOP-RTSG and EU-RHAB

The following section will give a short overview about different treatment options. Experience reported here is not solely based on experience with renal tumours but including experience with AT/RT and MRT too.

18.3.6.3.1 Surgery Total resection of MRT or AT/RT is significantly correlated with increased relapse free survival (41, 42), similarly advanced stage in MRTK has a significantly negative impact on survival (7, 8, 11). Van den Heuvel-Eibrink and colleagues reported 19% for stage III compared to 50% for stage I (7). Tomlinson compared stage I and II with stage III, IV and V resulting in 41% OS compared to 19% OS (11). Further discrimination into subgroups was not done by any of the authors. Preoperative treatment is advisable in non-completely resectable MRTK, instead of delaying systemic treatment due to prolonged postoperative recovery after complicated surgery. MRTK usually significantly shrinks on anthracyclin containing regimens (7), and intensive treatment is often providing a setting in which a complete resection becomes possible. However delaying surgery should be limited to a few cycles of treatment, as the biology of the disease tends to give rise to early progression. For further details of surgery see chapter 16.3.3, surgical guidelines. There is no difference to other renal tumours.

18.3.6.3.2 Chemotherapy Until now no randomized study comparing regimens has been conducted. However several hints concerning the effect of specific drugs have been published. Waldron, Wagner and colleagues reported, taken together, three stage IV patients successfully treated with combinations of doxorubicin, cyclophosphamide, vincristine, ifosfamide and etoposide (12, 13). Anthracyclin based treatment showed promising results in AT/RT in a recent report (41) and anthracyclins have shown to induce volume decrease in the preoperative setting of MRTK (7). However Tomlinson and colleagues did not find a difference in survival based on the use of doxorubicin (11). The report fails to give details on the

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different cohorts to rule out a selection bias due to probable accumulation of higher stages in the doxorubicin receiving cohort (11). Alkylating agents, especially ifosfamide seem to be important in the treatment of extracranial RT. In a series of 13 children from St. Judes, only those receiving ifosfamide survived (15). MRTK patients have been treated using different protocols over the last decades. The NWTSG treated their patients after upfront nephrectomy according to their unfavorable histology protocols during NWTS 1 to 4, largely based on vincristine, dactinomycin and doxorubicin with or without cyclophosphamide. In NWTS 1-5 Children having a local stage I or II had 41% OS, while higher stages showed 19.5% OS (11). Starting with NWTS 5 patients received a separate MRTK-protocol, based on carboplatinum, etoposide and cyclophosphamide, yielding 25.8% OS on 31 patients (AREN0321 High Risk Renal Tumours Trial Protocol). More recently, MRTK patients in North America were treated according to COG high risk protocol UH1 for localised MRTK. Stage IV patients and macroscopic residual stage III received an Irinotecan window treatment, which failed to show increased response (Dome J, preliminary data, personal communication). UH1 had to be closed several times for cardiac failures, VOD and respiratory distress syndromes. As a consequence total cumulative doses of doxorubicin had to be reduced from 375 mg/m2 to 225 mg/m2 and cyclophosphamide, etoposide cycles were reduced by 20% (Table 1 – Comparison of cumulative doses, Table 2 –Schedule of UH1). Also, despite significant treatment toxicity no convincing increase in survival was achieved so far. The COG regimens included radiation doses of 10.8 Gy flank for all stages. 22.5 Gy whole abdomen in specific cases.

Drug Cumulative Doses (mg/m2)

UH-1 Revised UH-1

Cyclophosphamide 17,000 14,800

Doxorubicin 375 225

Vincristine 22.5 22.5

Carboplatin 3000 3000

Etoposide 2500 2000

Duration (weeks) 28 28

Table 1

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Table 2

A similar approach was recently chosen by EpSSG using the same cumulative dosages and schedules (Compare Table 3 – EpSSG Schedule). In recent oral presentation, OS for liver and kidney RT was stated to be only 17.5% (B. Brennan, Oral presentation at the Rhabdoid Tumour meeting, Paris 2013). Proposed adjustment of treatment was shortening of intervals to two weeks and introducing ifosfamide. EpSSG recommended irradiating with a dose of 19.8 Gy irrespectively of age and stage, and 19.5 Gy for whole abdomen irradiation. EPSSG tends to perform abdominal surgery late, only after 4 cycles of intensive treatment, as recommend for non-resectable stages such as stage IV.

Table 3

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Starting with SIOP 93-01 MRTK was treated with alternating courses of carboplatinum and etoposide and doxorubicin and ifosfamide (SIOP93-01) or cyclophosphamide (SIOP2001). The cumulative dose of doxorubicin was 300mg/m2 (I-III) and 400mg/m2 for stage IV. 5y-EFS were 50%, 28%, 19% and 18% for stage I, II, III and IV respectively in 107 patients (7). The SIOP irradiation dose was: 30 Gy flank and 20 Gy whole abdomen. The EU-RHAB registry is a common treatment approach to cranial, renal and extra-cranial and extra- renal Rhabdoid tumours. It includes block treatment on a two week schedule with two cycles of alternating blocks of doxorubicin (70mg/48h), ICE (3x2g/m2 ifosfamide,1 x 500mg/m2 carboplatinum, 3 x 100mg/m2 etoposide) and VCA (vincristine 1x1.5mg/m2, cyclophosphamide 1 x 1.5g/m2 and dactinomycin 2 x 25µg/kg), followed by optional high dose treatment with carboplatinum and thiotepa or one more cycle of three blocks (Figure 1 – Overview of treatment schedule with or without HDSCT). Irradiation should be given as early as possible. Doses recommended are 19.8 Gy flank irradiation in children >12 months of age and 10.8 Gy for children <12 months of age. For further details use the EU- RHAB Registry protocol as stated above.

Figure 1

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With evolving knowledge about targeted therapies a variety of small molecules have been tested preclincally on rhabdoid tumour cell lines and/or xenograft models: Arsenic trioxid (14, 44); Aurora Kinase A radiosensitzing (32); cyclin D inhibition (14) by f.e. flavopiridol (34), Alvocidib or Fenretinide and Tamoxifen (45), HDAC inhibition by romidepsin (effective in AT/RT not in MRTK cell lines)(46) . A few have been tested in phase I and II studies: Aurorakinase A inhibitor Alisertib in a COG phase I and UK phase II study, results pending; HDAC inhibitor Vorinostat partly combined with cis-retionic acid in a phase I including two AT/RT, without objective response (47). However considering the etiology of MRTK it is unlikely a single agent will yield continuous response.

18.3.6.3.3 Radiotherapy The role of radiotherapy (RT) in MRTK still needs to be determined. (see section 17.17 of the SIOP-RTSG protocol and the EU-RHAB Registry) The young age of MRTK patients often encourages physicians to omit flank irradiation or lung irradiation in those patients, thus it remains uncertain whether different tumour biology in infant patients or omitting irradiation accounts for the significant difference in survival repeatedly reported for younger patients (7, 8, 11, 18). Looking at AT/RT cohorts, the association of survival and RT is compelling. In 1 series only one out of ten patients (43), including three infants, receiving RT in first line treatment died and one was alive with disease. In contrast three out of 21 without RT in first line, 19 younger than three years, survived without evidence of disease. Two of them received RT in second line treatment (43). In a report about MRTK treated in NWTS1-5, 100 of 142 had received RT. 4y OS was 28.5% in RT patients and 12.2% in non-RT patients (p=0.25). Again, RT was more frequently given to older patients or younger patients with advanced disease, thus rendering interpretation of the data difficult. Interestingly RT-dose of > 25 Gy might be of benefit in older patients (11). 52 patients having MRTK have been treated in the GPOH from 1993-2013. Patients with a local stage III, receiving RT had 36% OS whereas OS was 19% OS when not RT was not administered (Furtwängler, Rhabdoid Tumour meeting, Paris 2013). Sultan and colleagues, reporting on SEER data, showed a significant impact of RT on survival (HR 1.89; 1.29-2.78 95%CI; p=0.0012) in multivariate analysis adjusted for age and stage, both being of significant influence too (18). In 229 patients analysed, only 45 had MRTK and the remaining patients AT/RT or MRT. In summary RT seems to be justified even in infants with MRTKs. Further details are given in chapter 13 and the EU-Rhabdoid Registry.

18.3.7 Recommendations a. Registration Since most renal tumour patients will initially be registered through the common SIOP-RTSG UMBRELLA protocol, and do receive preoperative chemotherapy. As soon as the diagnosis MRTK becomes apparent patients should also be registered in the EU-RHAB Registry. Data exchange between the EU-Rhab Resitry and the SIOP-RTSG UMBRELLA protocol will be guaranteed. b. Diagnostics i. Imaging

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SIOP UMBRELLA - MRTK specific implications: Whole body MRI, cranial MRI ii. Histology SIOP UMBRELLA - MRTK specific implications: Recommend tru-cut biopsy in children younger than 24 months suffering from metastasis or familial predisposition (Family history for AT/RT; MRT or MRTK), without delaying the start of treatment. iii. Genetics Analysis of tumour tissue, patient’s and in case of germline mutation of parents’ samples for somatic and germline mutation of SMARCB1 and SMARCA4. iv. Research Blood samples as in SIOP UMBRELLA - Tumour material and ancillary studies according to EU- RHAB Registry 2015. c. Treatment The aim of this guideline is to facilitate best uniform treatment of all MRTK in the SIOP collaborative centers. i. Chemotherapy See the respective section in EU-RHAB Registry for Rhabdoid Tumours of the Kidney ii. Irradiation See the respective section in EU-RHAB Registry for Rhabdoid Tumours of the Kidney iii. Surgery See the respective section in EU-RHAB Registry for Rhabdoid Tumours of the Kidney d. Follow up Refer to SIOP-RTSG UMBRELLA and EU-RHAB – Additional cMRT/Whole body screening in germline mutation patients as a screening

18.3.8 References 1. Vujanic GM, Sandstedt B, Harms D, Boccon-Gibod L, Delemarre JF. Rhabdoid tumour of the kidney: a clinicopathological study of 22 patients from the International Society of Paediatric Oncology (SIOP) nephroblastoma file. Histopathology. 1996;28(4):333-40. 2. Hoot AC, Russo P, Judkins AR, Perlman EJ, Biegel JA. Immunohistochemical analysis of hSNF5/INI1 distinguishes renal and extra-renal malignant rhabdoid tumors from other pediatric soft tissue tumors. American Journal of Surgical Pathology. 2004;28(11):1485-91. 3. Hasselblatt M, Gesk S, Oyen F, Rossi S, Viscardi E, Giangaspero F, et al. Nonsense mutation and inactivation of SMARCA4 (BRG1) in an atypical teratoid/rhabdoid tumor showing retained SMARCB1 (INI1) expression. Am J Surg Pathol. 2011;35(6):933-5. 4. Versteege I, Sévenet N, Lange J, Rousseau-Merck MF, Ambros P, Handgretinger R, et al. Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature. 1998;394(6689):203-6.

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5. Schneppenheim R, Fruhwald MC, Gesk S, Hasselblatt M, Jeibmann A, Kordes U, et al. Germline nonsense mutation and somatic inactivation of SMARCA4/BRG1 in a family with rhabdoid tumor predisposition syndrome. American journal of human genetics. 2010;86(2):279-84. 6. Kordes U, Bartelheim K, Modena P, Massimino M, Biassoni V, Reinhard H, et al. Favorable outcome of patients affected by rhabdoid tumors due to rhabdoid tumor predisposition syndrome (RTPS). Pediatr Blood Cancer. 2013. 7. van den Heuvel-Eibrink MM, van Tinteren H, Rehorst H, Coulombe A, Patte C, de Camargo B, et al. Malignant rhabdoid tumours of the kidney (MRTKs), registered on recent SIOP protocols from 1993 to 2005: a report of the SIOP renal tumour study group. Pediatric blood & cancer. 2011;56(5):733-7. 8. Reinhard H, Reinert J, Beier R, Furtwängler R, Alkasser M, Rutkowski S, et al. Rhabdoid tumors in children: Prognostic factors in 70 patients diagnosed in Germany. Oncology reports. 2008;19(3):819-23. 9. Brennan B, Stiller C, Bourdeaut F. Extracranial rhabdoid tumours: What we have learned so far and future directions. The Lancet Oncology. 2013;14(8):e329-e36. 10. Verschuur A, Van Tinteren H, Graf N, Bergeron C, Sandstedt B, De Kraker J. Treatment of pulmonary metastases in children with stage IV nephroblastoma with risk-based use of pulmonary radiotherapy. Journal of Clinical Oncology. 2012;30(28):3533-9. 11. Tomlinson GE, Breslow NE, Dome J, Guthrie KA, Norkool P, Li S, et al. Rhabdoid tumor of the kidney in the National Wilms' Tumor Study: Age at diagnosis as a prognostic factor. Journal of Clinical Oncology. 2005;23(30):7641-5. 12. Waldron PE, Rodgers BM, Kelly MD, Womer RB. Successful treatment of a patient with stage IV rhabdoid tumor of the kidney: Case report and review. Journal of Pediatric Hematology/Oncology. 1999;21(1):53-7. 13. Wagner L, Ashley Hill D, Fuller C, Pedrosa M, Bhakta M, Perry A, et al. Treatment of metastatic rhabdoid tumor of the kidney. Journal of Pediatric Hematology/Oncology. 2002;24(5):385-8. 14. Lünenbürger H, Lanvers-Kaminsky C, Lechtape B, Frühwald MC. Systematic analysis of the antiproliferative effects of novel and standard anticancer agents in rhabdoid tumor cell lines. Anti- Cancer Drugs. 2010;21(5):514-22. 15. Gururangan S, Bowman LC, Parham DM, Wilimas JA, Rao B, Pratt CB, et al. Primary extracranial rhabdoid tumors: Clinicopathologic features and response to ifosfamide. Cancer. 1993;71(8):2653- 9. 16. Palmer NF, Sutow W. Clinical aspects of the rhabdoid tumor of the kidney: A report of the national Wilms' tumor study group. Medical and Pediatric Oncology. 1983;11(4):242-5. 17. Koga Y, Matsuzaki A, Suminoe A, Hatano M, Saito Y, Kinoshita Y, et al. Long-term survival after autologous peripheral blood stem cell transplantation in two patients with malignant rhabdoid tumor of the kidney. Pediatric Blood and Cancer. 2009;52(7):888-90. 18. Sultan I, Qaddoumi I, Rodriguez-Galindo C, Nassan AA, Ghandour K, Al-Hussaini M. Age, stage, and radiotherapy, but not primary tumor site, affects the outcome of patients with malignant rhabdoid tumors. Pediatr Blood Cancer. 2010;54(1):35-40. 19. Pastore G, Znaor A, Spreafico F, Graf N, Pritchard-Jones K, Steliarova-Foucher E. Malignant renal tumours incidence and survival in European children (1978-1997): Report from the Automated Childhood Cancer Information System project. European Journal of Cancer. 2006;42(13):2103-14. 20. Reinhard H, Semler O, Bürger D, Bode U, Flentje M, Göbel U, et al. Results of the SIOP 93-01/GPOH trial and study for the treatment of patients with unilateral nonmetastatic wilms tumor. Klinische Padiatrie. 2004;216(3):132-40. 21. Brennan BMD, Foot ABM, Stiller C, Kelsey A, Vujanic G, Grundy R, et al. Where to next with extracranial rhabdoid tumours in children [2]. European Journal of Cancer. 2004;40(4):624-6.

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22. Warmann SW, Nourkami N, Frühwald M, Leuschner I, Schenk JP, Fuchs J, et al. Primary lung metastases in pediatric malignant non-Wilms renal tumors: Data from SIOP 93-01/GPOH and SIOP 2001/GPOH. Klinische Padiatrie. 2012;224(3):148-52. 23. Fruhwald MC, Hasselblatt M, Wirth S, Kohler G, Schneppenheim R, Subero JI, et al. Non-linkage of familial rhabdoid tumors to SMARCB1 implies a second locus for the rhabdoid tumor predisposition syndrome. Pediatr Blood Cancer. 2006;47(3):273-8. 24. Biegel JA, Rorke LB, Emanuel BS. Monosomy 22 in rhabdoid or atypical teratoid tumors of the brain. New England Journal of Medicine. 1989;321(13):906. 25. Biegel JA, Allen CS, Kawasaki K, Shimizu N, Budarf ML, Bell CJ. Narrowing the critical region for a rhabdoid tumor locus in 22q11. Genes Chromosomes and Cancer. 1996;16(2):94-105. 26. Eaton KW, Tooke LS, Wainwright LM, Judkins AR, Biegel JA. Spectrum of SMARCB1/INI1 mutations in familial and sporadic rhabdoid tumors. Pediatric Blood and Cancer. 2011;56(1):7-15. 27. Bourdeaut F, Lequin D, Brugières L, Reynaud S, Dufour C, Doz F, et al. Frequent hSNF5/INI1 germline mutations in patients with rhabdoid tumor. Clinical Cancer Research. 2011;17(1):31-8. 28. Kordes U, Gesk S, Fruhwald MC, Graf N, Leuschner I, Hasselblatt M, et al. Clinical and molecular features in patients with atypical teratoid rhabdoid tumor or malignant rhabdoid tumor. Genes, chromosomes & cancer. 2010;49(2):176-81. 29. Bourdeaut F, Fréneaux P, Thuille B, Lellouch-Tubiana A, Nicolas A, Couturier J, et al. hSNF5/INII- deficient tumours and rhabdoid tumours are convergent but not fully overlapping entities. Journal of Pathology. 2007;211(3):323-30. 30. Birks DK, Donson AM, Patel PR, Sufit A, Algar EM, Dunham C, et al. Pediatric rhabdoid tumors of kidney and brain show many differences in gene expression but share dysregulation of cell cycle and epigenetic effector genes. Pediatric Blood and Cancer. 2013;60(7):1095-102. 31. Grupenmacher AT, Halpern AL, Bonaldo MDF, Huang CC, Hamm CA, De Andrade A, et al. Study of the gene expression and microRNA expression profiles of malignant rhabdoid tumors originated in the brain (AT/RT) and in the kidney (RTK). Child's Nervous System. 2013;29(11):1977-83. 32. Venkataraman S, Alimova I, Tello T, Harris PS, Knipstein JA, Donson AM, et al. Targeting Aurora Kinase A enhances radiation sensitivity of atypical teratoid rhabdoid tumor cells. Journal of Neuro- Oncology. 2012;107(3):517-26. 33. Venneti S, Le P, Martinez D, Eaton KW, Shyam N, Jordan-Sciutto KL, et al. P16INK4A and p14ARF tumor suppressor pathways are deregulated in malignant rhabdoid tumors. Journal of Neuropathology and Experimental Neurology. 2011;70(7):596-609. 34. Smith ME, Cimica V, Chinni S, Jana S, Koba W, Yang Z, et al. Therapeutically targeting cyclin D1 in primary tumors arising from loss of Ini1. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(1):319-24. 35. Algar EM, Muscat A, Dagar V, Rickert C, Chow CW, Biegel JA, et al. Imprinted CDKN1C is a tumor suppressor in rhabdoid tumor and activated by restoration of SMARCB1 and histone deacetylase inhibitors. PLoS ONE. 2009;4(2). 36. Hasselblatt M, Isken S, Linge A, Eikmeier K, Jeibmann A, Oyen F, et al. High-resolution genomic analysis suggests the absence of recurrent genomic alterations other than SMARCB1 aberrations in atypical teratoid/rhabdoid tumors. Genes Chromosomes and Cancer. 2013;52(2):185-90. 37. Lee RS, Stewart C, Carter SL, Ambrogio L, Cibulskis K, Sougnez C, et al. A remarkably simple genome underlies highly malignant pediatric rhabdoid cancers. Journal of Clinical Investigation. 2012;122(8):2983-8. 38. McKenna ES, Sansam CG, Cho YJ, Greulich H, Evans JA, Thom CS, et al. Loss of the epigenetic tumor suppressor snf5 leads to cancer without genomic instability. Molecular and Cellular Biology. 2008;28(20):6223-33.

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39. Kieran MW, Roberts CW, Chi SN, Ligon KL, Rich BE, Macconaill LE, et al. Absence of oncogenic canonical pathway mutations in aggressive pediatric rhabdoid tumors. Pediatric Blood and Cancer. 2012;59(7):1155-7. 40. Wilson BG, Wang X, Shen X, McKenna ES, Lemieux ME, Cho YJ, et al. Epigenetic antagonism between polycomb and SWI/SNF complexes during oncogenic transformation. Cancer Cell. 2010;18(4):316-28. 41. Chi SN, Zimmerman MA, Yao X, Cohen KJ, Burger P, Biegel JA, et al. Intensive multimodality Treatment for children with newly diagnosed CNS atypical teratoid rhabdoid tumor. Journal of Clinical Oncology. 2009;27(3):385-9. 42. Bourdeaut F, Fréneaux P, Thuille B, Bergeron C, Laurence V, Brugières L, et al. Extra-renal non- cerebral rhabdoid tumours. Pediatric Blood and Cancer. 2008;51(3):363-8. 43. Tekautz TM, Fuller CE, Blaney S, Fouladi M, Broniscer A, Merchant TE, et al. Atypical teratoid/rhabdoid tumors (ATRT): improved survival in children 3 years of age and older with radiation therapy and high-dose alkylator-based chemotherapy. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2005;23(7):1491-9. 44. Kerl K, Moreno N, Holsten T, Ahlfeld J, Mertins J, Hotfilder M, et al. Arsenic trioxide inhibits tumor cell growth in malignant rhabdoid tumors in vitro and in vivo by targeting overexpressed Gli1. International Journal of Cancer. 2014. 45. Alarcon-Vargas D, Zhang Z, Agarwal B, Challagulla K, Mani S, Kalpana GV. Targeting cyclin D1, a downstream effector of INI1/hSNF5, in rhabdoid tumors. Oncogene. 2006;25(5):722-34. 46. Graham C, Tucker C, Creech J, Favours E, Billups CA, Liu T, et al. Evaluation of the antitumor efficacy, pharmacokinetics, and pharmacodynamics of the histone deacetylase inhibitor depsipeptide in childhood cancer models in vivo. Clinical Cancer Research. 2006;12(1):223-34. 47. Fouladi M, Park JR, Stewart CF, Gilbertson RJ, Schaiquevich P, Sun J, et al. Pediatric phase I trial and pharmacokinetic study of vorinostat: A children's oncology group phase I consortium report. Journal of Clinical Oncology. 2010;28(22):3623-9.

18.3.9 Chairs and members of the MRTK Panel

Name Profession Country Email

Chair Rhoikos Oncologist Germany [email protected] Furtwängler

Tomás Acha Oncologist Spain [email protected]

Christophe Bergeron Oncologist France [email protected]

Beatriz de Camargo Oncologist Brazil [email protected]

Members Jan Godzinski Surgeon Poland [email protected]

Marry van den The m.m.vandenheuvel- Oncologist Heuvel-Eibrink Netherlands [email protected]

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Norbert Graf Oncologist Germany [email protected]

Ivo Leuschner Pathologist Germany [email protected]

Kathy Pritchard-Jones Oncologist UK [email protected]

Sara Stoneham Oncologist UK [email protected]

The Lieve Tytgat Oncologist [email protected] Netherlands

The Harm van Tinteren Statistician [email protected] Netherlands

Gordan Vujanic Pathologist UK [email protected]

Paolo D’Angelo Oncologist Italy [email protected]

Estelle Thebaud Oncologist France [email protected]

The Anne Smets Radiologist [email protected] Netherlands

Christian Rübe Radiotherapist Germany [email protected]

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18.4 Congenital Mesoblastic Nephroma (CMN) 18.4.1 Introduction / background Epidemiology Congenital mesoblastic nephroma (CMN) is a rare tumour that accounts for about 3% of all pediatric renal tumours [1]. However, it is the most common renal neoplasm in the first 3 months of life [2]. The median age is ≤ 1 month in most published series and CMN is frequently recognized before or at time of birth, illustrating the embryonal origin of the disease [3]. Clinical features The most common presentation of CMN is an abdominal mass/distention, followed by hypertension and gross haematuria [4]. Patients are usually asymptomatic and the mass is detected as an incidental finding. In some patients, the tumour is diagnosed on prenatal ultrasound [5-7]. Other very rare findings are hypercalcaemia and hyperreninaemia [3, 8]. Metastatic disease at time of initial presentation has not been described. Among 101 patients in two of the largest series, there were no cases of lymph node involvement or distant metastatic disease [3, 4]. However, cases of metastasis to the brain and lung have been documented at time of recurrence [9-11]. Histology There are two main histologic subtypes of CMN: classic (or conventional) and cellular [9, 12]. Some CMNs have a mixed pattern with features of both subtypes. Classic CMN tends to present in very young infants and neonates, whereas cellular CMN is seen in older infants [3]. Genetics Cellular CMN is morphologically similar to infantile fibrosarcoma (IFS), underscored by the completely similar chromosomal translocation t(12;15)(p13;q25), which results in a fusion of the ETV6 (TEL) gene with the NTRK3 gene [13-15]. ETV6 encodes a transcription factor with a helix-loop-helix protein dimerization domain and NTRK3 encodes a receptor tyrosine kinase. The chimeric ETV6-NTRK3 protein is postulated to have constitutively active tyrosine kinase growth pathway signaling [16]. A recent gene expression analysis of CMN showed that these tumours have a distinct gene expression profile compared to other pediatric renal tumours [17]. The expression pattern was consistent with receptor tyrosine kinase activation, with evidence of PI3-AKT, SRC, and MAPK activation. Interestingly, 4/14 cellular CMN manifested the gene expression pattern of CMN, but did not have detectable ETV6-NTRK6 transcript, indicating that molecular mechanisms other than the ETV6-NTRK6 fusion are responsible for the development of some cellular CMN [17]. Another aberration commonly described in cellular CMN is trisomy 11 [18]. In most cases, trisomy 11 occurs together with t(10;15)(p13;q25) [18]. CMN has never been reported with germline trisomy 11 nor other genetic predisposition syndromes. Also, familial CMN cases have not been reported so far. Treatment and outcome Outcomes for patients with CMN are generally excellent when treated with nephrectomy only, with overall survival rates of about 95% [1, 3, 4, 19]. The few tumours that recur are almost exclusively of the cellular subtype [3]. It remains to be established whether patients with stage III cellular CMN benefit from adjuvant chemotherapy. In a series published by the German Pediatric Oncology Group (GPOH), two of five patients with stage III cellular CMN developed recurrent disease (no recurrences in four patients with stage III classic CMN), whereas only one of the remaining 45 patients with other disease stages had a recurrence [3]. Three of five patients with cellular stage III CMN were treated with adjuvant chemotherapy at initial diagnosis; these patients remained free of disease after initial treatment. In a

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report of the United Kingdom Children’s Cancer and Leukaemia Group (CCLG) no recurrences occurred in 6 patients with stage III disease (2 cellular, 3 mixed and 1 classic type), only 1 of these 6 patients was treated with adjuvant chemotherapy because of tumour spill [1].

Report Study N Surgery Post- Radio- EFS OS Cause of only operative therapy death chemotherapy Howell 1982 NWTS 51 23 28 (ACT, 4 98% (1y) 98% (1y) Sepsis (1) [20] ACT/VCR) Sandstedt SIOP 29 (9 24 5 (all 1 93% (4y) 93% (4y) Sepsis (2) 1983 [21] classic, cellular) 20 cellular) Furtwängler SIOP 93-01 50 (29 41 9 (3 VCR, 3 1 94% (4.2y) 95% (4.2y) Tumour 2006 [3] (GPOH) classic, ACT / VCR, (extensive (3 (2 dead, all 21 2 AVD, 1 abdominal recurrences, cellular, 1 cellular) VP16 / CYC infiltration all cellular, 1 stage I, 1 / DOX / cellular stage I and 2 stage III) CARBO) CMN) stage III) Chaudry Children’s 30 (13 NA NA NA 93% NA NA 2009 [22] Hospital classic, 3 (Boston), The mixed, Hospital of 14 Sick Children cellular) (Toronto) England CCLG 50 (23 46 2 0 100% (4.4y) 100% - 2011 [1] classic, (ACT/VCR, (4.4y) 10 ACT/VCR/C mixed, YC) (mixed 14 type stage cellular) III) Table 1: CMN series > 25 patients:

Studies of cellular CMN have shown that these tumours respond to regimens containing different combinations of vincristine, dactinomycin, doxorubicin, and cyclophosphamide [3, 23]. This is not unexpected based on the sensitivity of infantile fibrosarcoma to similar sarcoma-directed therapy [24- 26]. Responses to ifosfamide/carboplatin/etoposide (ICE) have also been noted in patients with tumours refractory to the other agents [23].

18.4.2 3.4.2 Aims and endpoints Aims Primary aim:  To provide the best available care for CMN in children, including a harmonised diagnostic and treatment guideline.

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Secondary aims:  To determine the prevalence, clinical characteristics and outcome of paediatric CMN patients based on a prospective registration of a full cohort of European paediatric CCSK patients.  To study specific biological and genetic characteristics in paediatric CMN Endpoints Primary endpoint:  Outcome of paediatric CMN patients. Secondary endpoints:  The prevalence of paediatric CMN based on a European cohort registry of renal tumours (SIOP- RTSG).  The biology of CMN

18.4.3 Treatment recommendations

* Before starting chemotherapy for 28 weeks discuss with the National PI!

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Surgery For surgical recommendations, see chapter 16.3.4 Complete nephrectomy is the treatment of choice in localized disease. The perirenal fat should be removed, as CMN tends to infiltrate in the surrounding tissue. Re-resection should be performed in case of incomplete tumour resection or incomplete removal of the perirenal fat. Metastectomy is advised in exceptional cases with solitary metastasis.

Chemotherapy Pre-operative chemotherapy: < 7 months: immediate surgery > 7 months: pre-operative chemotherapy (actinomycin-D and vincristine) Post-operative chemotherapy (Do always contact the National PI before starting chemotherapy): In case of progression under treatment with actinomycin-D and vincristine, second line treatment with ICE (see treatment schedule below) is recommended. Other possible drugs are cyclophosphamide and doxorubicin.

Dosing ICE chemotherapy: Drugs Administration Dosing

Ifosfamide IV over 2 hours 66.7 mg/kg/day for infants < 12 months 2,000 mg/m2 for children ≥ 12 months Carboplatin IV over GFR Dose 1 hour > 150 560 mg/m2 2 mL/min/1.73m (18.7 mg/kg for infants) 100-150 500 mg/m2 2 mL/min/1.73m (16.6 mg/kg for infants) 75-99 370 mg/m2 2 mL/min/1.73m (12.3 mg/kg for infants) 50-74 290 mg/m2 2 mL/min/1.73m (9.7 mg/kg for infants) 30-49 200 mg/m2 (6.7 mg/kg for infants) mL/min/1.73m2 <30 Hold carboplatin mL/min/1.73m2 Etoposide IV 3.3 mg/kg/day for infants < 12 mo. over 1 hour 100 mg/m2/day for children ≥ 12 mo.

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18.4.4 Follow-up guideline for surveillance The follow-up surveillance should be done according to section 11.6.5.

18.4.5 Treatment recommendations of relapsed CMN Local relapse / metastatic relapse: 1. Surgery if possible 2. No complete surgical resection:  Chemotherapy naïve patients: combination of vincristine, dactinomycin  Chemotherapy non-naïve patients: ifosfamide, carboplatin, etoposide (ICE) +/- radiotherapy, or treatment with combinations of doxorubicin and cyclophosphamide.

18.4.6 References 1. England, R.J., et al., Mesoblastic nephroma: a report of the United Kingdom Children's Cancer and Leukaemia Group (CCLG). Pediatr Blood Cancer, 2011. 56(5): p. 744-8. 2. van den Heuvel-Eibrink, M.M., et al., Characteristics and survival of 750 children diagnosed with a renal tumor in the first seven months of life: A collaborative study by the SIOP/GPOH/SFOP, NWTSG, and UKCCSG Wilms tumor study groups. Pediatr Blood Cancer., 2008. 50(6): p. 1130- 1134. 3. Furtwaengler, R., et al., Mesoblastic nephroma--a report from the Gesellschaft fur Padiatrische Onkologie und Hamatologie (GPOH). Cancer, 2006. 106(10): p. 2275-83. 4. Howell, C.G., et al., Therapy and outcome in 51 children with mesoblastic nephroma: A report of the National Wilms' Tumor Study. J Pediatr Surg, 1982. 17: p. 826-831. 5. Kelner, M., et al., The vascular "ring" sign in mesoblastic nephroma: report of two cases. Pediatr Radiol, 2003. 33(2): p. 123-8. 6. Kotani, T., et al., Elevated levels of aldosterone in the amniotic fluid in two cases of congenital mesoblastic nephroma. Ultrasound Obstet Gynecol, 2010. 36(2): p. 256-8. 7. Portugal, R. and H. Barroca, Clear cell sarcoma, cellular mesoblastic nephroma and metanephric adenoma: cytological features and differential diagnosis with Wilms tumour. Cytopathology, 2008. 19(2): p. 80-5. 8. Bayindir, P., et al., Cellular mesoblastic nephroma (infantile renal fibrosarcoma): institutional review of the clinical, diagnostic imaging, and pathologic features of a distinctive neoplasm of infancy. Pediatr Radiol, 2009. 39(10): p. 1066-74. 9. Joshi, V.V., J. Kasznica, and T.R. Walters, Atypical mesoblastic nephroma. Pathologic characterization of a potentially aggressive variant of conventional congenital mesoblastic nephroma. Arch Pathol Lab Med, 1986. 110(2): p. 100-106. 10. Ali, A.A., et al., Congenital mesoblastic nephroma with metastasis to the brain: a case report. Am J Pediatr Hematol Oncol, 1994. 16(4): p. 361-364. 11. Heidelberger, K.P., et al., Congenital mesoblastic nephroma metastatic to the brain. Cancer, 1993. 72(8): p. 2499-2502. 12. Pettinato, G., et al., Classical and cellular (atypical) congenital mesoblastic nephroma: a clinicopathologic, ultrastructural, immunohistochemical, and flow cytometric study. Human Pathol, 1989. 20(7): p. 682-690.

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13. Knezevich, S.R., et al., ETV6-NTRK3 gene fusions and trisomy 11 establish a histogenetic link between mesoblastic nephroma and congenital fibrosarcoma. Cancer Res, 1998. 58(22): p. 5046- 5048. 14. Knezevich, S.R., et al., A novel ETV6-NTRK3 gene fusion in congenital fibrosarcoma. Nat Genet, 1998. 18(2): p. 184-187. 15. Rubin, B.P., et al., Congenital mesoblastic nephroma t(12;15) is associated with ETV6-NTRK3 gene fusion: cytogenetic and molecular relationship to congenital (infantile) fibrosarcoma. Am J Pathol, 1998. 153(5): p. 1451-1458. 16. Wai, D.H., et al., The ETV6-NTRK3 gene fusion encodes a chimeric protein tyrosine kinase that transforms NIH3T3 cells. Oncogene, 2000. 19(7): p. 906-915. 17. Gadd, S., et al., Mediators of Receptor Tyrosine Kinase activation in infantile fibrosarcoma: a Children's Oncology Group study. J Pathol, 2012. 228: p. 119-130. 18. Watanabe, N., et al., Duplication of the paternal IGF2 allele in trisomy 11 and elevated expression levels of IGF2 mRNA in congenital mesoblastic nephroma of the cellular or mixed type. Genes Chromosomes Cancer, 2007. 46(10): p. 929-35. 19. Chan, H.S., et al., Congenital mesoblastic nephroma: a clinicoradiologic study of 17 cases representing the pathologic spectrum of the disease. J Pediatr 1987. 111(1): p. 64-70. 20. Howell, C.G., et al., Therapy and outcome in 51 children with mesoblastic nephroma: a report of the National Wilms' Tumor Study. J Pediatr Surg, 1982. 17(6): p. 826-31. 21. Sandstedt, B., et al., Mesoblastic nephromas: a study of 29 tumours from the SIOP nephroblastoma file. Histopathology, 1985. 9(7): p. 741-50. 22. Chaudry, G., et al., Imaging of congenital mesoblastic nephroma with pathological correlation. Pediatr Radiol, 2009. 39(10): p. 1080-6. 23. Loeb, D.M., D.A. Hill, and J.S. Dome, Complete response of recurrent cellular congenital mesoblastic nephroma to chemotherapy. J Pediatr Hematol Oncol, 2002. 24(6): p. 478-481. 24. Grier, H.E., A.R. Perez-Atayde, and H.J. Weinstein, Chemotherapy for inoperable infantile fibrosarcoma. Cancer, 1985. 56(7): p. 1507-10. 25. Kurkchubasche, A.G., et al., The role of preoperative chemotherapy in the treatment of infantile fibrosarcoma. J Pediatr Surg, 2000. 35(6): p. 880-3. 26. Orbach, D., et al., Infantile fibrosarcoma: management based on the European experience. J Clin Oncol, 2010. 28(2): p. 318-23.

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18.4.7 Chairs and members of the CMN Panel

Name Profession Country Email

Chair Marry van den Oncologist The m.m.vandenheuvel- Heuvel-Eibrink Netherlands [email protected]

Tomás Acha Oncologist Spain [email protected]

Christophe Bergeron Oncologist France [email protected]

Beatriz de Camargo Oncologist Brazil [email protected]

Rhoikos Furtwängler Oncologist Germany [email protected]

Jan Godzinski Surgeon Poland [email protected]

The Martine van Groet Oncologist [email protected] Netherlands

Norbert Graf Oncologist Germany [email protected]

Ivo Leuschner Pathologist Germany [email protected]

Kathy Pritchard-Jones Oncologist UK [email protected]

Members Sara Stoneham Oncologist UK [email protected]

The Lieve Tytgat Oncologist [email protected] Netherlands

The Harm van Tinteren Statistician [email protected] Netherlands

Gordan Vujanic Pathologist UK [email protected]

Andrea Di Cataldo Oncologist Italy [email protected]

Estelle Thebaud Oncologist France [email protected]

NN Radiologist

Christian Rübe Radiotherapist Germany [email protected]

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18.5 Other Kidney Tumours Other non-WT kidney tumours include soft tissue sarcomas/PNET, neuroblastoma of the kidney, non- Hodgkin lymphoma (primary site in the kidney), teratoma and angiomyolipom. Together, they account for less than 2% of all primary renal tumours in children. As these are all very rare renal tumours, it is advised to contact the national coordinator in such rare cases.

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18.6 Follow-up and late effects Despite being associated with fewer late effects compared to most other childhood malignancies, renal tumour survivors still have an increased risk of severe chronic and life-threatening health conditions in adult life when compared to the general population [1,9]. Most frequently the late sequelae include secondary malignancies, renal, cardiac, pulmonary and gonadal dysfunction, musculoskeletal abnormalities, impaired fertility and hypertension [10]. However, studies on long-term outcomes of WT survivors, as summarised below, are mainly based on patient cohorts treated in the 1980/90s. Since then, overall treatment intensity has been reduced and refined leading to a lower likelihood of long- term sequelae.

Mortality: Comparing observed with expected number of deaths (<2%) 30 years after diagnosis indicates a 4-5 times increased mortality risk, with the main causes being secondary malignancies, and cardiac and pulmonary diseases. Furthermore, approximately 20-25% of survivors are found to have a severe chronic health condition [2, 10-12].

Secondary malignancies: The cumulative incidence of secondary malignancies is estimated as 0.5-1% and 2-3%, at 10 and 30 years after diagnosis, respectively. The type of secondary malignancies varies (sarcomas, breast cancer, lymphomas, etc.) but as the majority of these cancers occur within the radiotherapy field, radiotherapy is the main contributing risk factor. Doxorubicin may further potentiate the adverse effects related to radiotherapy due to the drug’s radiosensitisation of cells [1,2,13]. For surveillance of breast cancer in pulmonary irradiated renal tumour survivors, it is advised to follow the recommendations of the IGHG group [14].

Cardiotoxicity: Doxorubicin increases the risk of congestive heart failure, with an overall cumulative risk of about 5% at 20 years after treatment. The risk is related to the cumulative dose, and many guidelines recommend that survivors who received ≥ 250 mg/m2 should undergo long-term cardiac surveillance. Cardiotoxicity risk, as well as the occurrence of heart valve disease, is potentiated by concurrent use of radiotherapy (left flank, whole abdominal or pulmonary RT) and both females and very young patients seem to be more susceptible [2,15]. For cardiac surveillance it is advised to follow the recommendations of the IGHG group [16].

Renal function: End stage renal failure is reported in 1% of unilateral WT and about 10% of patients with bilateral disease after 20 years of follow-up. Irradiation to the remaining kidney and the use of high risk chemotherapy (e.g. ifosfamide and carboplatin) may further increase the risk of end stage renal failure [17, 18].

Musculoskeletal function: Radiotherapy reduces the growth of normal tissue with severity depending on the dose, radiation field and the patient’s age. The younger the patient, the more severe the developmental abnormalities become. Abnormal growth of the spinal column and ribs, breast hypoplasia and impaired muscle development of the torso are the most commonly observed sequelae [1].

Chronic lung disease: Approximately 5% of WT patients treated with lung RT develop pulmonary disease within 15 years of treatment, with the majority classified as pulmonary fibrosis or unspecified lung disease. Chronic lung disease occurs infrequently in patients not receiving radiotherapy [1,19].

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Ototoxicity: It is advised to perform audiological screening in children that have been exposed to carboplatin and cisplatin, and for the few patients that have received radiotherapy to the head and neck region, after discontinuation of therapy [20].

Gonadal impairment: Gonadal impairment has been documented after whole body irradiation, alkylating agents and high dose chemotherapy. For surveillance of gonadal impairment in female renal tumour survivors, it is advised to follow the recommendations of the IGHG group [21,22]. The IGHG guideline for male survivors will be launched shortly.

18.6.1 References 1. Wright KD, et al. Late effects of treatment for wilms tumor. Pediatr Hematol Oncol 2009;26:407–13. 2. Levit AL and Green DM. Late effects and QOL, Renal Tumors of Childhood (p229-p243) Biology and Therapyy | Kathy Pritchard-Jones | Springer 2015 3. Curry HL, et al. Caring for survivors of childhood cancers: The size of the problem. Eur J Cancer. 2006;42:501-8. 4. Diller L, et al. Chronic disease in the Childhood Cancer Survivor Study cohort: A review of published findings. J Clin Oncol. 2009;27:2339-55. 5. Oeffinger KC, et al. Chronic health conditions in adult survivors of childhood cancer. N Engl J Med. 2006;355:1572-82. 6. Hudson MM, et al. Clinical ascertainment of health outcomes among adults treated for childhood cancer. JAMA. 2013;309:2371-81. 7. Skinner R, et al. Long-term follow-up of children treated for cancer: why is it necessary, by whom, where and how? Arch Dis Child. 2007;92:257-60. 8. Skinner R, et al. Long-term follow-up of people who have survived cancer during childhood. Lancet Oncol. 2006;7:489-98. 9. Armenian SH, et al. Strategies to Prevent Anthracycline-Related Congestive Heart Failure in Survivors of Childhood Cancer. Cardiol Res Pract. 2012;Article ID 713294. 10. Termuhlen AM, et al. Twenty-five year follow-up of childhood Wilms tumor: a report from the Childhood Cancer Survivor Study. Pediatr Blood Cancer 2011;57:1210–6. 11. Cotton CA, et al. Early and late mortality after diagnosis of Wilms tumor. J Clin Oncol. 2009 10;27(8):1304-9. 12. Amstrong GT, et al. Late mortality among 5-year survivors of childhood cancer. J Clin Oncol. 2009:10;27(14):2328-38. 13. Lee JS, et al. Second malignant neoplasms among children, adolescents and young adults with Wilms tumor. Pediatr Blood Cancer 2015;62:1259–64. 14. Mulder RL, et al. International Late Effects of Childhood Cancer Guideline Harmonization Group. Recommendations for breast cancer surveillance for female survivors of childhood, adolescent, and young adult cancer given chest radiation: a report from the International Late Effects of Childhood Cancer Guideline Harmonization Group. Lancet Oncol. 2013 ;14(13):e621-9. 15. Green DM, et al. Congestive heart failure after treatment for Wilms’ tumor: a report from the National Wilms' Tumor Study group. J Clin Oncol 2001;19:1926–34. 16. Armenian SH, et al. International Late Effects of Childhood Cancer Guideline Harmonization Group. Recommendations for cardiomyopathy surveillance for survivors of childhood cancer: a report from the International Late Effects of Childhood Cancer Guideline Harmonization Group. Lancet Oncol. 2015;16(3):e123-36.

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17. Breslow NE, et al. End stage renal disease in patients with Wilms tumor: results from the National Wilms Tumor Study Group and the United States Renal Data System. J Urol. 2005;174(5): 1972-5. 18. Dekkers IA, et al. Long-term nephrotoxicity in adult survivors of childhood cancer. Clin J Am Soc Nephrol. 2013;8(6):922-9 19. Green DM, et al. Pulmonary disease after treatment for Wilms tumor: a report from the national wilms tumor long-term follow-up study. Pediatr Blood Cancer. 2013;60(10):1721-6. 20. Lee JW,et al. Clinical practice guidelines for the management and prevention of cisplatin-induced hearing loss. Therapeutic Drug Monitoring 2016 (in press). 21. Marjolein van Waas, et al. Treatment factors rather than genetic variation determine metabolic syndrome in childhood cancer survivors. Eur J Cancer, 2013;49(3):668-75. 22. Wendy van Dorp, et al. Recommendations for Premature Ovarian Insufficiency Surveillance for Female Childhood, Adolescent and Young Adult Cancer Survivors: A Report from the International Late Effects of Childhood Cancer Guideline Harmonization Group, JCO, 2016 (in press).

18.6.2 Chairs and members of the Late Effects Group

Name Profession Country Email

Chair Annelies Mavinkurve- Oncologist The A.M.C.Mavinkurve- Groothuis Netherlands [email protected]

Dennis Ku Oncologist China [email protected]

Monica Cypriano Oncologist Brazil [email protected]

Leo Kager Oncologist Austria [email protected]

Patrick Melchior Radiotherapist Germany [email protected]

Marry van den Heuvel- The m.m.vandenheuvel- Oncologist

Eibrink Netherlands [email protected] Members Monica Terenziani Oncologist Italy [email protected]

Catriona Duncan Oncologist UK [email protected]

Helene Sudour Oncologist France [email protected]

Carlota Calvo Oncologist Spain [email protected]

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19 Appendices 19.1 Appendix 1: Contract form for participation in the SIOP 2016 UMBRELLA protocol Every hospital that wants to participate in the SIOP 2016 UMBRELLA protocol needs to be a member of a National Group registered in SIOP-RTSG or - in the absence of a National Group - a single centre adhering to the ‘Structures and Standards’ given by the SIOP-RTSG (Appendix 8). Each participating centre needs to register every patient with a renal tumour and provide information about responsible persons (paediatric oncologist, radiologist, pathologist, radiotherapist, surgeon) dealing with these patients and all of them have to sign that they do adhere to the UMBRELLA protocol and that they provide all requested material (imaging, pathology, biomaterial) and data.

Minimal requirements for participating as a partner institution are 1. Providing full data sets by registration in ALEA/ObTiMA 2. Organised pathology review on a national / regional level 3. Organised radiology review (national / regional level) 4. Abdominal MRI (alternatively CT abdomen) and CT scan of the chest as a standard diagnostic approach for each child and for assessment of response in patients with metastatic tumours Countries that can only meet criteria 1 and 2, are welcome to register patients and to use the therapeutic guidelines. For each patient, quality criteria are collected for further analysis. Central pathology review is mandatory for inclusion in any kind of analysis. Further analyses will not be possible due to selection bias if a central radiology review of images is not done.

The requested form is given on the next page:

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Participation in the UMBRELLA protocol Centre Name Participant of the Address / Country following National Telephone / Fax Group: Email Herewith our centre confirms by signature that 4. We adhere to all regulations of the protocol 5. All patients with a renal tumour diagnosed at our centre will be enrolled in the UMBRELLA protocol if the patient or the parents or the legal representative provides informed consent 6. All requested baseline and serial information and data, imaging studies, biomaterial and pathological material will be provided from all patients National Principal Investigator Signature Name Address / Country Telephone / Fax Email Responsible Oncologist Signature Name Address / Country Telephone / Fax Email Responsible Radiologist Signature Name Address / Country Telephone / Fax Email Responsible Surgeon Signature Name Address / Country Telephone / Fax Email Responsible Pathologist Signature Name Address / Country Telephone / Fax Email Responsible Radiotherapist Signature Name Address / Country Telephone / Fax Email Responsible Biologist or external Signature biobanking contact Name Address / Country Telephone / Fax Email Responsible Data Manager Signature Name Address / Country Telephone / Fax Email

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19.2 Appendix 2: Information and Consent forms All documents provided in this Appendix are templates using the English language. All participatring countries need to translate these documents into their own language and provide these documentas as an extra document for ethical approval in their country.

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19.2.1 Parent / Guardian Information Sheet UMBRELLA SIOP 2016 study A prospective clinical study by the International Society of Paediatric Oncology Renal Tumour Study Group (SIOP-RTSG)

National Chief Investigator:

Name of your doctor:

PARENT / GUARDIAN INFORMATION SHEET

(Version 1.0, 30th December 2015)

Your child has been diagnosed as having a kidney (renal) tumour. This means a growth or lump in the kidney. Nine out of ten renal tumours in children are Wilms tumours (also called nephroblastoma). A few tumours will be of a different type and need different treatment. The precise treatment your child will need depends on the type of tumour and how far it has spread. Treatment is usually very successful. We would like to ask you to allow your child to take part in an international clinical research study called UMBRELLA Protocol SIOP 2016. Taking part in this study will not affect your child’s treatment, which will be the best current standard of care. We are asking your permission to collect clinical information about your child’s diagnosis and treatment and how the tumour responds to treatment and to include this in an international database. We are also asking your permission to collect and use samples of your child’s tumour, blood and urine at diagnosis and during treatment, which are left over after the necessary tests have been done to guide treatment. These will be used in the linked laboratory studies that look at changes in the genetic material (DNA) and proteins of the tumour and in the blood and urine. This important biological information will be put together with the clinical information (including any CT or MRI scans your child may have as part of standard treatment). All information we collect will be made anonymous (coded) so that your child cannot be personally identified. We aim to collect information on a few thousand children and adolescents with kidney tumours across Europe and beyond. The data will be used to develop new biomarkers that help to find new and better treatment approaches. Taking part in this study is entirely voluntary. Before you decide, it is important for you to get information about the disease and treatment and to understand, why the research is being done and what it will involve. Please take time to read the following information carefully and discuss it with your doctor and others if you wish. Please ask your doctor if there is anything that is not clear or if you would like more information. Thank you for taking the time to read this information leaflet.

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1. What is a kidney (renal) tumour? Kidney or renal tumours are rare cancers in childhood. Wilms tumour or nephroblastoma is the most common of these tumours. Approximately one in ten thousand children get Wilms tumour, usually in only one kidney (unilateral), although occasionally there may be tumours in both kidneys (bilateral disease). A few children with a suspected Wilms tumour turn out to have another type of kidney tumour once the pathologist has looked at a piece of the tumour down the microscope. Some of these tumours are treated almost the same as Wilms tumour, but some require different treatments. Your doctor will tell you if they think your child do not have a typical Wilms tumour. Such tumours are Clear Cell Sarcoma, Mesoblastic Nephroma, Rhabdoid Tumour, Renal Cell Carcinoma and a few others. In most cases we do not know what causes kidney tumours. In Wilms tumour a small number of children have a faulty gene. This is usually obvious, either because other members of the family have had Wilms tumour or because your child have had certain growth or development problems that have been present from birth. If this is the case, your doctor will discuss it with you. However, please note that for the majority of children with kidney or renal tumours, there is no such faulty gene nor is there any increased risk in your family.

2. What is the best treatment for Wilms tumour? Wilms tumour can be treated very successfully using a combination of chemotherapy (drugs), surgery (an operation) and, sometimes, radiotherapy (X-rays). About 90% of children can be cured of their Wilms tumour using a treatment without much risk of long-term side effects. The length and intensity of treatment depends on what the tumour looks like under a microscope (‘histology’) and something called tumour ‘stage’ – this is a measure of whether the tumour is only in the kidney or if it has spread beyond the kidney. Tumour stage can only be decided finally after an operation to remove the affected kidney with the tumour.  Stage I is when the tumour is only in the kidney and is completely removed by an operation.  Stage II is when the tumour has broken through the kidney lining but is still completely removed.  Stage III is when the tumour is left behind, either because it has spread to lymph nodes in the abdomen or splits open before or during the operation or because it is not possible to remove it completely.  Stage IV is when the tumour has spread to other parts of the body, usually the lungs.  Stage V is the special case of tumours in both kidneys (bilateral tumours) There are two main ways to start treatment for kidney tumours that have been in use around the world for over 30 years. The approach taken by the International Society of Paediatric Oncology (SIOP) is to give chemotherapy first to shrink the tumour and reduce the risk of it rupturing during the operation, then to remove it after 4 – 6 weeks. The other approach, used in North America, is to do the operation to remove the tumour first. In UMBRELLA, we use the SIOP approach of starting chemotherapy before surgery, as it leads to more tumours being of low stage, requiring less treatment after surgery. 3. What is the purpose of the UMBRELLA SIOP 2016 study? The study will provide information on the outcome of patients treated according to the best available treatment for children with all renal tumours. It is asking two main questions: 5. How do changes seen in the genetic material (DNA) and proteins of kidney tumours and in the

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blood and urine of the patients predict their response to treatment? 6. Will more accurate imaging of the tumour before chemotherapy starts and just before surgery is done, help doctors to decide how much treatment is needed for the different sub-types of Wilms tumour and other renal tumours of childhood? To answer these biological questions, we would like to store a small sample of your child’s urine, blood and tumour (1 - 2 teaspoonfuls or sugar lump sized) for use in laboratory research. Each sample is stored with a coded number so that the researcher is not given any personal identifiable information about your child.

4. Why has my child been asked to take part in this study? Because your child has been diagnosed with a renal (kidney) tumour.

5. Does my child have to take part? It is up to you and your child whether or not to take part. If you do decide to take part, you will be given these information sheets to keep and asked to sign a consent form. You are free to withdraw your consent at any time without having to give a reason. This would not affect the standard of your child’s care or the relationship with your child’s doctor. We would like to obtain a complete set of information about each child. However, if you wish, you can just give permission for registration of clinical information about your child (including copies of their imaging (CT/MRI) scans) to be used in the study without having to agree to samples of your child's blood, urine and normal kidney/tumour collected and used for the laboratory studies. All information will be registered in a coded way according to ethical rules and regulations.

6. What will happen to my child if we take part? With your permission, tissue samples from your child that are left over after normal tests, will be analysed for changes in the tumour’s genetic material (DNA) and protein. We will ask for a blood and urine sample at five timepoints: at diagnosis, during initial chemotherapy (week 2), before the main operation to remove the tumour, immediately afterwards and once again at the end of post-operative treatment. We will only ask for these samples at routine visits with your doctor and they will be taken at the same time as your child is having blood taken to monitor their chemotherapy. We will not ask for additional visits and there will not be any additional blood tests. The extra volume of blood taken for research at each timepoint is only 2-5 ml. This is less than half the volume taken at the same time for routine tests. In total, the maximum blood volume taken for research over a period of several months would be less than 25 ml depending on the age and size of your child. In addition we would like to take also few milliliters of blood from you as the parents to compare this with the results from your child. The laboratory information will be put together with the clinical information (including any CT or MRI scans your child may have) to see if this approach to use integrated data could help doctors plan treatment that is better designed for each individual child in the future. At the end of the study, if you agree, we would like to keep your child’s samples, including any extracted DNA and protein, to use in future research into childhood renal tumours or other cancers. Approved researchers, whose projects had undergone independent scientific and ethical review, would only use these samples and your child would not be identified to them.

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7. What are the side effects? There are no side effects from taking part in this study, as it does not affect your child’s treatment. Your doctor, who explains the details of your child’s treatment and its possible side effects, will give you a separate leaflet about the treatment of your child.

8. Will there be any inconveniences? There should not be any inconveniences resulting from taking part. The blood samples (2-5 ml) are taken at the same time as your child needs other blood tests so there will be no extra procedures involved. The urine samples are only taken if it is easy for them to be collected.

9. What are the possible benefits of taking part? There will be no direct benefit to you/your child but by allowing your child’s clinical details and their blood, urine and tumour to be studied in this way, we hope we will learn more about why some tumours respond better to treatment than others. This information should help doctors to improve treatment for children with Wilms and other renal tumours in the future.

10. What if something goes wrong? It is extremely unlikely that anything will go wrong, as this study does not affect your child’s treatment. If taking part in this research project causes a problem for your child, there are no special compensation arrangements. If your child is harmed due to someone’s negligence, then you may have grounds for legal action. Regardless of this, if you wish to complain, or have any concerns about any aspect of the way you have been approached or treated during the course of this study, your treating physician or your National Principal Investigator of the UMBRELLA SIOP 2016 study can be approached at any time to solve the problem.

11. If my child takes part in this study will his/her details remain confidential? Your child’s personal details (name, date of birth (DOB), local hospital record number) will be collected by your doctor and sent online to the National Coordinating office for this study in each country. The records for each child are then allocated a unique code number. This unique code number is used when information is given to the researchers and they will be unable to access any personally identifiable information. The original information provided by your doctor will be stored securely at the National Coordinating office only in line with national Data Protection legislation. All the data collected about your child, identified only by the unique code number, and including the results of tumour DNA testing, will be entered into a clinical research database. It will not be possible for researchers using data from this database to identify any child individually. Coded data about your child may also be combined with similar data on children with renal tumours treated by other research groups, for example in North America, so that we can learn more about the best treatment approaches for children diagnosed with renal tumours in the future. With your consent, we will inform your family doctor about your child’s participation in this study. Authorised professionals other than those involved directly in your child’s care may inspect your child’s medical notes in order to collect information for the study. Unrelated to this study, details of your child’s diagnosis will be made available by your child’s hospital as part of national cancer registration procedures. This information is used mainly for epidemiological research and to describe the overall anonymised outcome for children with cancer. You will be asked to

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give consent for your child’s information to be transferred to the National Cancer Registry, and to be used in this way.

12. What will happen to the results of the research studyy? The information collected will be stored and analysed in a central and international database that is located at the Netherlands Cancer Institute in Amsterdam. The data will be used to design computer- based tools that help to predict how individual tumours will behave. We believe this new approach will help future clinical decision-making. The results of the study will be published in medical and scientific journals and also be presented at international conferences. Your child would not be identified in any publication.

13. Who is organising and funding the research? This research is being organised by experts from a number of countries throughout Europe and beyond, who have considerable experience in the treatment of kidney cancer in children and adolescents (International Paediatric Oncology Society (SIOP) Renal Tumours Study Group). The study is coordinated in Germany by Professor Norbert Graf, Saarland University chair of the SIOP-Renal Tumour Study Group and Marry van den Heuvel-Eibrink, co-chair. This study has been approved by the Ethical Committee of the ‘Ärztekammer des Saarlandes’ in Germany as well as the Ethical Committees of participating centers. The project is funded by different National funding organizations. If you have any concerns or other questions about this study or the way it has been carried out, you should contact the principal investigator responsible for the registry at your hospital. Details are provided below: Local Hospital Contact Details

Hospital Name

Contact Name

Phone:

☐ I am thoroughly informed about the disease of my child and the UMBRELLA SIOP 2016 study. I could ask all my questions and do not have any further questions.

______Place, date, signature (mother) Place, date, signature (father)

______Place, date, signature (child, if > 10 years) Name of the child, birthdate

______Place, date, name, signature Place, date, name, signature Local Principal Investigator Witness

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19.2.2 Information sheet for young adults To be printed on Institution Header Paper

UMBRELLA SIOP 2016 study A prospective clinical study by the International Society of Paediatric Oncology Renal Tumour Study Group (SIOP-RTSG)

National Chief Investigator:

Name of your doctor:

INFORMATION SHEET FOR YOUNG ADULTS (Version 1.0, 30th December 2015)

You have been diagnosed as having a kidney (renal) tumour. This means a growth or lump in the kidney. Nine out of ten renal tumours in children are Wilms tumours (also called nephroblastoma). In young adults a Wilms tumour is rare and should therefore be treated as in childhood. Some tumours will be of a different type and need different treatment. The precise treatment you will need depends on the type of tumour that you have and how far it has spread. Treatment is usually very successful. We would like to ask you to take part in an international clinical research study called UMBRELLA Protocol SIOP 2016. Taking part in this study will not affect your treatment, which will be the best current standard of care. We are asking your permission to collect clinical information about your diagnosis and treatment and how the tumour responds to treatment and to include this in an international database. We are also asking your permission to collect and use samples of your tumour, blood and urine at diagnosis and during treatment, which are left over after the necessary tests have been done to guide treatment. These will be used in the linked laboratory studies that look at changes in the genetic material (DNA) and proteins of the tumour and in the blood and urine. This important biological information will be put together with the clinical information (including any CT or MRI scans you may have as part of standard treatment). All information we collect will be made anonymous (coded) so that you cannot be personally identified. We aim to collect information on a few thousand patients with these kidney tumours across Europe and beyond. The data will be used to develop new biomarkers that help to find new and better treatment approaches. Taking part in this study is entirely voluntary. Before you decide, it is important for you to have information about the disease and treatment as well as to understand why the research is being done and what it will involve. Please take time to read the following information carefully and discuss it with your doctor and others if you wish. Please ask your doctor if there is anything that is not clear or if you would like more information. Thank you for taking the time to read this information leaflet.

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UMBRELLA Protocol SIOP 2016

1. What is a kidney (renal) tumour? Kidney or renal tumours are rare cancers in childhood. Wilms tumour or nephroblastoma is the most common of these tumours in childhood but rare in young adults. Approximately one in ten thousand children get Wilms tumour, usually only in one kidney (unilateral), although occasionally there may be tumours in both kidneys (bilateral disease). A few children with a suspected Wilms tumour turn out to have another type of kidney tumour once the pathologist has looked at a piece of the tumour down the microscope. Some of these tumours are treated almost the same as Wilms tumour, but some require different treatments. Your doctor will tell you if they think you do not have a typical Wilms tumour. Such tumours are Clear Cell Sarcoma, Mesoblastic Nephroma, Rhabdoid Tumour, Renal Cell Carcinoma and a few others. In most cases we do not know what causes kidney tumours. In Wilms tumour a small number of patients have a faulty gene. This is usually obvious, either because other members of the family have had Wilms tumour or because you have had certain growth or development problems that have been present from birth. If this is the case, your doctor will discuss it with you. However, please note that for the majority of patients with kidney or renal tumours, there is no such faulty gene nor is there any increased risk in your family.

2. What is the best treatment for Wilms tumour? Wilms tumour can be treated very successfully using a combination of chemotherapy (drugs), surgery (an operation) and, sometimes, radiotherapy (X-rays). About 90% of patients can be cured of their Wilms tumour using a treatment without much risk of long-term side effects. The length and intensity of treatment depends on what the tumour looks like under a microscope (‘histology’) and something called tumour ‘stage’ – this is a measure of whether the tumour is only in the kidney or if it has spread beyond the kidney. Tumour stage can only be decided finally after an operation to remove the affected kidney with the tumour.  Stage I is when the tumour is only in the kidney and is completely removed by an operation.  Stage II is when the tumour has broken through the kidney lining but is still completely removed.  Stage III is when the tumour is left behind, either because it has spread to lymph nodes in the abdomen or splits open before or during the operation or because it is not possible to remove it completely.  Stage IV is when the tumour has spread to other parts of the body, usually the lungs.  Stage V is the special case of tumours in both kidneys (bilateral tumours) There are two main ways to start treatment for kidney tumours and these have been in use around the world for over 30 years. The approach taken by the International Society of Paediatric Oncology (SIOP) is to give chemotherapy first to shrink the tumour and reduce the risk of it rupturing during the operation, then to remove it after 4 – 6 weeks. The other approach, used in North America, is to do the operation to remove the tumour first. In UMBRELLA, we use the SIOP approach of starting chemotherapy before surgery, as it leads to more tumours being of low stage, requiring less treatment after surgery. As Wilms tumour is rare in young adults, most young adults with this tumour are primarily operated to confirm the diagnosis by pathological examination.

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3. What is the purpose of the UMBRELLA SIOP 2016 study? The study will provide information on the outcome of patients treated according to the best available treatment for children and young adults with a renal tumours. It is asking two main questions: 7. How do changes seen in the genetic material (DNA) and proteins of kidney tumours and in the blood and urine of the patients predict their response to treatment? 8. Will more accurate imaging of the tumour before chemotherapy starts and just before surgery is done, help doctors to decide how much treatment is needed for the different sub-types of Wilms tumour and other renal tumours of childhood? To answer these biological questions, we would like to store a small sample of your urine, blood and tumour (1 - 2 teaspoonfuls or sugar lump sized) for use in laboratory research. Each sample is stored with a coded number so that the researcher is not given any personal identifiable information about you.

4. Why have I been asked to take part in this study? Because you have been diagnosed with a renal (kidney) tumour.

5. Do I have to take part? It is up to you whether or not to take part. If you do decide to take part, you will be given these information sheets to keep and asked to sign a consent form. You are free to withdraw your consent at any time without having to give a reason. This would not affect the standard of your care or the relationship with your doctor. We would like to obtain a complete set of information about each patient. However, if you wish, you can just give permission for registration of clinical information about yourself (including copies of your imaging (CT/MRI) scans) to be used in the study without having to agree to samples of your blood, urine and normal kidney/tumour collected and used for the laboratory studies. All information will be registered in a coded way according to ethical rules and regulations.

6. What will happen to me if I take part? With your permission, tissue samples from you that are left over after normal tests, will be analysed for changes in the tumour’s genetic material (DNA) and protein. We will ask for a blood and urine sample at five timepoints: at diagnosis, during initial chemotherapy (week 2), before the main operation to remove the tumour, immediately afterwards and once again at the end of post-operative treatment. We will only ask for these samples at routine visits with your doctor and they will be taken at the same time as you having blood taken to monitor your chemotherapy. We will not ask for additional visits and there will not be any additional blood tests. The extra volume of blood taken for research at each timepoint is only 5 ml. This is less than half the volume taken at the same time for routine tests. In total, the maximum blood volume taken for research over a period of several months would be less than 50 ml. In addition we would like to take also few milliliter of blood from your parents to compare this with the results from you. The laboratory information will be put together with the clinical information (including any CT or MRI scans you may have) to see if this approach to use integrated data could help doctors plan treatment that is better designed for each individual patient in the future.

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At the end of the study, if you agree, we would like to keep your samples, including any extracted DNA and protein, to use in future research into childhood renal tumours or other cancers. Approved researchers, whose projects had undergone independent scientific and ethical review, would only use these samples and you would not be identified to them.

7. What are the side effects? There are no side effects from taking part in this study, as it does not affect your treatment. Your doctor, who explains the details of your treatment and its possible side effects, will give you a separate leaflet about the treatment you will receive.

8. Will there be any inconveniences? There should not be any inconveniences resulting from taking part. The blood samples (5 ml) are taken at the same time as you need other blood tests so there will be no extra procedures involved. The urine samples are only taken if it is easy to be collected.

9. What are the possible benefits of taking part? There will be no direct benefit to you but by allowing your clinical details and your blood, urine and tumour to be studied in this way, we hope we will learn more about why some tumours respond better to treatment than others. This information should help doctors to improve treatment for patients with Wilms and other renal tumours in the future.

10. What if something goes wrong? It is extremely unlikely anything will go wrong, as this study does not affect your treatment. If taking part in this research project causes a problem for you, there are no special compensation arrangements. If you are harmed due to someone’s negligence, then you may have grounds for legal action. Regardless of this, if you wish to complain, or have any concerns about any aspect of the way you have been approached or treated during the course of this study, your treating physician or your National Principal Investigator of the UMBRELLA SIOP 2016 study can be approached at any time to solve the problem.

11. If you take part in this study will your details remain confidential? Your personal details (name, date of birth (DOB), local hospital record number) will be collected by your doctor and sent online to the National Coordinating office for this study in each country. The records for each patient are then allocated a unique code number. This unique code number is used when information is given to the researchers and they will be unable to access any personally identifiable information. The original information provided by your doctor will be stored securely at the National Coordinating office only in line with national Data Protection legislation. All the data collected about you, identified only by the unique code number, including the results of tumour DNA testing, will be entered into a clinical research database. It will not be possible for researchers using data from this database to identify any patient individually. Coded data about you may also be combined with similar data on patients with renal tumours treated by other research groups, for example in North America, so that we can learn more about the best treatment approaches for patients diagnosed with renal tumours in the future. With your consent, we will inform your family doctor about your participation in this study. Authorised professionals other than those involved directly in your care may inspect your medical notes in order to collect information for the study.

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UMBRELLA Protocol SIOP 2016

Unrelated to this study, details of your diagnosis will be made available by your hospital as part of national cancer registration procedures. This information is used mainly for epidemiological research and to describe the overall anonymised outcome for patients with cancer. You will be asked to give consent for your information to be transferred to the National Cancer Registry, and to be used in this way.

12. What will happen to the results of the research study? The information collected will be stored in a central and international database that is located at the Netherlands Cancer Institute in Amsterdam and analysed. The data will be used to design computer- based tools that help to predict how individual tumours will behave. We believe this new approach will help future clinical decision-making. The results of the study will be published in medical and scientific journals and also be presented at international conferences. You would not be identified in any publication.

13. Who is organising and funding the research? This research is being organised by experts from a number of countries throughout Europe and beyond, who have considerable experience in the treatment of kidney cancer in children, adolescents and young adults (International Paediatric Oncology Society (SIOP) Renal Tumours Study Group). The study is coordinated in Germany by Professor Norbert Graf, Saarland University chair of the SIOP-Renal Tumour Study Group and Marry van den Heuvel-Eibrink, co-chair. This study has been approved by the Ethical Committee of the ‘Ärztekammer des Saarlandes’ in Germany and by the Ethical Committees of participating centers. The project is funded by different National funding organizations. If you have any concerns or other questions about this study or the way it has been carried out, you should contact the principal investigator responsible for the registry at your hospital. Details are provided below: Local Hospital Contact Details

Hospital Name

Contact Name

Phone:

☐ I am thoroughly informed about the disease of my child and the UMBRELLA SIOP 2016 study. I could ask all my questions and do not have any further questions.

______Place, date, signature (patient) Name of the patient, birthdate

______Place, date, name, signature Place, date, name, signature Local Principal Investigator Witness

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19.2.3 Information Sheet for patients over 14 years and below 18 years To be printed on Institution Header Paper

UMBRELLA SIOP 2016 study A prospective clinical study by the International Society of Paediatric Oncology Renal Tumour Study Group (SIOP-RTSG)

National Chief Investigator:

Name of your doctor:

INFORMATION SHEET FOR PATIENTS AGED 14 YEARS OR OLDER (Version 1.0, 30th December 2015)

You have been diagnosed as having a kidney tumour. Treatment for kidney tumours in children and adolescents is usually very successful. Nine out of ten kidney tumours found in children are Wilms tumours (also called nephroblastoma). A few tumours will be of a different type and require different treatment. We would like to invite you to take part in an international clinical research study called UMBRELLA Protocol SIOP 2016. Taking part in this study will not affect your treatment, which will be the best current standard of care. We are asking your permission to collect clinical information about your diagnosis and treatment and how the tumour responds to treatment and to include this in an international database. We are also asking your permission to collect and use samples of your tumour, blood and urine, which are left over after the necessary tests have been done to guide treatment. These will be used in the linked laboratory studies that look at changes in the genetic material (DNA) and proteins of the tumour and in the blood and urine. This important biological information will be put together with the clinical information (including any CT or MRI scans you may have as part of standard treatment). All information we collect will be made anonymous (coded) so that you cannot be personally identified. We aim to collect information on a few thousand children and adolescents with kidney tumours across Europe and beyond. The data will be used to develop new biomarkers that help to find new and better treatment approaches. Taking part in this study is entirely voluntary. Before you decide, it is important for you to understand why the research is being done and what it will involve. Please take time to read the following information carefully and discuss it with your doctor and others if you wish. Please ask your doctor if there is anything that is not clear or if you would like more information. Thank you for reading this information leaflet.

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UMBRELLA Protocol SIOP 2016

2. What is the best treatment for Wilms tumour? Wilms tumour can be treated very successfully using a combination of chemotherapy (drugs), surgery (an operation) and, sometimes, radiotherapy (X-rays). About 85% of children can be cured of their Wilms tumour using relatively simple treatment without much risk of long-term side effects. The length of treatment and how strong it is depends on what the tumour looks like under a microscope (‘histology’) and something called tumour ‘stage’ – this is a measure of whether the tumour is only in the kidney or if it has spread beyond the kidney. Tumour stage can only be decided finally after an operation to remove the affected kidney with the tumour.  Stage I is when the tumour is only in the kidney and is completely removed by an operation.  Stage II is when the tumour has broken through the kidney lining but is still completely removed.  Stage III is when the tumour is left behind, either because it has spread to lymph nodes in the abdomen or splits open before or during the operation or because it is not possible to remove it completely.  Stage IV is when the tumour has spread to other parts of the body, usually the lungs.  Stage V is the special case of tumours in both kidneys (bilateral tumours) There are two main ways to start treatment for kidney tumours that have been in use around the world for over 30 years. The approach taken by the International Society of Paediatric Oncology (SIOP) is to give chemotherapy first to shrink the tumour and do the operation to remove the kidney after 4 – 8 weeks more safely. The other approach, used in North America, is to do the operation to remove the tumour first. In UMBRELLA, we use the SIOP approach of starting chemotherapy before surgery, as it leads to more tumours being of low stage, requiring less treatment after surgery.

3. What is the purpose of the UMBRELLA SIOP 2016 study? The study is asking two main questions: a) How do changes seen in the genetic material (DNA) and proteins of kidney tumours predict their response to treatment? b) Will more accurate measurement of the tumour using the imaging done before chemotherapy starts and just before surgery is done, help doctors to decide how much treatment is needed for the different sub-types of Wilms tumour and other renal tumours of childhood. To answer these biological questions, we would like to store a small sample of your urine, blood and tumour (1 - 2 teaspoonfuls or sugar lump sized) for use in laboratory research. Each sample is stored with a coded number so that the researcher is not given any personal identifiable information about you.

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4. Why am I asked to take part in this study? Because you have been diagnosed with a renal (kidney) tumour.

5. Do I have to take part? It is up to you and your parents whether or not to take part. If you do decide to take part, you will be given these information sheets to keep and asked to sign a consent form. You are free to withdraw your consent at any time without having to give a reason. This would not affect the standard of your care or the relationship with your doctor. We would like to obtain a complete set of information about each patient. However, if you wish, you can just give permission for clinical information about yourself (including copies of your imaging (CT/MRI) scans) to be used in the study without having to agree to samples of your blood, urine and tumour collected and used for the laboratory studies.

6. What will happen to me if I take part? With your permission, tissue samples from your tumour, that are left over after normal tests, will be analysed for changes in the tumour’s genetic material (DNA) and protein. We will ask for a blood and urine sample at five timepoints: at diagnosis, during initial chemotherapy (week 2), before the main operation to remove the tumour, immediately afterwards and once again at the end of post-operative treatment. We will only ask for these samples at routine visits with your doctor and they will be taken at the same time as you are having blood taken to monitor your chemotherapy. We will not ask for additional visits and there will not be any additional blood tests. The volume of blood taken for research at each timepoint is only 5 ml. This is less than half the volume taken at the same time for routine tests. In total, the maximum blood volume taken for research over a period of several months would be less than 50 ml. The laboratory information will be put together with the clinical information (including any CT or MRI scans you may have) to see if this approach to use integrated data could help doctors plan treatment that is better designed for each individual child in the future. At the end of the study, if you agree, we would like to keep your samples, including any extracted DNA and protein, to use in future research into childhood renal tumours or other cancers. Approved researchers, whose projects had undergone independent scientific and ethical review, would only use these samples and you would not be identified to them.

8. Will there be any inconveniences? There should not be any inconveniences resulting from taking part. The blood samples (5 ml) are taken at the same time, as you need other blood tests. There will be no extra procedure involved.

9. What are the possible benefits of taking part? There will be no direct benefit to you. But by allowing to study your clinical details, blood, urine and tumour, we hope we will learn more about why some tumours respond better to treatment than others.

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This information should help doctors to improve treatment for children with kidney tumours in the future.

10. What if something goes wrong? It is extremely unlikely anything will go wrong, as this study does not affect your treatment. If taking part in this research project causes a problem for you, there are no special compensation arrangements. If you wish to complain, or have any concerns about any aspect of the way you have been approached or treated during the course of this study, your treating physician or your National Principal Investigator of the UMBRELLA SIOP 2016 study can be approached at any time to solve the problem.

11. If I take part in this study will my details remain confidential? Your personal details (name, date of birth (DOB), local hospital record number) will be collected by your doctor and sent online to the National Coordinating office for this study in each country. The records for each patient are then allocated a unique code number. This unique code number is used when information is given to the researchers and they will be unable to access any personally identifiable information. The original information provided by your doctor will be stored securely at the National Coordinating office only in line with national Data Protection legislation. All the data collected about you, identified only by the unique code number, including the results of tumour DNA testing, will be entered into a clinical research database. It will not be possible for researchers using data from this database to identify any patient individually. Coded data about you may also be combined with similar data on children with renal tumours treated by other research groups, for example in North America, so that we can learn more about the best treatment approaches for patients diagnosed with renal tumours in the future. With your consent, we will inform your family doctor about your participation in this study. Authorised professionals, other than those involved directly in your care may inspect your medical notes in order to collect information for the study. Unrelated to this study, details of your diagnosis will be made available by your hospital as part of national cancer registration procedures. This information is used mainly for epidemiological research and to describe the overall anonymised outcome for children with cancer. You will be asked to give consent for your information to be transferred to the National Cancer Registry, and to be used in this way.

12. What will happen to the results of the research study? The information collected will be stored in a central database that is located at the Netherlands Cancer Institute in Amsterdam and analysed. The data will be used to design computer-based tools that help to predict how individual tumours will behave. We believe this new approach will help clinical decision- making. The results of the study will be published in medical and scientific journals and also be presented at international conferences. You would not be identified in any publication.

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This study has been approved by the Ethical Committee of the ‘Ärztekammer des Saarlandes’ in Germany and by the Ethical Committees of participating centers. The project is funded by funded by different National funding organizations.

If you have any concerns or other questions about this study or the way it has been carried out, you should contact the principal investigator responsible for the registry at your hospital. Details are provided below: Local Hospital Contact Details

Hospital Name

Contact Name

Phone:

☐ I am thoroughly informed about the disease of my child and the UMBRELLA SIOP 2016 study. I could ask all my questions and do not have any further questions.

______Place, date, signature (patient) Name of the patient, birthdate

______Place, date, name, signature Place, date, name, signature Local Principal Investigator Witness

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19.2.4 Information Sheet for children aged 8 – 14 years To be printed on Institution Header Paper

(Version Number 1 – December 2015)

Your doctors think you have a lump or tumour in your kidney. The majority of kidney tumours in children are Wilms tumours (also called nephroblastoma). A few tumours will be of a different type and need slightly different treatment. Most children get better with a mixture of treatments including medicine we call chemotherapy and an operation to remove the kidney with the lump in it. Some children also need a treatment we call radiotherapy, which is just like having a chest X-ray done except you have to come to hospital every day for a few weeks. We would like to ask you to take part in an international clinical research study called ‘UMBRELLA SIOP 2016’ (SIOP is the International Society of Paediatric Oncologists or childhood cancer doctors). This registry is trying to find better risk factors of kidney tumours by analysing genes and proteins in your tumour, blood and urine. This will help us to find even better and safer treatments for kidney tumours in children and adolescents. It is up to you and your parents whether or not you take part in this study. Please take time to read the following information carefully and talk about it with your parents or your nurse or doctor if you wish. Ask us if there is anything that you do not understand. Take time to decide whether or not you wish to take part.

1. What is a kidney (renal) tumour? Kidney or renal tumours are rare cancers in childhood and adolescence. Wilms tumour or nephroblastoma is the most common of these tumours. Approximately one in ten thousand children get Wilms tumour, usually in only one kidney (unilateral), although occasionally there may be tumours in both kidneys (bilateral disease). A few children with a suspected Wilms tumour turn out to have another type of kidney tumour once the pathologist has looked at a piece of the tumour down the microscope. Some of these tumours are treated almost the same as Wilms tumour, some require different treatments. Your doctor will tell you more about your tumour and the necessary treatment. In most cases we do not know what causes kidney tumours.

2. What question is the study asking? This study is trying to find out if the molecular analysis of your tumour, blood and urine will find new risk factors (biomarkers) to better classify kidney tumours in children and adolescents. Such genetic analysis might even find new targets for better treatments of these tumours. For that purpose we would like to store few milliliters of blood and urine from you and a small, sugar lump size piece of your tumour when it is removed, so that scientists can carry out these tests called ‘research’ to try to understand more about kidney tumour.

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3. Do I have to take part? No. It is up to you and your parents whether or not to take part. If you and your parents do decide to take part, you will be given these information sheets to keep and asked to sign a consent form. If you and your parents decide to take part you can still change your mind at any time. If you do change your mind, or decide not to take part at all, your doctor won’t mind.

4. What will happen to me if I take part? All children with kidney tumours will be given chemotherapy drugs for a few weeks to shrink the tumour. You will then have an operation to remove the kidney with the lump. The amount of chemotherapy you need after the operation can only be decided once we know what the tumour looks like down the microscope and whether the surgeon has been able to remove it all. All children with kidney tumours are asked to allow molecular analysis of their tumour after removal and also their blood and urine during treatment. We need to do this because sometimes we want to find better treatments for children and adolescents with these tumours.

5. Are there any other choices? If you or your parents decide not to take part in this study, then your doctor would just give you the treatment which is currently the best available: this will vary according to the type of tumour that you have.

6. What are the side effects? There are no side effects for taking part in this study. Blood and urine will be collected at routine visits to the hospital and the analysis of the tumour is done from the operated and removed tumour. Independent from the participation in the study you do need treatment against your tumour. This treatment is depending on the type of tumour you have. Your doctor will tell you in detail what you need and what are the side effects of treatment. Most of the treatments in use for cancer in children cause some side effects, for example, increased risk of infection and hair loss. Most of these go away once treatment is finished.

7. Will there be any other problems? No, we don’t think so.

8. What are the possible benefits of taking part? Whatever you decide, you will get the best possible treatment from your doctors and nurses. By taking part in this registry, we hope to make the treatment of these tumours even better for children and adolescents like yourself in the future.

9. What will happen if I decide not to take part? Whatever you decide, you will continue to get the best possible treatment.

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10. Will anyone else know that I am taking part in this study? The only people who will know that you are taking part in this registry will be the team of doctors and nurses looking after you and the members of the SIOP Renal Tumour Study Group, who collect information on all the patients like yourself in this study, but by anonymising the data beforehand.

11. What will happen to the results of the study? The results of this study will be looked at regularly. When the study is finished, the results will be printed in a special sort of newspaper for doctors. Your name will not appear in any report.

12. What if I have other worries? If you have any other worries, don’t be afraid to talk to somebody about them. Doctors and nurses get asked questions all the time, they won’t mind. There is a lot of information to take in.

Thank you for taking the time to read this information sheet and for taking part in the study, if you do decide that you want to.

Contact Details: (insert name of relevant local personnel)

☐ I am thoroughly informed about my disease and the UMBRELLA SIOP 2016 registry. I could ask all my questions and do not have any further questions.

______Place, date, signature (child, if > 8 years) Name of the child/adolescent, birthdate

______Place, date, name, signature Place, date, name, signature Local Principal Investigator Witness

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19.2.5 Information Sheet to be read to children less than 8 years old To be printed on Institution Header Paper

(Version Number 1 - December 2015)

You have a lump in your tummy, which maybe a “Wilms tumour”. We don’t know why it’s happened to you but we do know how to make you better. You will need a mixture of treatments. These will include medicines we call “chemotherapy” and an operation to remove the lump. You might also need a treatment we call “radiotherapy”. This is a bit like when you’ve had an X-ray picture done but you have to come to hospital every day for a few weeks.

We are always trying to make the treatment of Wilms tumour better by lots of doctors all around the world working together. At the moment, we are doing a study called “UMBRELLA SIOP 2016”. We keep a register (like at school) of all the children having treatment for Wilms tumour and how they are doing. We hope that by your joining in, we will be able to make the treatment of children with kidney tumours in the future, safer and better.

Thank you for listening to or reading this with your mum or dad. If you have any questions, you can ask your nurse, doctor or your mum or dad to explain it some more.

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19.2.6 Consent for enrolment in the UMBRELLA SIOP 2016 registry To be printed on Institution Header Paper

Centre Number: Study Number: Patient Identification Number for this trial:

PARENT/CHILD/ADOLESCENT/YOUNG ADULT CONSENT FORM (Version Number 1 - December 2015)

Title of Project: UMBRELLA SIOP 2016 study

Name of Treating Physician:

Please initial box

☐ I confirm that I have read and understand the information sheet(s) dated (version ) for the above study and have had the opportunity to ask questions.

☐ I understand that my/my child’s participation is voluntary and that I am/my child is free to withdraw at any time, without giving any reason, without my/his/her medical care or legal rights being affected.

☐ I understand that sections of any of my/my child’s medical notes may be looked at by responsible individuals from regulatory authorities, where it is relevant to my/my child’s taking part in research. I give permission for these individuals to have access to my/my child’s records.

☐ I agree for my child to take part in the above registry.

______Name of patient Date Signature

______Name of parent/guardian Date Signature

______Name of person taking consent Date Signature

1 for patient; 1 for researcher; 1 to be kept with hospital notes

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19.2.7 Consent for data storage and exchange To be printed on Institution Header Paper

Centre Number: Study Number: Patient Identification Number for this trial:

PARENT/CHILD/ADOLESCENT/YOUNG ADULT CONSENT FORM FOR DATA STORAGE AND EXCHANGE (Version Number 1 - December 2015)

Title of Project: UMBRELLA SIOP 2016 study

Name of Treating Physician:

Clinical data of me/your child and imaging and biological data of my/his/her tumour will be stored in a centralized database located at the Netherlands Cancer Institute in Amsterdam in an anonymized way. These data are needed to answer the research questions of the UMBRELLA SIOP 2016 registry.

In addition personal data will be exchanged with the reference centres for radiology, surgery, pathology and radiotherapy to get a centralized opinion about the tumour of me/your child. This is part of standard clinical care and enhances the quality of my/your child’s diagnosis and treatment. Sharing of personal data outside of your local hospital and reference centres will not happen. All physicians are bound to the medical confidentiality.

Please initial box

☐ I confirm that I have read and understand the information provided above and that I have had the opportunity to ask questions and that I have no further questions.

☐ I agree that clinical, imaging and biological data can be stored in a central clinical database in an anonymized way and can be shared to answer research questions related to the UMBRELLA SIOP 2016 study.

☐ I agree that personal data can be exchanged to reference centres of radiology, surgery, pathology and radiotherapy.

______Name of patient Date Signature

______Name of parent/guardian Date Signature

______Name of person taking consent Date Signature

1 for patient; 1 for researcher; 1 to be kept with hospital notes

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19.2.8 Consent for storing and usage of biomaterial To be printed on Institution Header Paper

Centre Number: Study Number: Patient Identification Number for this trial:

PARENT/CHILD/ADOLESCENT/YOUNG ADULT CONSENT FORM FOR STORING AND USAGE OF BIOMATERIAL (Version Number 1 – June 2016)

Title of Project: UMBRELLA SIOP 2016 study Within the UMBRELLA SIOP 2016 study biomaterial (tumour, blood and urine) of children and adolescents and young adults with kidney (renal) tumours will be stored and analysed. This allows us to get new knowledge about these tumours and may help to develop new therapies against kidney (renal) tumours. Tumour material is taken after surgery of the tumour from the tumour sample. In addition normal renal tissue will be sampled in case a tumour nephrectomy was done. This will not harm you/your child. We will only ask for blood and urine samples at routine visits with your doctor. The volume of blood taken for research at each timepoint is only 5 ml. In total, the maximum blood volume taken for research would be less than 25-50 ml depending on the age and size of your child. If you agree, we would also like to store blood samples from the parents to analyse a familiar genetic background. The tumour material will be stored in centralized biobanks in participating countries. Exchange of tumour material to answer specific research questions will be done after pseudonymizing all samples and corresponding data. In addition disclosure of your child’s identity is not allowed. The biobank in your country, where biomaterial is stored, is the following: Hospital Name

Contact Name

Phone:

☐ I do agree that biomaterial (tumour, blood and urine) of me/my child suffering from a kidney (renal) tumour can be stored in a biobank and that this biomaterial can be used in the biological research studies integrated into the UMBRELLA SIOP 2016 registry.

☐ I do agree that biomaterial (blood) of family members can be stored in a biobank and that this biomaterial can be used in the biological research studies integrated into the UMBRELLA SIOP 2016 registry.

______Place, date, signature (mother) Place, date, signature (father)

______Place, date, signature (child, if > 10 years) Name of the child, birthdate

______Place, date, name, signature Place, date, name, signature Local Principal Investigator Witness

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19.3 Appendix 2a: Information and Consent forms (Mother language version) To be added by each country!

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19.4 Appendix 3: CRFs of SIOP 2016 UMBRELLA protocol CRFs are provided in an extra document. All CRFs are translated into eCRFs to be used for remote data entry. All CRFs are clustered into 2 groups of CRFs: First group of CRFs for local participating centres to document and collect data: 1. For all patients: F1 (Registration), F2 (pre-operative Chemotherapy), F3 (Surgery), F4 (Pathology), F5 (Biomaterial), F7 (postoperative chemotherapy), F8 (SAE), F8C (Cardiotoxicity), F8N (Nephrotoxicity), F9 (Follow-up), F20A (End of treatment for unilateral cases [not for bilateral cases  F20B]) 2. Only for patients with minimal invasive surgery: F3MIS (Minimal invasive surgery) 3. Only for patients with radiotherapy: F6 (Radiotherapy) 4. Only for patients with metastatic disease: F3M (Metastatic surgery) 5. Only for patients with bilateral disease: F12A (Bilateral Wilms Tumour), F12B (Bilateral Wilms Tumour Treatment), F20B (End of treatment for bilateral Cases) 6. Only for patients with Non-Wilms Tumour: F11 (Non-Wilms Tumour) 7. Only for patients with relapse: F17 (Relapse, Progression), F17T (Treatment of relapse) Second group are CRFs for reference centres to collect data for quality control: F2R (Reference radiology), F15 (Reference Pathology), F16 (Panel Pathology)

A timeline for the different CRFs is given on the following page:

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19.5 Appendix 4: Technical details and guidelines regarding radiology 19.5.1 Abdominal ultrasound Abdominal ultrasound is mandatory. With the right equipment/settings and an experienced operator, it offers valuable non-invasive imaging of the abdomen. Representative images should be appropriately annotated and stored. Technical Requirements  curved array probe (≥5 MHz) o B-Mode with harmonic imaging o Colour-coded duplex sonography  High frequency linear probe (≥ 10MHz) Operator Requirements The operator should be a radiologist or paediatrician with training and experience in paediatric ultrasound and paediatric oncology. Documentation Requirements (see radiology CRF)  Each renal lesion size in three dimensions and location within the kidney  Lesion volume = a x b x c x 0.523 o In case of multiple lesions each lesion (with a maxiumum of 3) will be measured and the total volume assessed. Response must be assessed for each lesion separately. If the tumour cannot be delineated from the kidney, tumour and kidney should be measured as a whole.  Relation to remaining kidney, liver, spleen, pancreas, aorta and IVC  renal veins, vena cava – Duplex and B-mode to exclude thrombus  Suspicious lymph nodes, hepatic lesions or intraperitoneal implants: o Position o Size o Number  Abdominal Fluids: o Location (intra/retroperitoneal) o Haemorrhagic?  Contralateral kidney: screen for involvement or other abnormalities

19.5.2 Abdominal MRI In case of renal impairment, abdominal imaging should be done by ultrasound and non-enhanced MRI only.

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Initial pre-surgery and follow-up MRI should be carried out according to the following recommendations. Sedation or general anaesthesia is recommended in young children according to local practice.

Operator Requirements The protocol should be performed by MR-radiographers and radiologists with expertise in paediatric abdominal MRI. Technical Requirements  1.0 Tesla or higher Coils: should cover around 1.5 times the area of interest. Surface multi-channel coils are preferred, usually posterior spine coil and anterior body coil. Individual coil elements should be selected as appropriate for the field-of-view of individual pulse sequences. The whole peritoneal cavity should be examined, including liver and pelvis.  Motion artefacts can be minimized with respiratory triggering, echo-navigator, single-slice acquisition sequences, or high-temporal resolution sequences and motion correction such as BLADE (Siemens), Propeller (GE), MultiVane (Philips).  Recommended spatial resolution is ≤ 4 mm slice thickness, and ≤ 1 x 1 mm² in plane resolution (matrix and FOV to be adjusted to the size of the abdomen)  IV contrast is recommended but not mandatory. Basic Imaging Protocol Recommendation Mandatory: 1. Coronal and axial STIR (TE > 70 ms, TI 140-170 ms) or T2 with fat suppression (TE 50-80 ms) with respiratory gating or other compensation for respiratory motion. Cover the whole abdomen and pelvis. Slice thicknesses ≤ 4 mm. In-plane base-resolution, ≤ 1x1 mm. 2. T2-weighted SE non-fat suppressed with respiratory gating or other compensation for respiratory motion covering the entire kidneys/tumour in axial, coronal and sagittal plane. Slice thickness ≤ 4 mm and in-plane base-resolution, approximately 0.9 mm. 3. T1-weighted SE non-fat suppressed of the whole abdomen in axial plane. Slice thickness ≤ 4 mm. In-plane-base-resolution approximately 0.9 mm. Recommended: 1. If available, a volumetric acquisition with flip-angle optimisation can replace ‘mandatory 2’, e.g. SPACE (Siemens), CUBE (GE), VISTA (Philips). Resolution approximately 1 x 1 x 1 mm3. 2. Axial diffusion-weighted imaging (may be replaced by the corresponding pulse sequence as outlined in the research section below). Respiratory gating is not mandatory. Cover the entire lesion. EPI readout. Use parallel imaging. TR > 2300 ms; TE as short as possible; slice thickness, 6 mm; in-plane base resolution, approximately 2.7 mm; b values, 0, 50, 200 (250), 400 (500), 800 (1000) s/mm2. Apply (spectral pre-pulse) fat suppression. 3. If no contraindication for administration of gadolinium chelates: Instead of ‘mandatory 3’, Pre-contrast fat-suppressed T1 and contrast-enhanced fat- suppressedT1-W sequences in 2 planes or pre-contrast and porto-venous 3D-spoiled

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gradient-echo, 3D FLASH or VIBE (Siemens), LAVA (GE), THRIVE (Philips). Breathold is required (depiction of necrotic areas and small lesions within the normally enhancing renal parenchyma). Research: Optional recommended protocol (Diagnosis and pre-surgery only) This optional examination is aiming at increasing the knowledge about DWI in nephroblastoma patients. It therefore contains specific recommendations to facilitate comparability of results from different hospitals. It should be used initially and pre-surgery.  Axial diffusion-weighted imaging (may replace the corresponding pulse sequence in the basic protocol). Respiratory gating is not mandatory. Cover the entire lesion. Single-shot with EPI readout. Use parallel imaging (factor 2). Receiver band-width, 1,500 Hz (12 Hz/pixel) TR, 2800 ms; TE as short as possible (70 - 90 ms); slice thickness, 6 mm; FOV of 350 mm and phase field-of-view of 75% with matrix 128 x 96 gives an in-plane resolution of 2.7 x 2.7 mm2; b values: 0, 25, 50, 100, 150, 200, 250, 500, 750 and 1,000. Apply (spectral pre-pulse) fat suppression.  Dynamic contrast-enhanced imaging with T1-weighted spoiled gradient-echo performed during quiet breathing and covering the lesion. Flip angle, 25 degrees. Slice thickness, 6-8 mm; in-plane resolution, 2.5-4 mm. Temporal resolution, 2 s. Temporal resolution has highest priority, and the spatial resolution may need adjustment to achieve high temporal resolution. Consecutive 2-s acquisitions are run for 120 s in total with contrast medium injection 30 s into the run. Gadolinium chelate at 0.1 mmol/kg BW is injected as a bolus using an automated power injector. Please follow MHRA advice and local protocols for administration of gadolinium chelates. This sequence replaces step 3 in the recommended protocol.

19.5.3 Abdominal CT (only if MRI is not available) A volumetric acquisition should be performed during breath-hold in the porto-venous phase following intravenous administration of iodinated contrast medium (use of an automated power injector is strongly recommended). Cover the abdomen and pelvis. Oral contrast is not mandatory. Unenhanced or delayed images are reserved for specific situations such as renal or tumoural haemorrhage. Appropriate contention should be used according to age to avoid motion artefacts.

Technical Requirements

 Positioning of IV line; IV contrast is mandatory: 2mg/kg iodinated contrast agent (300-370 mg/l iodine concentration), 1.5-2 ml/s flow rate, 35 sec scan delay (porto-venous phase)  Scan area: from diaphragm to pubic bone  Tube potential: 80 – 100 kVp  FOV: adapt appropriately  Slice thickness: 1-1.5 mm  Kernel: standard  Position: supine, arms above head  Minimum rotation time (≤ 0.5 sec)  Pitch: 1.0 to 1.3  Dose reduction options recommended (tube current modulation, iterative reconstruction)

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 Tube current adjusted to body weight/age or BMI according to local practice and national DRLs  Reconstructions in COR and SAG plane.  NB: parameters are different with dual-source CT

19.5.4 Chest CT Is not to be performed immediately after a procedure under sedation or general anaesthesia (risk of persisting atelectasis). If in rare situations chest CT is to be performed under GA, controlled ventilation techniques are necessary to reduce the risk of atelectasis. Appropriate contention should be used according to age to avoid motion artefacts. Without IV contrast (unless in same session as abdominal CT)

Technical Requirements

 Scan area: All lung parenchyma, from superior thoracic aperture to and including posterior costophrenic angles  Tube potential: 80 – 100 kVp  FOV: adapt appropriately  Slice reconstruction: 1-1.5 mm  Kernel: lung (B60f 1 mm)  Position: supine, arms above head  Minimum Rotation time (≤ 0.5 sec)  Pitch: 1.0 to 1.3  Tube current adjusted to body weight/age or BMI according to local practice and national DRLs; the CT dose must provide adequate SNR and CNR to depict small nodules (too noisy images may reduce small lesion depiction)  Maximum intensity projection (MIP) reconstruction with a thickness of 5-7 mm is recommended to increase detection of lung nodules  N.B.: Parameters are different when dual-source CT is used)  N.B.: Lung lesions in patients <2 years at diagnosis are extremely rare in nephroblastoma and therefore suspicious for MRTK.

19.5.5 Chest X-Ray AP view at diagnosis is mandatory. This could be a chest x-ray made for positioning of the central venous line. During follow-up after end of treatment AP (PA) view will be performed.

19.5.6 Optional diagnostic imaging 19.5.6.1 Selective renal arteriography: Only in the very rare case of complex anatomy and MR or CT angiography being insufficient

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19.5.6.2 MIBG If neuroblastoma cannot be ruled out, because:  There is no destruction/deformation of calyces or renal pelvis  Urine-catecholamine is elevated  There is an atypical growth pattern: tumour encasing abdominal vessels  There are calcifications  There are very large tumours infiltrating the whole kidney 19.5.6.3 MRI of the head In case of:  MRTK - possible synchronous brain tumour(s)  CCSK (optional) - frequent site of metastatic relapse  Focal neurology or other clinical signs of cerebral metastasis Technique:  ≥ 1.5 Tesla  Head coil  Sequences: T1, T2, FLAIR, post-gadolinium T1-W  At least 2 acquisition planes or 3D sequences

19.5.6.4 Bone Scan  CCSK – frequent site of primary metastasis (alternatively whole body MRI or PET-scan)

19.5.6.5 Echocardiography Fractional shortening, ‘ejection fraction’ and ‘end systolic’ wall stress should be documented before the first dose of Doxorubicin in all patients planned to receive doxorubicin, i.e. Stage IV and high risk histology. In patients treated for localised disease, the measurements should be repeated after a total cumulative dose of 150 mg/m2 and again after a total dose of 250 mg/m2, within 3 month of end of treatment. For patients receiving a higher cumulative dose (high risk protocol and stage IV disease), the measurements should be repeated after a total cumulative dose of 150 mg/m2 and 250 mg/m2 and within 3 months of end of treatment.

19.5.6.6 MAG3-Scintigraphy Optional to evaluate the side-specific function of remaining renal tissue in specific cases before surgery:  Genetic predisposition syndromes  Stage V/Bilateral nephroblastoma  Bilateral nephroblastomatosis

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19.5.7 Imaging definitions 19.5.7.1 Pulmonary Metastasis: Chest-CT definition: Lung nodules with the following characteristics will be recorded  Non-calcified  Round shaped  Sharply marginated

Lung lesions with other characteristics:  “ground-glass,” ill-defined, or diffusely alveolar - they will be considered to be of inflammatory origin  Linear in shape – they will be considered to be atelectasis  Calcified - they will be considered to be granulomas  Triangular or trapezoidal and perifissural or subpleural – they will be considered lymph nodes

The largest nodule will be measured, classified according to diameter and followed-up for response:  1 – 2 mm  3 – 5 mm  6 – 10 mm  > 10 mm

The number of lung lesions will be recorded as following:  0  1 – 4  5 – 10  11 – 20  >20

Lung lesions will be documented in a table like below.

Right Lung Left Lung Total

Nodules 1-2 mm N= N= N=

Nodules 3-5 mm N= N= N=

Nodules 6-10 mm N= N= N=

Nodules > 10 mm N= N= N=

If > 10 mm, size of …….. mm …….. mm largest Nodule

All nodules N= N= N=

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19.5.8 Cutting Needle Biopsy 19.5.8.1 Indications According to local protocols cutting needle biopsy can be mandatory or optional. It should be considered when: Unusual clinical presentations:  Age > 6 years  Urinary infection or septicaemia  Psoas infiltration  Pulmonary metastasis < age of 2 years (suspicious for MRTK)  Extra-hepatic and extra-pulmonary metastases Unusual findings by imaging:  Numerous calcifications  Voluminous lymphadenopathies  Renal parenchyma not visible  Almost totally extra-renal process Biological findings  Hypercalcaemia (suspicious for MRTK)  LDH level > 4N (suspicious for neuroblastoma or malignant haemopathy)

19.5.8.2 Limitations Cutting needle biopsies are of limited use in the differentiation of:  Nephroblastoma vs. nephroblastomatosis  Diffuse anaplasia vs. focal anaplasia  Stromal subtype vs. embryonal rhabdomyosarcoma  Cystic Nephroma, cystic partially differentiated nephroblastoma (CPDN) vs. cystic nephroblastoma Cutting needle biopsies should not be used in:  Age 6 months and younger (upfront surgery)  Completely cystic tumours (consider upfront surgery)

19.5.8.3 Procedural Recommendations  Under general anaesthesia  Ultrasound or CT guidance – make sure to sample solid and viable tumour, avoid sampling of necrotic or cystic areas

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 Co-axial technique is mandatory  Retroperitoneal biopsy tract only (do not use transperitoneal access)  Use cutting needles with a size of 18 or 16 Gauge to guarantee sufficient tissue for pathologic differentiation.  No direct fixation of all specimens (part of specimens in culture medium like RPMI or immediate freezing)

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19.6 Appendix 5: Details of pathology 19.6.1 Histological classification 19.6.1.1 Definitions of Wilms’ tumour and its subtypes, and other renal tumours of childhood Based on the correlation between the histological features and survival, three prognostic groups of typical renal tumours of childhood were discerned in the previous SIOP Trials and Studies: low risk, intermediate risk and high risk tumours.

Mesoblastic nephroma, clear cell sarcoma of the kidney and rhabdoid tumour of the kidney represent separate entities from nephroblastoma but are typical renal tumours of childhood and are included in the SIOP classification and trial/study. Other, less common renal tumours, which may occur at any age including children, should be also registered through the SIOP as they may provide a useful clue in our understanding of renal tumours.

The SIOP (Stockholm) Working Classification of Renal Tumours of Childhood will be followed in this Study [9].

19.6.1.2 The SIOP Working Classification of Renal Tumours of Childhood [9]

Pretreated Cases Primary nephrectomy cases

Mesoblastic nephroma Mesoblastic nephroma Cystic partially differentiated nephroblastoma Cystic partially differentiated nephroblastoma

Low risk Low Completely necrotic nephroblastoma

Nephroblastoma - epithelial type Non-anaplastic nephroblastoma and its variants Nephroblastoma - stromal type Nephroblastoma - mixed type Nephroblastoma - regressive type

Nephroblastoma - focal anaplasia Nephroblastoma - focal anaplasia Intermediate risk Intermediate

Nephroblastoma - blastemal type Nephroblastoma - diffuse anaplasia Nephroblastoma – diffuse anaplasia

Clear cell sarcoma of the kidney Clear cell sarcoma of the kidney High risk High Rhabdoid tumour of the kidney Rhabdoid tumour of the kidney

Please note that nephroblastomas are treated according to their histological type and stage (and only stage I low risk tumours receive no postoperative therapy).

It is important to emphasise that for treatment purposes, in addition to anaplasia, only three major types of nephroblastoma need to be recognised: completely necrotic nephroblastoma (low risk tumours), blastemal (high risk tumour) and others (intermediate risk tumours), but pathologists should assess and record in their reports a percentage of different components (regressive changes, blastemal,

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epithelial and stromal) as we will be prospectively analysing these features in order to identify those that might have further prognostic significance. (Cystic partially differentiated nephroblastoma should be diagnosed on imaging studies and treated with surgery only).

Here follows a short description of the types of tumours that should be entered into this study. More detailed and extensive descriptions are given in the references given for each tumour.

The SIOP histological criteria for Wilms’ tumour subtyping are summarised in Table 1.

Table 1. Histological criteria for Wilms' tumour subtyping in SIOP WT 2001 Tumour type Histological features (% of a tumour) CIC Epithelium Stroma Blastema Completely necrotic 100% 0 0 0 Regressive >66% 0 - 33% 0 - 33% 0 - 33% Mixed <66% 0 - 65% 0 - 65% 0 - 65% Epithelial <66% 66% - 100% 0 - 33% 0 - 10% Stromal <66% 0 - 33% 66% - 100% 0 - 10% Blastemal <66% 0 - 33% 0 - 33% 66% - 100% CIC - chemotherapy-induced changes

I LOW RISK TUMOURS

MESOBLASTIC NEPHROMA Mesoblastic nephroma is a renal tumour that usually occurs in the first year of life. The oldest child with confirmed mesoblastic nephroma in the National Wilms’ Tumour Study (NWTS) files was diagnosed at the age of 29 months. Cases of ‘mesoblastic nephromas’ in older children and adults have been shown to be Metanephric Stromal Tumours or some other entities.

There are three histological subtypes of mesoblastic nephroma: the classical, cellular and mixed type. The distinction between the types has no implication for therapy so far. Classical mesoblastic nephroma is a monomorphous tumour composed of spindle cells with large, vesicular nuclei, noticable nucleoli and abundant cytoplasm. The cells are arranged in interlacing bundles and mitotic figures are usually present. The tumour-kidney border is irregular and long radial extensions (finger-like extensions) of tumour tissue into the adjacent renal tissue are a characteristic finding. Also, within the tumour small rests of connective tissue with entrapped tubules are usually seen. Cellular mesoblastic nephroma has a sharper, pushing tumour-kidney border, increased cellularity and numerous mitoses. Mixed type shows features of both classical and cellular type in any proportion. All types show infiltrative growth and may infiltrate the adjacent perirenal fat (but it is more commonly seen in the classical type) and spread into the renal sinus. Complete, wide surgical resection is the only recommended treatment for localised disease. Local recurrences and metastases have been described in a few cases, especially in children

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older than six months of age, although some children were < 1 month old at diagnosis. Virtually all relapses occur within 12 months of nephrectomy and in about 70% of relapsed cases the tumour is of the cellular type.

In the differential diagnosis, metanephric stromal tumour, blastemal and stromal nephroblastoma, clear cell sarcoma and rhabdoid tumour of kidney must be considered (in difficult cases, please consult excellent tables in 4th series of the AFIP Fascicle on ‘Tumours of the kidney, bladder, and related urinary structures’, 2004).

Cytogenetic abnormalities of chromosome 11 and a translocation involving chromosome 15 have been reported in cellular mesoblastic nephroma. The finding of ETV6-NTRK3 gene fusions has established a histogenetic link between cellular mesoblastic nephroma and congenital fibrosarcoma and in difficult cases, it is recommended to test for the presence of this translocation (FISH and RT-PCR). It is worth emphasising that only pure cellular mesoblastic nephroma shows the presence of ETV6 rearrangement and ETV6-NTRK3 fusion transcript.

CYSTIC PARTIALLY DIFFERENTIATED NEPHROBLASTOMA (CPDN) CPDN is a distinct variant of nephroblastoma that usually occurs in children less than 2 years of age. The histological criteria for making a diagnosis of CPDN are as follows: 1. It is composed entirely of cysts and their thin septa; 2. The thin septa are the only ‘solid’ portion of the tumour; 3. The tumour forms a discrete mass, well demarcated from the non-cystic renal parenchyma; 4. The cysts are lined by flattened, cuboidal or hobnail epithelium; and 5. The septa contain blastemal cells in any amount, with or without other embryonal stromal or epithelial cell types. Thus, variable differentiated glomeruli, tubules, mesenchyme, striated muscle, cartilage, fibrous tissue, and fat may be admixed with blastemal cells in septa. The presence of well-differentiated tubules only is not enough to make a diagnosis of this tumour and separate it from cystic nephroma. Recent biological studies showed that cystic nephroma and CPDN are not related tumours [10]. However, they are both treated with surgery only and both share the same, excellent prognosis. Intermediate risk nephroblastomas may present with numerous cysts but they also contain solid areas and septa are usually thicker and show chemotherapy-induced changes. Beware that other renal tumours such as clear cell sarcoma and rhabdoid tumour may have a predominantly cystic appearance.

COMPLETELY NECROTIC NEPHROBLASTOMA Pre-operative chemotherapy given in SIOP results in so-called ‘chemotherapy-induced change’ in many nephroblastomas. Depending on their initial histological pattern, some nephroblastomas are completely or almost completely necrotic, while others show less marked or minimal/moderate changes. The relationship between the percentage of chemotherapy-induced changes and prognosis has been shown in other tumours such as osteosarcoma as well as in previous SIOP studies on nephroblastoma in which completely necrotic nephroblastomas had excellent prognosis.

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The histological criteria for making a diagnosis of completely necrotic nephroblastoma are:

1. The absence of any viable tumour tissue, especially nests of blastemal, on gross and microscopical examination of multiple blocks taken from different areas of a tumour, according to the recommended protocol (see above); the presence of scattered mature tubules, stroma and tiny groups of blastemal cells is allowed as they may represent remnants of nephrogenic rests. 2. The presence of regressive and/or necrotic changes caused by chemotherapy.

Although complete tumour necrosis makes histological diagnosis of any tumour impossible, in many cases ‘ghost’ tumour structures (mainly blastema, occasionally epithelial elements) can be recognised, and are helpful in distinguishing nephroblastoma from other renal tumours. In addition, the presence of nephrogenic rests, which are virtually never associated with non-Wilms’ tumour is a very reliable clue that the tumour has been a nephroblastoma before chemotherapy. Finally, it is well known that regression of other renal tumours such as clear cell sarcoma, rhabdoid tumour or renal cell carcinoma, is minimal to moderate under the actinomycin D - vincristine protocol, and their histological features can be recognised even in treated cases.

The typical histological appearance of treated nephroblastoma is a mixture of necrosis, fibromyxomatous stroma containing lipid- and/or haemosiderin-laden macrophages, and haemorrhage. In some cases scattered mature tubules, stroma or tiny groups of blastemal cells may be seen within necrotic areas – these may represent remnants of pre-existing nephrogenic rests and should not be regarded as viable tumour tissue. The main pattern of the necrotic area is coagulative-type necrosis of small round cells or tubules, with the majority of ‘ghost’ structures consisting of large sheets of small, pink, necrotic nuclei, consistent with coagulative necrosis of blastemal cells or tubules. (If in doubt whether the necrotic tumour is a nephroblastoma, the reticulin staining may help to identify scarce epithelial or mesenchymal ‘ghost’ structures). The presence of identical changes in a lymph node is regarded as a proof of its involvement with a tumour and, therefore, it is very important to sample and microscopically examine all lymph nodes removed. Beware of Tamm-Horsfall protein which is sometimes accompanied by discrete tubules in a lymph node – this must not be interpreted as a metastasis (for other lesions and changes which may mimic lymph node metastases, see a paper by Weeks et al. 1990).

II INTERMEDIATE RISK TUMOURS

Beckwith and Palmer’s criteria for histological subtyping of nephroblastomas state that one component has to comprise at least 2/3 (66%) of a tumour mass for the tumour to be subclassified accordingly. However, pre-operative chemotherapy alters the original histological features of nephroblastomas and often results in areas of necrosis and regression. Therefore, the criteria applicable to subclassification of primarily operated tumours have to be modified to take these changes into account. The quantification of regression and viable tumour components is important because they might be of relevance for prognosis for patients. The presence of considerable amount of blastema after pre-operative chemotherapy clearly indicates its non-responsiveness to chemotherapy and has been shown to be associated with poorer outcome. Although the assessment of percentage of necrosis/regression is subjective, it is very important for subclassification of nephroblastomas, and it should be done on both gross and histological examination.

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Histological types of nephroblastoma from this group are described below, but a simple approach is as follows:

1. Assess the percentage of necrosis/regressive changes 2. If they comprise more than 2/3 of a tumour mass – it is a regressive type 3. If they comprise less than 2/3 of a tumour mass – look for a predominant histological component and subclassify a tumour accordingly (blastemal, epithelial or stromal type). If no component is predominant, it is a mixed type. 4. Even if you find focal anaplasia, subclassify the tumour according to its other components.

In the group of intermediate risk tumours, five subtypes of nephroblastoma have been recognised as follows:

NEPHROBLASTOMA - EPITHELIAL TYPE The histological criteria for making a diagnosis of epithelial type nephroblastoma are as follows:

1. The viable part of a tumour comprises more than 1/3 of a tumour mass; 2. The viable tumour consists of at least 2/3 of epithelial structures; 3. The stromal component may comprise the rest of the viable tumour; and 4. Only up to 10% of blastemal component is present (if >10% of blastema is present, such tumours should be subclassified as mixed type).

The epithelial elements are regarded as follows: a) tubules – spaces lined by columnar epithelial cells arranged in a fairly regular manner radially around the central space; cell margins are sharp, they have basal, crowded nuclei, and mitotic activity may be marked; tubules are usually back-to-back, with virtually no supporting stroma; b) rosettes – circular arranged tumour cells with elongated ovoid nuclei, but no central lumen is present; c) papillary structures – finger-like projections of a stroma covered with epithelial cells; d) glomerular structures – tuft-like masses of malignant cells surrounded by a well- formed capsule or rather flattened tumour cells. The stromal elements are regarded as follows: undifferentiated stromal cells, myxoid, fibroblastic, smooth muscle, skeletal muscle, adipose cells, cartilage and osteoid formations. The presence of genuine anaplasia classifies the tumour as anaplastic nephroblastoma (focal or diffuse) even if otherwise completely epithelial (see criteria for anaplasia in the chapter for high risk tumours).

Epithelial nephroblastoma usually occurs in younger children (median age 15 months in a SIOP series), and about 80% of cases are in stage I [11]. Beware of ‘pure’ epithelial nephroblastoma in older children, which may be confused with renal cell carcinoma.

NEPHROBLASTOMA – STROMAL TYPE Stromal nephroblastoma represents a subtype in which the stromal elements are a predominant component of the tumour. The fetal rhabdomyomatous nephroblastoma, which in the past was regarded as a nephroblastoma with better prognosis, is also included here. The histological criteria for making a diagnosis of stromal type nephroblastoma are as follows:

1. The viable part of a tumour comprises more than 1/3 of a tumour mass;

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2. The viable tumour consists of at least 2/3 of stromal structures; 3. The epithelial component may comprise the rest of the viable tumour; and 4. Only up to 10% of blastemal component is present (if >10% of blastema is present, such tumours should be subclassified as mixed type).

The stromal elements are regarded as follows: undifferentiated, myxoid, fibroblastic, smooth muscle, skeletal muscle, adipose cells, cartilage, bone, and osteoid. Stromal differentiation may be induced by preoperative chemotherapy as a stromal type nephroblastoma is far more common in children who have recieved preoperative chemotherapy. It is likely that other tumour components, especially blastema, are destroyed by preoperative chemotherapy while stromal elements are chemotherapy resistant and may even further differentiate and result in prominent skeletal muscle component, for example.

Stromal nephroblastoma usually occurs in younger children and usually shows minimal to moderate chemotherapy induced changes since stromal tissue is usually resistant to chemotherapy.

NEPHROBLASTOMA – MIXED TYPE The histological criteria for making a diagnosis of mixed type nephroblastoma are as follows:

1. The viable part of a tumour comprises more than 2/3 of a tumour mass; 2. The viable tumour consists of blastemal and/or epithelial and/or stromal elements but none of them comprise more than 2/3 of the viable tumour 3. Tumours which contain >10% of blastema, even if the predominant components are epithelial or stromal components

NEPHROBLASTOMA – REGRESSIVE TYPE Nephroblastoma – regressive type is a tumour in which chemotherapy-induced changes comprise more than 2/3 of the tumour mass, irrespective of what the viable part of tumour is (except for diffuse anaplasia). Please note that assessment of percentage of necrosis/regression is done on both gross and histological examination, so blocks should be taken not only from viable parts of the tumour mass but also from those that show necrotic/regressive changes.

The histological criteria for making a diagnosis of regressive type nephroblastoma are:

1. More than 2/3 of -viable tumour is non-viable (regressive and/or necrotic changes caused by chemotherapy) on gross and microscopical examination of multiple blocks taken from different areas of a tumour, according to the recommended protocol; 2. The viable tumour elements are histological components of nephroblastoma including blastemal, epithelial and stromal elements.

The typical histological appearance of treated nephroblastoma is a mixture of necrosis, fibro-myxo- sclerotic stroma containing lipid- and/or haemosiderin-laden macrophages, and haemorrhage. The main pattern of the necrotic area is coagulative-type necrosis of small round cells, with the majority of ‘ghost’ structures consisting of large sheets of small, pink, necrotic nuclei, consistent with coagulative necrosis of blastemal cells.

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NEPHROBLASTOMA WITH FOCAL ANAPLASIA Nephroblastoma - focal anaplasia type is nephroblastoma which contains one or two foci of anaplasia according to the established criteria (see below). Usually, the size of an anaplastic focus should not exceed 15mm. However, it is still important to determine tumour’s (underlying) type and if it is blastemal, it should be classified as high risk tumour.

III HIGH RISK TUMOURS

NEPHROBLASTOMA - BLASTEMAL TYPE This nephroblastoma type belong to high risk tumours but only if diagnosed after pre-operative chemotherapy. In cases diagnosed after primary nephrectomy, blastema predominant nephroblastoma remains in the Intermediate risk tumours.

The histological criteria for making a diagnosis of blastemal type nephroblastoma are as follows:

1. The viable part of a tumour comprises more than 2/3 of the tumour mass; 2. At least 2/3 of the viable tumour consists of blastema; 3. Epithelial and/or stromal components of nephroblastoma may be present in varying proportions.

The blastemal elements are regarded as undifferentiated round or elongated cells, which are usually closely packed and show no evidence of epithelial and/or stromal differentiation. There are several distinctive patterns in which blastemal cells may occur and it is not uncommon to find more than one pattern in the same tumour. They include the diffuse, serpentine, nodular, and basaloid patterns but they are of no prognostic or therapeutic significance. In rare cases it may be difficult to distinguish blastema from early epithelial differentiation – with no reliable immunohistochemical markers to help, the distinction has to be based on histological criteria only.

NEPHROBLASTOMA WITH ANAPLASIA Anaplasia was recognised as an unfavourable histological feature of nephroblastoma in earlier trials. The histological criteria for making a diagnosis of anaplasia are as follows:

1. The presence of large atypical tri/multipolar mitotic figures; 2. Marked nuclear enlargement, with diameters at least three times those of adjacent cells; and 3. The presence of hyperchromatic tumour cell nuclei.

Please note that all three criteria for anaplasia have to be met in order to make the diagnosis.

Anaplasia may occur in the blastemal, epithelial or stromal component of nephroblastoma and it can be focal or diffuse.

Focal anaplasia is defined as the presence of one or two clearly defined foci, sharply demarcated within a primary intrarenal tumour, without evidence of anaplasia or prominent nuclear atypia in other areas. The size of an anaplastic focus usually does not exceed 15mm.

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Diffuse anaplasia is defined if any of the following are present:

1. Non-localised anaplasia, and/or anaplasia beyond the original tumour capsule; 2. Anaplastic cells present in intrarenal or extrarenal vessels, renal sinus, extracapsular invasive sites, or metastatic deposits; 3. Anaplasia is focal, but nuclear atypia approaching the criteria for anaplasia (so-called 'unrest nuclear change') is present elsewhere in the tumour; 4. Anaplasia that is not clearly demarcated from non-anaplastic tumour; and 5. Anaplasia is present in a biopsy or other incomplete tumour sample.

Focal anaplasia Diffuse anaplasia

This topographic definition of focal anaplasia makes it mandatory that pathologists carefully document the exact site from which every section is obtained (e.g. on a diagram, specimen photocopy, and/or photograph of the gross specimen). Please use a pre-prepared diagram in the SIOP Institutional Pathology Form F4 or a photograph.

Anaplasia occurs in about 5-8% of patients with nephroblastoma. Preoperative chemotherapy does not obliterate or produce anaplasia but it makes its recognition easier since non-anaplastic areas are destroyed by chemotherapy whereas anaplastic foci remain unchanged. This provides further support to the hypothesis that anaplasia represents more resistant rather than a more aggressive cell line. Diffuse anaplasia excludes the diagnosis of any other type of intermediate or high risk (blastemal type) nephroblastoma (for example, it may occur in excessively regressive WT). The age distribution of anaplastic nephroblastoma differs from non-anaplastic nephroblastoma: anaplasia never occurs in the first six months of life, it is very rare between 6-12 months (1-2%), median age at diagnosis is 61 months and >50% of children are over five years of age (for non-anaplastic nephroblastoma median age is 45 months, and 25% of children are over five years of age).

Although the criteria for anaplasia have been well established, it still represents a diagnostic problem resulting in either missed or ‘overdiagnosed’ cases, while only in rare instances it is confused with other renal tumours. It is important to bear in mind that all three criteria for the diagnosis of anaplasia have to be met and that some histological changes may mimic anaplasia including calcification, fused or smudged masses of nuclear chromatin due to technical artefact, stain precipitate, circulating megakaryocytes, overlapping cells in thick sections, and bizarre nuclei resulting from chemotherapy with the formation of hyperchromatic multinucleated and bizarre macronucleated skeletal muscle cells in response to injury. However, the diagnosis of anaplasia in the skeletal muscle must be made if atypical mitoses and other histological criteria are present.

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CLEAR CELL SARCOMA OF THE KIDNEY This distinctive tumour comprises 5% of primary renal tumours of childhood. It is extremely rare in the first six months of life and in young adults, and the majority of patients are between 2 and 3 years of age. There is a male predominance, but no association with chromosomal defects, genetic abnormalities or specific malformations and syndromes has been reported. Unlike nephroblastoma, CCSK is always unilateral and unicentric.

Histologically, this tumour has a deceptively bland appearance and many histological subtypes. The classical pattern has a uniform appearance of a diffuse growth of relatively small cells with normochromatic nuclei, inconspicuous nucleoli, pale staining cytoplasm, and ill-defined cell membrane. In only 20% of the cases do the tumour cells have clear cytoplasm. The most characteristic feature is a peculiar vascular pattern consisting of arborising blood vessels that create an alveolar or trabecular pattern (best seen with the reticulin stain or CD34 marker).

The classical pattern of CCSK is relatively simple to diagnose, but others including the myxoid, sclerosing, cellular, epithelioid, palisading, spindle cell, storiform, and anaplastic pattern can cause problems in reaching the diagnosis. In some CCSKs, there can be extensive hyalinisation and these tumours may be confused with cases of nephroblastoma with sclerosis due to pre-operative treatment, or rhabdoid tumours. In differential diagnosis blastemal nephroblastoma, mesoblastic nephroma, PNET and rhabdoid tumour must be considered (in difficult cases, please consult excellent tables in the 4th series of AFIP Fascicle on ‘Tumours of the kidney, bladder, and related urinary structures’ [12] the paper by Argani et al. [6] and papers by Gooskens et al. [7] and Furtwängler et al. [8].

The histogenesis of the tumour is uncertain. The tumour cells are positive for vimentin and recent studies showed that NGFR and Cyclin D1 markers are strongly positive in CCSK and are very helpful in distinguishing them from other renal tumours [13]. CCSK is generally negative for cytokeratin, factor VIII associated antigen, epithelial membrane antigen, desmin, and S100 protein.

RHABDOID TUMOUR OF THE KIDNEY Rhabdoid tumour of kidney (RTK) is rare, constituting 2% of paediatric renal tumours. It typically occurs in early childhood, with about 80% of patients younger than 2 years, whereas it is extremely rare after 5 years of age. Two characteristic associations of RTK are hypercalcaemia and the development of synchronous or metachronous primary brain tumours. On the other hand, it is never associated with conditions predisposing to nephroblastoma or with nephrogenic rests.

Histological criteria for diagnosis of rhabdoid tumour include the finding of its characteristic histological features and unique immunohistochemical profile. Typical histological features comprise non-cohesive sheets of cells with abundant eosinophilic cytoplasm and large eccentric nuclei with prominent eosinophilic central nucleoli - these are regarded as the most characteristic feature of the tumour and they are always present at least in some areas of the tumour. Another characteristic feature is the presence of large oval intracytoplasmic hyaline inclusions composed of whorled masses of intermediate filaments. Both of these features may only be focal, and should be specifically looked for in any undifferentiated renal tumour of childhood. In addition to the classical pattern of rhabdoid tumour, many other patterns have been described including sclerosing, clear cell sarcoma-like, epithelioid, spindled, lymphomatoid, vascular, pseudopapillary and cystic patterns.

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The diagnostic immunohistochemical marker for RTK is INI1, which is lost in tumour cells. Molecular studies showed that genetic abnormalities of the hSNF5/INI1 tumour suppressor gene on chromosome 22 are characteristic for both renal and extra-renal rhabdoid tumours. The presence of an hSNF5/INI1 mutation results in a marked reduction in nuclear expression of the gene product, detectable immunohistochemically. There is now no need to do other immunohistochemical markers in order to confirm the diagnosis (but one should bear in mind that some other tumours, such as renal medullary carcinoma and epitheliod sarcoma are also negative for INI1).

19.6.1.3 Nephrogenic Rests Nephrogenic rests are foci of embryonal cells which persist after 36 weeks of gestation and they are considered as potential precursors of nephroblastoma. They have been found in 35-40% of patients with nephroblastoma and very rarely in routinely examined perinatal postmortem kidneys. They have not been described in association with other typical renal tumours of childhood and their finding in problematic cases is a clue that the tumour is nephroblastoma. Two main types of nephrogenic rests have been recognised: perilobar and intralobar rests. They can be further subclassified as dormant, sclerosing, or hyperplastic, and all these appearances may be present in an individual case. The rests may regress to fibrous tissue or progress to nephroblastoma. Hyperplastic rests may be difficult to distiguish from a small nephroblastoma. Perilobar nephrogenic rests occur in hemihypertrophy and Beckwith-Wiedemann syndrome while intralobar rests are associated with WAGR and Denys-Drash syndromes.

19.6.2 Differential diagnosis of renal tumours of childhood The results of the SIOP Trials and Studies showed that there are significant discrepancies in diagnosis and staging of renal tumours between the institutional pathologists and central pathology review panels. For this reason, rapid central pathology review is now being introduced and all centres participating in the study should submit their cases urgently.

Here are some clinical, macroscopical and histological features of renal tumours of childhood, which might be a useful for reaching a correct diagnosis. Age at diagnosis is a rather reliable criterion. Anaplastic nephroblastoma has never been described in the first six months and is extremely rare in the first year of life, but after 5 years of age it comprises 10% of nephroblastomas. Clear cell sarcoma of kidney hardly occurs in the first 6 months of life, while mesoblastic nephroma and rhabdoid tumour of kidney are extremely rare in children over 3 years of age. Grossly, many renal tumours may show areas with cysts but only CPDN and cystic nephroma are entirely cystic neoplasms, with no solid areas. There are some unique features of nephroblastoma, which are very useful in distinguishing it from other renal tumours including: 1. Nephroblastoma is the only typical renal tumour of childhood which may be bilateral (in 5% of cases) or multifocal (cases of bilateral renal cell carcinoma have been exceptionally reported) 2. Nephrogenic rests are commonly present in nephroblastoma but not in other tumours (there is only one report of nephrogenic rests associated with mesoblastic nephroma and CCSK, respectively)

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3. Skeletal muscle, adipose tissue and genuine neoplastic tubules are only seen in nephroblastoma 4. Nephroblastoma and nephroblastomatosis are the only tumour lesions that occurs in syndromes known to be predisposing to nephroblastoma (WAGR, Beckwith-Wiedemann, Denys-Drash syndrome) Immunohistochemical and molecular markers may be helpful in diagnosing renal tumours of childhood.

19.6.3 Other tumours included in the study: In addition to more common renal tumours of childhood discussed above, there are numerous other tumours, which may occur at any age. All these tumours should be registered and submitted for central pathology review as they may provide important information in our understanding of renal tumours in general. These include:

1. Metanephric tumours (metanephric stromal tumour, metanephric adenofibroma, metanephric adenoma) 2. Adenomas (all other types) 3. Cystic nephroma 4. Renal cell carcinoma (all variants) 5. Transitional cell carcinoma 6. Neuroepithelial tumours (renal neuroblastoma, renal Ewing sarcoma/PNET, renal carcinoid) 7. Miscellaneous sarcomas (without evidence of blastemic cells and/or epithelial component in five different blocks) 8. Renal lymphoma 9. Angiomyolipoma 10. Other tumours (adrenal tumours, teratoma) and lesions (xanthogranulomatous pyelonephritis, etc.), if preoperative chemotherapy for nephroblastoma has been given 11. Metastases from other sites

19.6.4 Histological staging 19.6.4.1 SIOP WT staging criteria for renal tumours of childhood apart from RCC Stage I a) The tumour is limited to kidney or surrounded with a fibrous (pseudo)capsule if outside of the normal contours of the kidney. The renal capsule or pseudocapsule may be infiltrated by the tumour but it does not reach the outer surface. b) The tumour may be protruding (‘bulging’) into the pelvic system and ‘dipping’ into the ureter but it is not infiltrating their walls. c) The vessels or the soft tissues of the renal sinus are not involved. d) Intrarenal vessel involvement may be present.

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Notes: - Be aware of mature tubules within the sinus or hilar region, which usually represent perilobar nephrogenic rests. Intralobar nephrogenic rest may grow within the sinus, too. Genuine infiltration of the sinus/hilar structures is usually seen as blastemal foci closely related to nerves. - Fine needle aspiration or percutaneous core needle (‘tru-cut’) biopsy does not upstage the tumour. - The presence of necrotic tumour or chemotherapy-induced change in the renal sinus, renal veins and/or within the perirenal fat should not be regarded as a reason for upstaging a tumour - Infiltration of the adrenal gland does not upstage tumour if the external capsule of the adrenal gland is intact - Liver: tumour might be attached to the liver capsule and this should not be regarded as infiltration of the adjacent organ; only if clear infiltration of the liver parenchyma is present, tumour should be regarded as stage III

Stage II

a) Viable tumour penetrates through the renal capsule and/or fibrous pseudocapsule into perirenal fat but is completely resected (resection margins ‘clear’) b) Viable tumour infiltrates the soft tissues of the renal sinus c) Viable tumour infiltrates blood and lymphatic vessels of the renal sinus or renal veins or is present in the perirenal tissue but it is completely resected. d) Viable tumour infiltrates the ureter’s wall. e) Viable tumour infiltrates adjacent organs or vena cava but is completely resected.

Stage III a) Viable tumour extends to the resection margins. If there is only non-viable tumour at inked resection line, it is regarded as stage III only if viable tumour is <5 mm to the inked margin. If the viable tumour is >5 mm from the resection line and only regressive changes are found at inked margin it does not upstage the tumour (the minimal distance of 5 mm tissue without viable has to be documented with several blocks of this area). b) Any abdominal lymph nodes are involved with either viable or non-viable tumour. c) Pre- or intra-operative tumour rupture, if visable at pathological examination (irrespective of other criteria for staging). d) Tumour thrombus is present at resection margins of ureter, renal vein or vena cava inferior (always discuss resection margins with a surgeon) e) Tumour thrombus which is attached to the IVC wall is removed piecemeal by surgeon f) Tumour has been biopsied (wedge/open biopsy) prior to preoperative chemotherapy or surgery g) Tumour implants (viable and/or non-viable) are found anywhere in the abdomen h) Tumour (viable and/or non-viable) has penetrated through the peritoneal sureface

Notes: - Renal vein retraction issue: Often a thrombus bulges out of the resection line of the renal vein. This is even more emphasised by retraction of the vein after resection and fixation. Such cases

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have to be discussed with surgeon in order to establish whether the thrombus was removed easily or was attached within the IVC and they had to use instruments or enforced power to remove the thrombus. If the surgeon could just pull out the thrombus out of the vessel, a portruding thrombus at the vessel resection margin does not mean stage III. Only in cases with piecemeal removal or very difficult removal of the thrombus, a stage III has to be considered. - The presence of necrotic tumour or chemotherapy-induced changes in a lymph node is regarded as proof of previous tumour with microscopic residue and therefore the tumour is assigned stage III (because of the possibility that some viable tumour is left behind in the adjacent lymph node). The regressive changes in the lymph nodes should have an appearance of a tumour-like area having the shape of previous tumour infiltration. Groups of macrophages in the sinus should not be regarded as previous tumour infiltration. - Mature tubules can be found in lymph nodes often associated with Tamm-Horsfall protein deposits, but also without it. This should not be regarded as lymph node metastasis. - The presence of ruptures at diagnosis is only considered as pathological stage III if it can be seen at nephrectomy. If not, tumour should be staged on the basis of other criteria seen, and the final treatment stage should be decided after discussion at multidisciplinary team /tumour board meeting.

Stage IV Haematogenous metastases (lung, liver, bone, brain, etc.) or lymph node metastases outside the abdominal-pelvic region.

Stage V Bilateral renal tumours at diagnosis. Each side should be sub-staged according to the above criteria.

19.6.4.2 Staging in cases of Nephron Sparing Surgery On the local pathology request form, it should be stated that nephron sparing surgery (NSS) was done. In such cases it is important to investigate the resection margins very carefully. Often the tumour nodules are resected with a small rim of renal parenchyma, especially in cases with multipe nodules within one kidney. Surgeons have agreed to use the following surgical classification for operation: - NSS (A) = Partial Nephrectomy (resection of tumour with a rim of normal renal parenchyma) - NSS (B) = Enucleation (resection of tumour without a rim of normal renal parenchyma) Histopathological examination should include evaluation of the complete circumference of the lesion. In small lesions, they should be embedded completely. The minimal distance to the resection line/margin has to be measured. A ‘safe rim‘ should not be less than 1mm. The finding of nephrogenic rests at the resection margin is not regarded as positive resection margins, and it should not be taken into account for staging purposes. Histopathological assessment should clearly state one of the following findings:  Safe rim of renal parenchyma on resection margin, except nephroblastomatosis (0)  Intact pseudo-capsule along the resection margin (1)  Tumour breach (2)

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19.6.4.3 Staging for Renal Cell Carcinoma (according to the TNM system) For staging of Renal Cell Carcinoma please see part B of the UMBRELLA protocol!

19.6.5 References 1. Murphy WM, Grignon DJ and Perlman EJ: Tumours of the Kidney, Bladder, and related urinary structures. In AFIP Atlas of tumor pathology, series 4. 2004. American Registry of Pathology. ISBN: 978-1-881041-88-7. 2. Weeks DA, Beckwith JB and Mierau GW: Benign nodal lesions mimicking metastases from pediatric renal neoplasms: a report of the National Wilms' Tumor Study Pathology Center. Hum Pathol, 1990. 21(2): p. 1239-44 3. Weirich A, Leuschner I, Harms D, et al.: Clinical impact of histologic subtypes in localized non- anaplastic nephroblastoma treated according to the trial and study SIOP-9 / GPOH. Annals of Oncology, 2001. 12: p. 311-19 4. Marry M. van den Heuvel-Eibrink, Harm van Tinteren, et al.: Outcome of blastemal type Wilms tumour patients treated according to intensified treatment in the SIOP WT 2001 protocol, a report of the SIOP renal tumor study group (SIOP-RTSG). Eur J Cancer, 2015. 51: p. 498-506 5. Graf N, et al., Is the absolute blastemal volume after pre-operative chemotherapy relevant for prognosis? Pediatr Blood Cancer 2011. 57(5). 6. Argani P, Perlman EJ, Breslow NE et al.: Clear cell sarcoma of the kidney: a review of 351 cases from the National Wilms Tumor Study Group Pathology Center. Am J Pathol, 2000. 24(1): p. 4-18 7. Gooskens SL, Furtwängler R, Spreafico F et al.: Outcome of patients with relapsed Clear Cell Sarcoma of the Kidney (CCSK) registered on recent SIOP, UK-CCLG and AIEOP study. Br J Cancer, 2014. 111: p. 227-233 8. Furtwängler R, Gooskens SL, van Tinteren H et al.: Clear cell sarcomas of the kidney registered on International Society of Pediatric Oncology (SIOP) 93-01 and SIOP 2001 protocols: a report of the SIOP Renal Tumour Study Group. Eur J Cancer, 2013. 49(16): p. 3497-506 9. Vujanic GM, Sandstedt B, Harms D et al., Revised International Society of Paediatric Oncology (SIOP) Working Classification of Renal Tumors of Childhood. Med Pediatr Oncol, 2002. 38: p. 79-82 10. Cajaiba MM, Khanna G, Smith EA et al., Pediatric cystic nephroma: distinctive features and frequent DICER1 mutations. Hum Pathol, 2016. 48: p. 81-87 11. Verschuur AC, Vujanic GM, van Tinteren H et al., Stromal and epithelial predominant Wilms tumours have an excellent outcome – the SIOP 93 10 experience. Pediatr Blood Cancer, 2010. 55: p. 233-238 12. Murphy WM, Grignon DJ, Perlman EJ: AFIP Atlas of Tumor Pathology, Series 4 Tumors of the kidney, bladder, and related urinary structures. 2004. ISBN: 978-1-881041-88-7 13. Mirkovic J, Calicchio M, Fletcher CD, Perez-Atayde AR. Diffuse and strong cyclin D1 immunoreactivity in clear cell sarcoma of kidney. Histopathology, 2015. 67: p. 306-312 14. Boccon-Gibod L, Rey A, Sandstedt B, et al. Complete necrosis induced by preoperative chemotherapy in Wilms’ tumor as an indicator of low risk: Report of the International Society of Paediatric Oncology (SIOP) Wilms’ tumor trial and study. Med Pediatr Oncol 2000; 34: 183-190

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15. Weirich A, Leuschner I, Harms D, et al.: Clinical impact of histological subtypes in localized non-anaplastic nephroblastomatreated according to the Trial and Study SIOP-9/GPOH. Ann Oncol 2001; 12: 311-319 16. Vujanic GM, Kelsey A, Mitchell C, Shannon RS, Gornall P: The role of biopsy in the diagnosis of renal tumors of childhood: results of the UKCCSG Wilms tumour study 3. Med Pediatr Oncol 2003; 40: 18-22 17. Vujanić GM, Harms D, Bohoslavsky R, Leuschner I, de Kraker J, Sandstedt B: Non-viable tumour tissue should not upstage Wilms’ tumours from stage I to stage II – a report from the SIOP 93-10 nephroblastoma trial and study. Pediatr Develop Pathol 2009; 12: 111-115 18. Vujanic GM, Sandstedt B, Kesley A, Sebire N. Central pathology review in multicentre trials and studies: lessons from the nephroblastoma trials. Cancer 2009; 115: 1977-83 19. Vujanic GM, Sandstedt B. The pathology of nephroblastoma: the SIOP approach. J Clin Pathol 2010; 63: 102-109. 20. Popov SD, Sebire NJ, Pritchard-Jones K, Vujanic GM. Renal tumours in children aged 10-16 years – a report from the UKCCLG. Pediatr Develop Pathol 2011; 14: 189 – 193

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19.7 Appendix 6: Possible treatment regimen for Stage IV group D in exceptional cases Recent unpublished but presented data from the COG Renal Tumour Group suggests significant activity of Vincristine and Irinotecan (VI) in anaplastic Wilms tumour [1]. Thus VI regimen has been integrated in the current COG protocol for stage IV HR histology including all drugs with proven activity. The current treatment recommendation of SIOP RTSG for stage IV high risk histology integrates VI chemotherapy in a backbone of dose-intensive alternating courses of well-known and efficacious courses of chemotherapy (CCE, VDCy), together with flank and pulmonary radiotherapy and high-dose (HD) chemotherapy. The efficacy of this schedule has however not yet been investigated by a prospective clinical trial for high risk stage IV nephroblastoma. The schedule is proposed based on the best available evidence. But it is advised to always contact the international PI for stage IV first in order to validate this treatment recommendation. The use of this regimen is always an individual choice of the centre. Please contact the (inter)national PI before starting this treatment. A randomised clinical trial concept for stage IV is under discussion and will most likely use the treatment regimen here proposed as one possible postoperative treatment arm for the category of patients with high risk histology. The role of upfront use of HD chemotherapy for high risk metastatic disease is under debate. A meta- analysis on the role of HD chemotherapy in relapsed Wilms tumours – most of them with pulmonary metastasis – has shown a favourable hazard ratio for patients with high risk histology consolidated with HD chemotherapy [2]. Several other groups reported a trend to favourable outcome [3, 4]. Melphalan as single drug has a moderate toxicity profile. It was standard of care for cases with high risk relapse of nephroblastoma in the UKW-R study [5] and can be recommended as efficient treatment component as part of a dose-intensive treatment. Centers/countries that cannot adhere to HD-chemotherapy consolidation may use the dose intensive VI/CCE/VDCy regimen without applying HD-chemotherapy. Centers/countries that cannot adhere to the dose-intensive VI/CCE/VDCy regimen, may propose the 4- drug regimen as in SIOP 2001. However with this comes the likelihood of insufficient efficacy. The regimen will be piloted in the framework of a stage IV trial. A randomised trial for stage IV is just under development and may use this postoperative treatment for this group of patients. Note: Local stage II tumours with diffuse anaplasia receive flank radiotherapy (of note: not for stage II blastemal type). All local stage III tumours receive abdominal or flank radiotherapy. All patients with high-risk histology receive lung irradiation or radiotherapy to metastasis of other sites. In diffuse anaplasia flank radiotherapy should be given as early as possible and pulmonary irradiation can be delayed to analyse response to VI (see chapter 17).

19.7.1 High risk and high dose regimen (HR & HD) for group D patients This treatment is a possible suggestion. Please contact always the (inter)national PI for stage IV. Total duration of the treatment is 25 weeks. The first course starts as soon as the patient has recovered from surgery and clinical condition allows. This should be the case within 21 days after the end of the last preoperative course of chemotherapy. Each cycle commences when absolute neutrophil count is > 1.0 x 109/l and platelet count >100 x 109/l provided rising WBC values. After the first cycle of VI, imaging of metastasis is recommended to document the disease status. It includes seven cycles of standard chemotherapy with combinations of vincristine/, vincristine and irinotecan/, cyclophosphamide, carboplatin and etoposide/, vincristine, cyclophosphamide and doxorubicin/ and high dose melphalan:

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 Single Vincristine week 7, 8, 17 and 18 o Vincristine 1.5mg/ m2 IV Bolus day 1  Vincristine/Irinotecan (VI) week 1 and 10 o Vincristine 1.5mg/ m2 IV Bolus days 1 and 8 o Irinotecan 50 mg/m2 IV 2h days 1 to 5 with supportive care including use of prophylactic cefixime  Carboplatin/Cyclophosphamide/Etoposide (CCE) week 4, 16, 22 o Carboplatin 200 mg/m2 IV 1h day 1-3 o Cyclophosphamide 1 g/m2 IV 1h day 1-3 o Etoposide 100 mg/m2 IV 1h day 1-3  Vincristine/Doxorubicin/Cyclophosphamide (VDCy) week 13, 19 o Vincristine 1,5 mg/m2 IV Bolus day 1 o Doxorubicin 50 mg/m2 IV 6h day 1 o Cyclophosphamid 1,2 g/m2 IV 1h day 1  Melphalan high dose week 25 o Melphalan 200mg/m2 IV 2h day -2

DOX 50 mg/m2  CYCLO 1,2 g/m2  VP16 100 mg/m2  CARBO 200 mg/m2  CYCLO 1,0 g/m2  2 IRI 50 mg/m   VCR 1,5 mg/m2         -RT-    SCH -OP- < -----RT- ---- >      CT CT Weeks 1 2 3 4 5 6 7 8 9 10+ 11 12 13* 14 15

Melphalan 200 mg/m2  DOX 50 mg/m2  CYCLO 1,2 g/m2  VP16 100 mg/m2   CARBO 200 mg/m2   CYCLO 1,0 g/m2   VCR 1,5 mg/m2              SCT   CT Weeks 16# 17 18 19* 20 21 22# 23 24 25 26 27

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SCH: Stem Cell Harvest, in case of unsuccessful harvesting repeat CCE and try second harvesting before irradiation of lung or metastatic site(s); SCT: Stem Cell Transplantation; RT: Radiotherapy (week 2-3: flank, week 7-10 metastasis/lung); OP: metastatic surgery; *: replace VCR/DOX/CYCLO with CARBO/CYCLO/VP16 (CCE) if no response to preoperative AVD in week 13 and 19 #: consider 2/3 dose reduction of CCE in case of hematoxic delay in week 16 and 22 +: replace IRI/VCR (VI) with CCE in week 10 in case of PD in week 3 evaluation CT: evaluation of metastasis with CT in case of lung metastasis, with MRI all other sites of metastasis (week 3, 6 and 24)

Dose reductions (see table in section 14.5.3): Treatment should be delayed if the absolute neutrophil count is <1.0 x109/l or platelet count <100 x 109/l. G-CSF can be given if delays of treatment or grade 4 neutropenia occurs and is indicated after CCE and VDCy cycles. Use of Cotrimoxazole is recommended for this regimen as PCP prophylaxis.

19.7.2 References

1. Daw NC, Anderson JR, Hoffer FA, et al.: A phase 2 study of vincristine and irinotecan in metastatic diffuse anaplastic Wilms tumor: Results from the Children’s Oncology Group AREN0321 study. J Clin Oncol, 2014. 32(15 Suppl): p.10032 2. Ha TC, Spreafico F, Graf N, et al.: An international strategy to determine the role of high dose therapy in recurrent Wilms’ tumour. Eur J Cancer, 2013. 49: p. 194-210 3. Kremens B, Gruhn B, Klingebiel T, et al.: High-dose chemotherapy with autologous stem cell rescue in children with nephroblastoma. Bone Marrow Transplant, 2002. 30:893-8 4. Furtwängler R, Nourkami N, Alkassar, et al.: Update on relapses in unilateral nephroblastoma registered in three consecutive SIOP/GPOH studies – a report from the GPOH-Nephroblastoma study group. Klin Paediatr, 2011. 223: p. 113-119 5. Hale J, Hobson R, Moroz V, Sartori P. Results of UK Children’s Cancer and Leukemia Group (CCLG) protocol for relapsed Wilms tumor (UKWR): unified relapse strategy improves outcome. 40th meeting of International Society of Paediatric Oncology. 2008. O154: p. 62

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19.8 Appendix 7: Integrated Research Projects and list of laboratories All these research projects are regularly updated in the Intranet of the website of SIOP-RTSG (http://siop-rtsg.eu).

19.8.1 Wilms Tumour 19.8.1.1 Copy number variation by Multiplex ligation-dependent probe amplification (MLPA) assay (UK: RW, KPJ) Tumour samples will be analysed with a custom MLPA assay measuring copy number status of 1q, 1p, 16q, TP53, WT1, WTX, MYCN and FBXW7. All the loci measured have previously been shown to be disrupted by copy number aberrations in WT. MLPA will be carried out on multiple spatially distinct samples (typically 3 per case) to allow assessment of spatial intra-tumour heterogeneity as a potential confounding factor (if multiple frozen specimens are not available, DNA from FFPE blocks may be substituted). The MLPA assay is suitable for rapid determination of biomarker status in a GLP-compliant clinical setting. Assays will be carried out in the current project according to established methods based on the optimised MRC-Holland protocol, and analysed using the MRC-Holland Coffalyser MLPA application. On-going studies, including methylation and expression profiling and whole exome sequencing of selected samples, will complement these assays and allow a multi-level approach towards a better understanding of the molecular basis of ‘blastemal type’ WT and improved diagnostic tests to assist pathologists.

19.8.1.2 Copy number variation and allelic imbalances by SNP array analysis (UK: RW, KPJ) Each tumour will be profiled with an Illumina CytoSNP array, according to their standard SOPs. Copy number aberrations and allelic imbalances will be detected using OncoSNP (http://www.well.ox.ac.uk /ogc/arrays-analysis-tools). This data set will provide rigorous validation of the MLPA assay, and serve as a valuable resource for identifying any novel potential biomarkers in a large, well-characterised tumour series. Samples will be analysed in batches corresponding to a fully populated Illumina BeadChip (12 arrays). SNP data will also be analysed to measure local clonal diversity, as modelled from shifts in allelic imbalance within individual SNP profiles [3, 4]. These results will be analysed with respect to clinical outcome to determine if heterogeneity is a promising prognostic biomarker in WT. The SNP array micro- heterogeneity analysis will be carried out in collaboration with David Gisselsson Nord, using methods developed in his laboratory.

19.8.1.3 TP53 sequencing and 17p status in anaplastic tumours by Sanger sequencing (Würzburg: MG) Recent evidence suggests that TP53 mutational status has the potential to select diffuse anaplastic cases with wild-type TP53 for treatment reduction [1, 2, 5]. At least 60 additional anaplastic WT samples sourced from SIOP 2001 aqnd UMBRELLA will be analysed for TP53 mutational status by Sanger sequencing and for 17p copy number analysis. These data will be compared to fatal non-anaplastic cases to deduce risk estimates.

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19.8.1.4 TP53 sequencing and 17p status in anaplastic tumours by Sanger sequencing (Würzburg: MG) Recent evidence suggests that TP53 mutational status has the potential to select diffuse anaplastic cases with wild-type TP53 for treatment reduction [1, 2, 5]. At least 60 additional anaplastic WT samples sourced from SIOP 2001 and UMBRELLA will be analysed for TP53 mutational status by Sanger sequencing and for 17p copy number analysis. This data will be compared to fatal non-anaplastic cases to deduce risk estimates.

19.8.1.5 Mutation screening and epigenetic analysis of candidate Wilms tumor genes and models for their in vitro analysis (Würzburg: MG) Our genome-wide analyses have identified a series of novel Wilms tumor genes that allow subclassification of Wilms tumors (Wegert et al., 2015). We will expand our mutation and expression analysis to characterize the clinical relevance of resulting subgroups and explore their potential to provide diagnostic or therapeutic guidelines in future. The primary targets for these analyses are WT1, CTNNB1, WTX, TP53, MYCN, MAX, FBXW7, AURKA, HACE1, SIX1, SIX2, DROSHA, DGCR8, DICER1, LIN28B, DIS3L2, CHD4, GLI3, RERE, REST, CTR9, MLLT1 and IGF2. These will be tested in DNA/RNA from either frozen tumor tissue or microdissected FFPE material. In addition, we will characterize the methylation status of candidate genes and regions to check if epigenetic alterations may have effects similar to mutation or loss of expression. Our preliminary data already points to a significant contribution of methylation states. Analysis of Wilms tumor genes still suffers from the paucity of models to characterize the biological effects of tumor-specific mutations and the possible role of novel treatment options. We are continuously expanding our bank of viable frozen tumor material and we test novel methods to cultivate the different histological components of Wilms tumors, either in 2D cultures or as 3D spheroids that better recapitulate the in vivo situation.

19.8.1.6 Classification of CCSK and functional models (Würzburg: MG, & Dublin: MOS) In a large scale screen of 159 CCSKs we have shown that these tumors mostly present with an internal tandem duplication affecting the C-terminus of the BCOR gene (Kenny et al., 2016). Only rarely YWHAE- NUTM2B/E fusion transcripts were detectable. On the other hand, there is a significant fraction of cases (12 %) that carry neither of these changes. We will analyse such cases for additional genetic drivers that allow further subclassification. We will also use expression profiling and protein mass spectrometry to identify additional genes and proteins that fall into the BCOR pathway to further our understanding of the biological basis of CCSK.

19.8.1.7 Mutation analysis of candidate WT genes by MiSeq (UCL: WM, RW) We will determine the mutation status of the known WT genes WT1, CTNNB1, WTX and TP53 for all the tumours in the study. We have previously conducted this analysis by standard Sanger sequencing, but have now carried out a successful TP53 pilot project using the Illumina MiSeq instrument, and intend to transition to this method for all genes during the course of the proposed project. This panel of genes may be expanded as results become available from on-going whole genome and whole exome sequencing analyses of WT.

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19.8.1.8 Sequencing ‘discovery’ of new WT genes (collaboration Heidelberg/PMC: MK, SP, MvH, HC) While sequencing studies of very small tumour series have proved informative in some cases, unless the putative tumour-associated variants are very frequent, somewhat larger groups are required to detect recurrent aberrations. We propose to analyse a series of up to 50 (100 tumour/normal pairs) collected and analysed over ~3 years. This is larger in size than cohorts used in a recent analysis of breast cancer genomes, where the mutation frequencies of three commonly affected genes ranged from 10-43% [6]. We will select cases from which high quality tumour and germline DNA for sequencing have been extracted from frozen material. In parallel, the DNA methylation profiles of both tumours and nephrogenic rests will be determined by microarray analysis. In a nested single national centre nested WT subset (Utrecht, PMC) we will perform NGS after micro dissection of the tumour components in order to unravel the driving oncogenic biological characteristics of the subtypes of WT.

19.8.1.9 Epigenetic analysis of Wilms tumour (UK: RW, KPJ) The term epigenetics is used to describe heritable genetic modifications that are not attributable to changes in the primary DNA sequence. Epigenetic mechanisms of gene regulation can be broadly classified into: chromatin remodelling by covalent histone modification or protein binding, DNA cytosine methylation, and non-coding RNA. They are initially set during embryogenesis, but then they are remodelled throughout the development to define embryo patterning and for organ and cell-type specification. Upon terminal differentiation, the epigenome is maintained to sustain cell identity. Aberrant epigenetic events have been considered as the earliest events in tumourigenesis whereby epigenetic disruption results in a pool of progenitor cells prior to a gene-specific genetic or epigenetic initiating mutation [7]. The tumours then acquire epigenetic and genetic plasticity as a later event that is proposed to lead to tumour heterogeneity [8, 9]. Therefore, while this adjustable architecture normally allows the timely/regulated expression of signalling pathways, when disrupted (during development or somatically) it may play a role in cancer initiation and progression. The same effect of a “classical” DNA mutation has been reported in nephrogenic rests (specifically PLNR) and within cells of the surrounding normal kidney in a mosaic pattern [10, 11]. Although LOI at 11p15 is the most common aberration in WT [12] it does not represent the only epigenetic abnormality found, as point aberrations and vast changes in epigenetic architecture have been described (Table 19.7.1). In cancer, these changes in DNA methylation and chromatin accessibility are associated with the silencing or the overexpression of tumour suppressor genes and oncogenes, respectively. Epigenetic modifications have a crucial role in gene expression, underpinning the regulation, maintenance and development of the normal cell. We will apply epigenetic methodology to the analysis of normal and tumour tissues currently being collected for this study.

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Aberration Finding within the tumour

Global hypo methylation Results in genome instability in tumour cells [13, 14]. RASSF1 hyper methylation Frequency of 54% [15] HACE1 hyper methylation Frequency of 73% [16] P16 hyper methylation Frequency of 23% [17] GLIPR1/RTVP hypo methylation Frequency of 87.5%, results in overexpression [18] WT1-antisense transcript hypo Results in biallelic expression [19] methylation LOI 11p15 Frequency of 69%, results in overexpression of IGF2 and down- regulation of H19 [12] Hyper methylation of protocadherin Results in expression loss of these cluster at 5q31 proteins at the cell surface [20]

Table 19.7.1: Epigenetics alterations found in Wilms tumours.

19.8.1.10 Analysis of circulating cell-free DNA (PMC: LT) Circulating blood biomarkers constitute non-invasive real-time surrogates for diagnosis, prognosis, therapeutic response or resistance monitoring, and as tools for assessing intratumour heterogeneity [21]. Circulating extracellular DNA derived from body fluids such as blood are commonly analysed to assess malignant diseases (circulating tumour DNA) [22], as they potentially carry tumour-specific sequence alterations [23-25]. Advances in sequencing technologies have enabled the rapid identification of somatic genomic alterations in individual tumours, and circulating tumour DNA present the opportunity to analyse the genomic landscape of tumours in a non-invasive way. Recent studies have shown the feasibility of using circulating tumour DNA to monitor tumour dynamics in a limited number of patients with various solid cancers [26, 27]. Blood samples collected in EDTA tubes will be processed after collection and centrifuged to separate the plasma from the peripheral-blood cells. DNA will be extracted from aliquots (2 ml) of plasma with the use of the QIAamp circulating nucleic acid kit (Qiagen).

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19.8.1.11 microRNA expression profiling of kidney tumours (Homburg: EM, NG) The project “microRNA expression profiling of embryonic tumours” will provide microarray-based microRNA (miRNA) expression analysis including the computational miRNA data evaluation. miRNAs are a highly conserved family of small non-coding RNAs (17-22 nt) that regulate the expression of their target genes on the post-transcriptional level, through binding to complementary sequences on target messenger RNA transcripts (mRNAs) mostly resulting in gene silencing. Recently, miRNAs were also shown to up-regulate target gene expression [28]. Since the first description of miRNAs in 1993 in C. elegans more than 2000 different human miRNAs have already been identified (http://www.mirbase.org). miRNAs play an essential role in development, proliferation, and apoptosis ensuring the cellular homeostasis of healthy human cells. An alteration of this cellular homeostasis through aberrant expression of miRNAs likely contributes to many human pathologies including cancer [29]. Previously, the group of Eckart Meese analysed the miRNA expression profiles in different tumour and non-tumour pathologies. This project is to investigate the miRNA expression in embryonic tumour tissue and body fluids of the embryonic tumours patients including whole blood, serum, and urine. Scientific problems to be solved - Standardized analysis of the miRNA expression profile of embryonal tumours tissue - Standardized analysis of the miRNA expression profile of body fluids of patients with embryonal tumours including serum, whole blood, and urine - Comparative analysis of the miRNA profiles in tissues and body fluids Preliminary work done by the group of Eckart Meese, Homburg / Germany Recently, the group determined the “miRNome” of 863 miRNAs by microarray-based analysis of whole blood samples that were derived from patients with different tumour and non-tumour diseases, including lung cancer, prostate cancer, pancreatic ductal adenocarcinoma, melanoma, ovarian cancer, gastric tumours, pancreatic tumours, Chronic Obstructive Pulmonary Disease (COPD), pancreatitis as well as from unaffected controls. The miRNA signatures showed a high disease-specificity. Pathway analysis confirmed disease association of the respective miRNAs. Genome-wide association studies identified significant correlations between the genomic localization of genetic variants and deregulated miRNAs [30]. microRNA expression changes after lung cancer resection was analyzed in a follow-up study [31]. In a retrospective study we analysed the miRNA expression pattern in serum obtained from lung cancer patients before and after cancer diagnosis. We identified specific miRNAs that show an altered pattern several years prior to diagnosis. Our retrospective study supports the idea that serum- derived miRNAs could be valuable biomarkers for cancer diagnosis [32]. We also applied high- throughput SOLiD transcriptome sequencing of miRNAs expressed in human peripheral blood and solid tissue of patients with lung cancer [30]. To this end, we developed a bioinformatics pipeline to generate profiles of miRNA markers and to detect novel miRNAs with diagnostic information [33]. Towards the identification of genetic changes in chemotherapeutically treated Wilms tumour we previously identified treatment-independent miRNA signatures in blood cells of WT patients by comparing untreated children with Wilms tumour and children treated according to the SIOP protocol [34]. Presently, we analyse if the expression of certain miRNAs in WT tissue is correlated to known risk factors. Using a 1205 miRNA profile we are able to separate blastemal WTs from WTs of other histological subtypes (unpublished data). Hierarchical clustering using the expression of selected miRNAs yielded a cluster with only regressive WTs and a cluster that includes all blastemal WTs (also unpublished data). Besides this specific work, we have a long-standing experience in generation and

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analysis of multi-level gene expression data and the development of several in silico tools to analyse mRNA and miRNA expression profiles (GeneTrail, miRTrail, ILP) [35, 36].

State of the art There are several reports on miRNA expression analyses in embryonal tumour tissue. However, overall the results of the present studies do not provide a coherent picture of the miRNome neither in tumour tissue nor in body fluids of patients with embryonic tumours. The discrepancies might be due to different methods applied for miRNA analysis (i.e., microarray, qRT-PCR, sequencing), to varying numbers of analysed miRNAs, and to patient cohorts with differences in ethnicity, clinic-pathological features, therapy etc. Therefore, we will apply a highly standardized protocol to generate embryonic tumours specific miRNA signatures.

Specific goals, workflow and methods The main goal of this project is to provide miRNA expression profiles of all patients with kidney tumours. The signatures will be generated both from cancer tissue samples and from patients’ body fluids including whole blood, serum and urine. Using a comprehensive bioinformatics’ workflow that was developed in our previous studies, we will identify miRNAs specific for embryonal tumours and their subtypes. To this end, we will apply a standardized procedure for all samples to be analysed. We will also predict target genes of specific miRNAs. - RNA isolation from tissue and body fluids o We will isolate total RNA including small RNA from tissue samples and body fluids using the miRNeasy Mini Kit (Qiagen). Quality control is done by the Bioanalyzer 2100 (Agilent). - Microarray analyses o miRNA profiles will be established for tissue, whole blood, serum, and urine using the Agilent miRNA microarray platform. We will analyse all known human miRNAs as annotated in the version 16.0 of the Sanger miRBase. - Bioinformatics data analyses o Signature analyses will be performed using comprehensive bioinformatics approaches as developed in our previous work. For analysis of miRNA in blood PAXgeneTM Blood RNA tubes are needed. Fresh blood can be directly injected into these tubes. PAXgeneTM tubes need to be sent at room temperature immediately to the coordinating laboratory in Homburg.

19.8.1.12 Discriminating tumour types by Urine proteomics using TST (UCL: WM) As urine can be easily obtained, even in children using paediatric urine bags, it presents an ideal bio fluid to assess for non-invasive prediction of histological subtype and tumour response during pre- nephrectomy chemotherapy [37]. This underscores the unique applicability for the SIOP RTSG setting, where children are treated before obtaining histology in the majority of cases. Urine has been studied successfully as a source of biomarkers and, as a direct product of the kidney; it is particularly relevant to renal tumours [38, 39]. In children this could potentially discriminate children with WT and non-WT, or alternatively identify WT-subtypes at high risk of progression or relapse. In addition, serial sampling may help prioritise proteomic signatures that reflect the response to chemotherapy [40-43]. The collection of

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a urine specimen is simple, reliable, cost effective and acceptable to children, parents and carers. Previous work has established that urine collected from nappies can be used for biomarker discovery [44]. A variety of methods are used in primary care, predominantly ‘clean catch’, urine collection pads or bags. Although the easiest urine collection method is by clean catch, this has been shown to be impractical in the setting of the timing and sample processing requirements of the UMBRELLA study in the UK [45]. Hence, this study recommends the use paediatric urine collection bags for children who are in nappies and clean catch for those who are able to co-operate with this method. Any deviations from this collection method must be noted on sample collection sheets.

Fig. 19.7.2: Schematic of urine profiling strategy. Two strategies utilising immunodepletion and isobaric labelling are proposed. Controls will be age-matched samples available from parallel projects and/or a proportional pool of all samples across all time points. Tryptic (or other) digests will be analysed by LC- MS/MS using CID and HCD for peptide/protein identification. Relative quantitation will be based on TMT reporter ion intensities using Proteome Discoverer software for data analysis. Exclusion lists generated in initial analytical runs may be applied to second analytical runs to improve proteomic coverage. A further fractionation may be introduced to increase proteomic coverage.

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Qualitative and quantitative proteomic characterisation of serial urine (and blood) samples will be performed as a pilot study to assess the feasibility of collection and processing methodology in all centres. No additional cost for transporting the locally processed, deep frozen serum, plasma and urine samples is expected as each patient’s set of samples can be transferred in batches, together with their frozen tumour. Methods for the pre-processing and quantitative proteomic analysis of urine will follow methods established over the last ten years. Initially two operational methods will be investigated to define the method best suited to the provision of reproducible quantitative data from the samples collected. Thermo Scientific TMT cysteine-reactive Isobaric Mass Tagging Kits enable quantitative tandem labelling of proteins extracted from cells/tissues and bio fluids for identification and analysis by mass spectrometry (Fig. 19.7.2). The primary research aim is to define differences in circulating and secreted proteins released by chemo-sensitive versus chemo resistant blastemal cells in WT during pre-nephrectomy chemotherapy. Defining such proteomic markers and assessing them in combination with known genetic markers in the corresponding tumours should gain a greater understanding of the disrupted biological pathways that influence chemo resistance.

19.8.2 Non-Wilms Tumour Non-Wilms tumours comprise about 6% of all renal tumours in children. Systematic standard biological sample collection as for Wilms tumours (RNA and DNA) has not been pursued in the past. In the current project, collection of urine, blood and tumour as well as germline material of all non-WT patients is aimed for in order to be able to look for discriminating molecular characteristics, to compare these findings with radiologic images and to identify novel driving oncogenic events in non-WT by next generation sequencing (NGS), thereby unravelling not only the heterogeneity of non-WT but also the molecular signatures of rare renal tumour subtypes, such as CCSK, MRTK, RCC and CMN. For that purpose, DNA and RNA as well as blood, urine and tumour tissue of all non-Wilms renal tumours and their matched normal kidney tissue will be collected and stored. RNA/WGS-sequencing will be applied, which enables identification of novel transcripts, and which will identify potential differentially expressed genes of interest. RNA-sequencing provides insight in transcriptome alterations such as coding mutations, as well as gene expression, impaired alternative splicing and gene fusions. To be able to perform RNA and WGS sequencing, frozen tumour and matched normal kidney are required from all newly diagnosed non-Wilms tumour cases immediately after nephrectomy. All samples will be assessed for tumour cell content, attached with histology slides and the derived DNA will be subjected to stringent quality control. In addition DNA will be stored to study the underlying genetic and epigenetic mechanisms that will be identified by RNA Sequencing and to identify novel aberrations by deep sequencing methods such as Whole Genome Sequencing (WGS). The aim is to discover recurrent pathogenetic changes that result in the development or progression of the different types of non-Wilms tumours and also to identify genetic changes that are associated with relapse. The 4 subtypes of non-WT that will be focussed on are described below, more in detail: 19.8.2.1 Clear Cell Sarcoma of the Kidney (CCSK) The only recurring molecular marker, which has been identified in patients with CCSK is a balanced translocation involving t(10;17)(q22;p13) [46-48]. Recently, O’Meara et al. found a rearrangement of YWHAE on chromosome 17 and FAM22 on chromosome 10; the YWHAE-FAM22 transcript was detected in 6 of 50 CCSKs tested [49]. The influence of this translocation on clinical outcome is unknown so far. In

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the current proposal, all CCSKs will be tested for this translocation using RNA-sequencing in order to answer this question. In addition, whole transcriptome sequencing identified BCOR internal tandem duplication as a common feature of CCSK [50, 51]. In addition, 90% of the cases do not show a recurrent molecular aberration. The aim of this project is to identify other important oncogenic molecular factors and drivers for relapse.

19.8.2.2 Renal Cell Carcinoma (RCC) Genetic and molecular differences between different histological subtypes of RCC are poorly specified. This includes papillary, clear-cell, combined, collecting duct, translocation (TFE3 / TFEB genes), chromophobe, pure sarcomatoid subtype RCC [52, 53], but also renal medullary carcinoma, RCC post- neuroblastoma, RCC arising from Wilms tumour, and RCC not otherwise specified. Associated genetic syndromes are Von Hippel-Lindau (VHL) syndrome, tuberous sclerosis (TSC1 / TSC2), hereditary papillary RCC (MET), and Birt-Hogg-Dubé syndrome (FLCN). In the current project, all paediatric RCCs will be documented for the first time in a prospective setting by clinical, histological and molecular typing and the data will be combined with information on the underlying syndromes through standard clinical genetic counselling.

19.8.2.3 Malignant Rhabdoid Tumour of the Kidney (MRTK) A few recurring genetic and molecular aberrations are known for MRTK: SMARCB1/SMARC A4/INI1 mutations, aurora kinase changes, IGF2 overexpression, overexpression of the Sonic Hedgehog pathway, Cyclin D1 mutations, overexpression of the WNT pathway and others. In the current project, all MRTKs will be checked for these aberrations and the relevance for clinical outcome will be studied in multivariate setting with other prognostic factors, such as advanced stage disease (stage III/IV), relapse and younger age [54]. The frequency of SMARCB1 will be determined by using FISH analyses and mutational analysis of SMARCB1. There is a close collaboration with the European Rhabdoid Registry ‘EU-RHAB’6.

19.8.2.4 Congenital Mesoblastic Nephroma (CMN) Two histological subtypes can be identified, the cellular and classic subtype. So far, no information is available on the molecular background of the histological signature, apart from the recurring balanced translocation involving t(12;15) (ETV6-NTRK3) which occurs in cellular CMN. Still, so far it is not well understood what the driving event is in CMNs that show clinical progression. In the current project we aim to identify molecular markers that are associated with a trend to progress.

19.8.3 Novel models This research will be done at the Princess Maxima Center, Utrecht, The Netherlands. Adult stem or progenitor cell organoids are 3D adult-organ-derived epithelial structures that contain self-reneweing and organ-specific stem or progenitor cells, as well as differentiated cells. Organoid

6 http://www.rhabdoid.de/

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cultures have proved to be of value for basic research and for the study of healthy tissue homeostatis and the biology of disease. Kidney organoid systems (both for Wilms tumour as well as non-Wilms tumour) are currently being developed from tumour tissue as well as urine, to be used for preclinical studies of paediatric renal tumours. For that purpose, fresh material can be send to the Princess Maxima Center by FEDEx. Contact [email protected] in advance.

19.8.4 Radiological Projects 19.8.4.1 LUNGS  Quantification of superiority of CT compared to CXR and impact on management  Frequency of pulmonary lymph nodes (defined by morphology and location) in children with WT and their behavior under chemotherapy  Development of a mathematical risk model for the presence of lung metastases using multiple parameters such as age, tumour size, lymph nodes, invasion of IVC, histology, left/ right tumour  Optimization of CT technique with respect to minimum signal to noise / contrast to noise characteristics to ensure reliable detectability of nodules at the lowest dose (ALARA principle)  Prognostic value of lung disease volume as assessed by CAD  Extranodular lung findings and pulmonary complications of therapy

19.8.4.2 PRIMARY TUMOUR  Assessment of tumour heterogeneity by MRI (and US): can it help in targeting biopsies (and/or molecular sampling of the excised kidney) and identify genetically different components  Evaluation of radiological response to chemotherapy of primary tumour as assessed by automatic- semiautomatic software (change of volume and contrast enhancement)  Assessment of different modalities of MRI as a clinically useful functional imaging tool to predict tumour histology and response, in unilateral tumours from centres that use MRI as routine, and in all bilateral tumours, where MRI is already recommended as best practice to differentiate nephrogenic rests from Wilms tumour

19.8.4.3 BILATERAL DISEASE  the prognostic value of DWI in the follow-up of bilateral disease (Wilms/nephroblastomastosis), especially for multifocal lesions when surgery is questionable.

19.8.4.4 RUPTURE  Correlation between local or peritoneal relapse and peritoneal fluid or distant nodules

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19.8.5 Radiotherapeutic Projects 19.8.5.1 Impact of a reduced target volume in flank irradiation on acute and late toxicity and tumour control

Purpose:

 To correlate highly conformal radiotherapy techniques with loco-regional control.  To correlate highly conformal radiotherapy techniques with late toxicity (kidney damage, hepatic damage, cardiac effects, growth abnormalities, diabetes, vascular events, treatment induced neoplasms).

The current standard technique of flank irradiation is the inclusion complete ipsilateral hemiabdomen taking into account of the volume of the preoperative kidney size. This target volume may be treated by simple AP-PA fields (2D-technique). This includes large amounts of small and large bowl as well as parts of the lateral and anterior abdominal wall, which may not be contaminated by tumour cells. Even though more modern 3D conformal radiation techniques and the use of intensity modulated radiotherapy (IMRT) approaches increase the conformality, still a large volume of normal tissue is irradiated. The multicenter prospective trial will examine a new target volume definition, which only includes the tumour bed areas (medial and dorsal abdominal wall, adherences of preoperative kidney extension); this results in a reduction of the standard target volume of about ½ to 2/3. For treatment planning highly conformal techniques as IMRT and VMAT- approaches will be used. It is expected to reduce the dose to normal tissues by 20-40% using these techniques. Primary endpoints are acute, subacute and late toxicities as well as loco-regional recurrence rates. Secondary endpoint could be the incidence of second malignancies in a long-term follow-up. As those techniques deserve an excellence experience in pediatric radiation oncology as well as the technical requirements the study will be limited of 8 to 10 radiotherapy centers.

19.8.6 Surgical Projects The following surgical projects will take place within the protocol: 1. Surgical access to WT nephrectomy. This will be re-done on the whole data of SIOP-RTSG 2. Surgical implications in case of chemoresistant WT 3. Timing of the lung metastasectomy and its clinical implications 4. Surgery for WT relapse 5. Surgery for liver metastasis 6. The impact of the surgical guideline violoations on the outcome of ET patients This will be a comparison with the review published by Peter Ehrlich of COG data (Ehrlich PF et al.: Surgical protocol violations in children with renal tumors provides an opportunity to improve pediatric cancer care: a report from the Children's Oncology Group. Pediatr Blood Cancer, 2016, doi: 10.1002/pbc.26083) Further projects wil be defined during the run of the UMBRELLA protocol.

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19.8.7 References

1. Popov, S.D., et al., Bilateral Wilms tumour with P53 related anaplasia. Pediatr Dev Pathol, 2013. 2. Gadd, S., et al., Are there anaplastic Wilms tumours that retain an intact P53 pathway, in 8th International conference on pediatric remal tumour biology2013, CHMC: Bethedsa Washington USA. 3. Gisselsson, D., et al., Genetic bottlenecks and the hazardous game of population reduction in cell line based research. Exp Cell Res, 2010. 316(20): p. 3379-86. 4. Lundberg, G., et al., Intratumour diversity of chromosome copy numbers in neuroblastoma mediated by on-going chromosome loss from a polyploid state. PLoS One, 2013. 8(3): p. e59268. 5. Maschietto, M., et al., TP53 mutation status defines two distinct classes of diffuse anaplastic Wilms tumours, in 8th Inetrnational conference on pediatric renal tumour biology2013, CNMC: Bethedsa Washington USA. 6. Ellis MJ, et al. Analysis of luminal-type breast cancer by massively parallel sequencing. in AACR Annual Meeting 2011 2011. Orlando, Florida. 7. Feinberg, A.P., R. Ohlsson, and S. Henikoff, The epigenetic progenitor origin of human cancer. Nat Rev Genet, 2006. 7(1): p. 21-33. 8. Gerlinger, M., et al., Intratumour heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med, 2012. 366(10): p. 883-92. 9. Feinberg, A.P. and B. Tycko, The history of cancer epigenetics. Nat Rev Cancer, 2004. 4(2): p. 143- 53. 10. Ohlsson, R., et al., Mosaic allelic insulin-like growth factor 2 expression patterns reveal a link between Wilms' tumourigenesis and epigenetic heterogeneity. Cancer Res, 1999. 59(16): p. 3889- 92. 11. Vuononvirta, R., et al., Perilobar nephrogenic rests are nonobligate molecular genetic precursor lesions of insulin-like growth factor-II-associated Wilms tumours. Clin Cancer Res, 2008. 14(23): p. 7635-44. 12. Scott, R.H., et al., Stratification of Wilms tumour by genetic and epigenetic analysis. Oncotarget, 2012. 3(3): p. 327-35. 13. Ehrlich, M., et al., Satellite DNA hypomethylation in karyotyped Wilms tumours. Cancer Genet Cytogenet, 2003. 141(2): p. 97-105. 14. Ludgate, J.L., et al., Global demethylation in loss of imprinting subtype of wilms tumour. Genes Chromosomes Cancer, 2012. 15. Wagner, K.J., et al., Frequent RASSF1A tumour suppressor gene promoter methylation in Wilms' tumour and colorectal cancer. Oncogene, 2002. 21(47): p. 7277-82. 16. Zhang, L., et al., The E3 ligase HACE1 is a critical chromosome 6q21 tumour suppressor involved in multiple cancers. Nat Med, 2007. 13(9): p. 1060-9. 17. Arcellana-Panlilio, M.Y., et al., Decreased expression of the INK4 family of cyclin-dependent kinase inhibitors in Wilms tumour. Genes, Chromosomes and Cancer, 2000. 29(1): p. 63-69. 18. Chilukamarri, L., et al., Hypomethylation and aberrant expression of the glioma pathogenesis- related 1 gene in Wilms tumours. Neoplasia, 2007. 9(11): p. 970-8. 19. Malik, K., et al., Identification of Differential Methylation of the WT1 Antisense Regulatory Region and Relaxation of Imprinting in Wilms’ Tumour. Cancer Res, 2000. 60(9): p. 2356-2360.

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20. Dallosso, A.R., et al., Frequent long-range epigenetic silencing of protocadherin gene clusters on chromosome 5q31 in Wilms' tumour. PLoS Genet, 2009. 5(11): p. e1000745. 21. De Mattos-Arruda, L., et al., Circulating tumour cells and cell-free DNA as tools for managing breast cancer. Nat Rev Clin Oncol, 2013. 10(7): p. 377-89. 22. Sunami, E., et al., Analysis of methylated circulating DNA in cancer patients' blood. Methods Mol Biol, 2009. 507: p. 349-56. 23. Schwarzenbach, H., et al., Evaluation of cell-free tumour DNA and RNA in patients with breast cancer and benign breast disease. Mol Biosyst, 2011. 7(10): p. 2848-54. 24. Schwarzenbach, H., et al., Genomic profiling of cell-free DNA in blood and bone marrow of prostate cancer patients. J Cancer Res Clin Oncol, 2011. 137(5): p. 811-9. 25. Gormally, E., et al., Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance. Mutat Res, 2007. 635(2-3): p. 105-17. 26. Diehl, F., et al., Circulating mutant DNA to assess tumour dynamics. Nat Med, 2008. 14(9): p. 985- 90. 27. Bettegowda, C., et al., Detection of circulating tumour DNA in early- and late-stage human malignancies. Sci Transl Med, 2014. 6(224): p. 224ra24. 28. Bruno, I.G., et al., Identification of a microRNA that activates gene expression by repressing nonsense-mediated RNA decay. Mol Cell, 2011. 42(4): p. 500-10. 29. Calin, G.A., et al., Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A, 2002. 99(24): p. 15524-9. 30. Keller, A., et al., Toward the blood-borne miRNome of human diseases Nature Methods, 2011. 31. Leidinger, P., et al., MicroRNA expression changes after lung cancer resection: a follow-up study. RNA Biol, 2012. 9(6): p. 900-10. 32. Keller, A., et al., Stable serum miRNA profiles as potential tool for non-invasive lung cancer diagnosis. RNA Biol, 2011. 8(3). 33. Keller, A., et al., Next-generation sequencing identifies novel microRNAs in peripheral blood of lung cancer patients. Mol Biosyst, 2011. 7(12): p. 3187-99. 34. Schmitt, J., et al., Treatment-independent miRNA signature in blood of Wilms tumour patients. BMC Genomics, 2012. 13: p. 379. 35. Backes, C., et al., GeneTrail--advanced gene set enrichment analysis. Nucleic Acids Res, 2007. 35(Web Server issue): p. W186-92. 36. Backes, C., et al., A dictionary on microRNAs and their putative target pathways. Nucleic Acids Res, 2010. 38(13): p. 4476-86. 37. Weeks, M.E., et al., Analysis of the urine proteome in patients with pancreatic ductal adenocarcinoma. Proteomics Clin Appl, 2008. 2(7-8): p. 1047-57. 38. Kennedy, M.J., et al., Urine collected from diapers can be used for 2-D PAGE in infants and young children. Proteomics Clin Appl, 2009. 3(8): p. 989-99. 39. Pieper, R., et al., Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots. Proteomics, 2004. 4(4): p. 1159-74. 40. Spahr, C.S., et al., Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest. Proteomics, 2001. 1(1): p. 93-107. 41. Humphryes, P.C., et al., Analysis of multiple Leptospira interrogans serovar Canicola vaccine proteomes and identification of LipL32 as a biomarker for potency. Clin Vaccine Immunol, 2012. 19(4): p. 587-93. 42. Sinclair, J., et al., Profiling signatures of ovarian cancer tumour suppression using 2D-DIGE and 2D- LC-MS/MS with tandem mass tagging. J Proteomics, 2011. 74(4): p. 451-65.

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43. Weeks, M.E., Urinary proteome profiling using 2D-DIGE and LC-MS/MS. Methods Mol Biol, 2010. 658: p. 293-309. 44. Kennedy, M.J., et al., Urine collected from diapers can be used for 2-D PAGE in infants and young children. Proteomics Clinical Applications, 2009. 3(8): p. 989-999. 45. Urinary tract infection in children: diagnosis, treatment and long-term management, NHS, Editor 2007, Royal College of Obstetricians and Gynaecologists: UK. p. 1-178. 46. Brownlee, N.A., et al., Recurring translocation (10;17) and deletion (14q) in clear cell sarcoma of the kidney. Arch Pathol Lab Med, 2007. 131(3): p. 446-51. 47. Punnett, H.H., et al., Translocation 10;17 in clear cell sarcoma of the kidney. A first report. Cancer Genet Cytogenet, 1989. 41(1): p. 123-8. 48. Rakheja, D., et al., Translocation (10;17)(q22;p13): a recurring translocation in clear cell sarcoma of kidney. Cancer Genet Cytogenet, 2004. 154(2): p. 175-9. 49. O'Meara, E., et al., Characterization of the chromosomal translocation t(10;17)(q22;p13) in clear cell sarcoma of kidney. J Pathol, 2012. 227(1): p. 72-80. 50. Ueno-Yokohata H, Okita H, Nakasato K, et al.: Consistent in-frame internal tandem duplications of BCOR characterize clear cell sarcoma of the kidney. Nat Genet, 2015. 47(8): p. 861-3 51. Astolfi A, Melchionda F, Perotti D, et al.: Whole transcriptome sequencing identifies BCOR internal tandem duplication as a common feature of clear cell sarcoma of the kidney. Oncotarget, 2015. 6(38): p. 40934-9 52. Ross, H. and P. Argani, Xp11 translocation renal cell carcinoma. Pathology, 2010. 42(4): p. 369-73. 53. Winarti, N.W., et al., Pediatric renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion. Int J Surg Pathol, 2008. 16(1): p. 66-72. 54. van den Heuvel-Eibrink, M.M., et al., Malignant rhabdoid tumours of the kidney (MRTKs), registered on recent SIOP protocols from 1993 to 2005: a report of the SIOP renal tumour study group. Pediatr Blood Cancer, 2011. 56(5): p. 733-7

19.8.8 Table of laboratories doing molecular genetic analyses The following table provides the information about the laboratories doing molecular genetic analyses:

Investigators France Aurore Coulomb, Linda Dainese Hôpital Armand-Trousseau 26 avenue du Docteur A. Netter – 75571 PARIS Cedex 12 France Phone: ++33 1 44 73 63 38 Fax: ++33 1 44 73 62 82 Email: [email protected] [email protected] Germany Manfred Gessler Theodor-Boveri-Institut für Biowissenschaften / Biocenter, Lehrstuhl Entwicklungsbiochemie, Universität Wuerzburg, Am Hubland D-97074 Würzburg,

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Germany Phone: ++49 931 31 84159 / 84160 (Sekr.) Fax: ++49 931 31 87038 Email: [email protected] Eckart Meese, Andreas Keller, Norbert Graf Universität des Saarlandes, Campus Homburg, Institut für Humangenetik, Gebäude 60, 66421 Homburg, Germany Phone: ++49 6841 16 26038 Fax: ++49 6841 16 26185 Email: [email protected] Italy Daniela Perotti, Filippo Spreafico, Paolo Radice Fondazione IRCCS Istituto Nazionale per lo Studio e la Cura dei Tumori Via Amadeo 42 20133 Milan, Italy Phone: ++39 02 23902644 Fax: ++39 02 23903073 Email: [email protected] [email protected] [email protected] Netherlands Marry M. van den Heuvel-Eibrink, Hans Clever, Jarno Drost Laboratory HUBRECHT Institute Princess Màxima Center for Pediatric Oncology, Lundlaan 6, Room KE 01129.2, 3584EA Utrecht, The Netherlands Phone: ++31 88 972 72 72 Fax: ++31 30 2121801 Email: [email protected] Please call (+31-6-10947882) or email before sending material Sweden David Gisselsson, Linda Holmquist Mengelbier Department of Clinical Genetics BMC C13 Lund University SE 221 84 Lund Sweden Phone: +4646733914036 Fax: +4646131061 Email: [email protected] UK Kathy Pritchard-Jones, Richard Williams UCL Institute of Child Health, 30 Guilford Street, LONDON WC1N 1EH, United Kingdom Phone: ++44 207 8138495 Fax: ++44 207 8138588 Email: [email protected]

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[email protected]

This list will continuously updated and will be found in the Intranet of the SIOP-RTSG website.

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19.9 Appendix 8: Rules and regulations of SIOP-RTSG 19.9.1 Structure of SIOP-RTSG Since 1971, SIOP (International Society of Paediatric Oncology) has conducted 7 prospective clinical trials (SIOP 1, SIOP 2, SIOP 5, SIOP 6, SIOP 9, SIOP 93-01 and SIOP 2001) for children with nephroblastoma (Wilms Tumour). The number of participating centres and countries has progressively increased. In December 2007 the SIOP Renal Tumour Study Group (SIOP-RTSG) was initiated to take forward the planning and implementation of new studies in children and adolescents affected by kidney tumours. SIOP-RTSG has an intergroup structure and it is acknowledged that the existing National Societies and Regional Groups will retain their existing structures in order to mange the contribution of centres in their own group towards new trials and studies. The purpose of this paragraph is:  To describe the structure of SIOP-RTSG and its committees and subgroups  To define the roles and responsibilities of its members and  To describe the conduct of SIOP-RTSG trials an studies

Fig. 19.8.1: Overall structure of SIOP-RTSG

Within SIOP-RTSG a Board and a Coordination Group are established.

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19.9.1.1 SIOP-RTSG Board The SIOP-RTSG Board is a small group responsible for the general management of the SIOP-RTSG. The size of the Board should be sufficiently small enough to ensure that business is carried out efficiently but large enough to ensure appropriate expertise (fig. 19.8.2).

Fig. 19.8.2: Overall structure of SIOP-RTSG with highlighted Board

The Board will elect two members to act as the chair and the vice-chair. It will meet at least twice a year both separately and/or in conjunction with the SIOP-RTSG Coordination Group (CG). One of the meetings can be organised as a webconference. More frequent meetings may be organised if required, inviting people from other SIOP-RTSG Committees, if necessary. The Board will be responsible for the organisation and content of SIOP-RTSG meetings.

Role of the Board:  To plan the strategic direction and organisation of SIOP-RTSG  To provide overall supervision for SIOP-RTSG trials and studies  To nominate members of the trial committees  To review the progress of trials and provide advice to the Trial Management Committee (TMC) on all aspects of the conduct of a trial.  To consider any new information relevant to a trial and the results of other trials that may have a direct bearing on the conduct of a SIOP-RTSG trial

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 To review the progress of the different disciplines panels and provide advice to the chairs of the Discipline Panels (DC)  To close a trial on proposal of the TMC  To ensure that all SIOP-RTSG trials are conducted according to the principles of good clinical practice  To apply for money for the overall structure of the SIOP-RTSG and the International Data Centre

19.9.1.2 SIOP-RTSG Coordination Group The SIOP-RTSG Coordination Group (CG) is a larger Group, which includes representatives of all countries contributing to SIOP-RTSG trials and studies and the Chairs of the Discipline Panels. Board members are automatically members of the Coordination Group, as are all members of the trial management committees. Additional experts may be invited to join as considered necessary (fig. 19.8.3).

Fig. 19.8.3: Overall structure of SIOP-RTSG with highlighted Coordination Group

Defined groups of people have to work on specific therapeutic areas. Six such areas have been defined so far (unilateral non-metastatic nephroblastoma, unilateral metastatic nephroblastoma, bilateral nephroblastoma, relapsed nephroblastoma, non-nephroblastoma renal Tumour, adult patients with nephroblastoma). Every effort will be undertaken to combine therapeutic questions into a minimum number of clinical trials to reduce bureaucracy as much as possible. The Trial management committee

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(TMC) will not be responsible for an individual clinical trial, but will take the responsibility for developing ideas in therapeutic areas. A clear description of the TMC is given below.

The CG is expected:  to collaborate with the TMCs to support the SIOP-RTSG trials and studies in the different countries  to review the progress of trials  to provide advice to the TMC on all aspects of the conduct of a trial  to propose, discuss and approve protocol modifications  to discuss and approve the proposals made by the Board

The CG will meet at least once per year and will be chaired by the Chair(s) of the Board. Members of the CG are asked to participate in all CG meetings, if possible, and may be asked to withdraw from the group if they are unable to attend two consecutive meetings without explanation to the Board.

19.9.1.3 Trial Management Committee Therapeutic areas and members of the teams that will develop therapeutic questions will be initially suggested from the meetings of the coordinating group, and will be approved by the Board. Such a small group of individuals (normally 6 – 10 members) will form the Trial management committee (TMC). The TMC will elect a Chief Investigator. It is the duty of the TMC to develop, implement and manage the conduct of each SIOP-RTSG trial and study. Each Trial and Study will be led by a Coordinating Investigator, who acts as the chair and is appointed by the Board.

The TMC has ultimate responsibility for the safe and effective conduct of the trial and will provide overall supervision by:  Monitoring and supervising the progress of the trial towards its interim and overall objectives  Ensuring that the trial is conducted according to the principles of good clinical practice  Considering recommendations from the Board and the CG, paying particular attention to the progress of the trial, adherence to the trial protocol and patient safety and to recommendations of the IDMC (which should include comments on any relevant evidence external to the trial)  Taking appropriate action in light of new information (this could include changes to the protocol, additional patient information or a proposal to suspend or stop the trial)  Informing the relevant trials organizations and research boards of the progress of the trial  Advising on publicity and the presentation of all aspects of the trial.  Taking action to obtain timely and complete data submission  Ensuring that trial results will be published in an accurate and timely manner.

Each Trial and Study has to have its own Independent Data Monitoring Committee (IDMC). The role of the Chief Investigators is to direct and coordinate the activity of the TMC and take action, on behalf of the TMC, to guarantee the trial success according to the trial objectives. Decisions should usually be possible by consensus between the members of the TMC but where this is not possible, advice should be taken from the Coordinating Group and, if necessary by reference to the Board and the ISC. It is important that all implications (i.e. ethical, statistical, practical, financial, etc) for the trial are considered when any decision is made.

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The Chair of the TMC will call and organize TMC meetings, also informing the Board members. All meetings of a TMC have to be minuted and the minutes have to be distributed to the TMC, and to the Board and CG members. The TMC should also respond in writing to any involvement of the IDMC. The minutes will be posted in the SIOP-RTSG website. Six Trial Management Committees have been identified so far:  Unilateral non-metastatic nephroblastoma Trial Committee  Unilateral metastatic nephroblastoma Trial Committee  Bilateral nephroblastoma Trial Committee  Relapsed nephroblastoma Trial Committee  Non-nephroblastoma kidney Tumour Trial Committee  Adults with nephroblastoma

19.9.1.4 National coordinators SIOP-RTSG trials and studies recruit patients across a wide range of countries, each of which have their own structures, languages and legal requirements. The role of the National Coordinator (NC) is critically important to the successful conduct of a study in each participating country / regional group. The NC will be a member of the Coordination Group and will liaise with the SIOP-RTSG to address the problems in the implementation of the study in his/her own country / group. All countries participating in SIOP-RTSG trials and studies must be represented by a NC. The NC may work within, or collaborate with a national data centre, but personal commitment is essential to address and resolve challenges to the effective conduct of the studies, particularly in smaller countries where there may be less direct support available. Specifically, the NC is expected to:  Promote the study and obtain commitment to participate in SIOP-RTSG studies by the clinical centres in his / her country / group,  Be responsible for all necessary regulatory and ethical approval processes required in their country, including phamacovigilance and annual safety reports  Be responsible for the distribution of the protocol, all clinical research forms and other pertinent materials to the participating centres within their group  Manage the data collection and implement procedures for data quality control within their group  Act as a point of reference for the clinicians from participating centres to address clinical questions  Act as the representative of his / her country / group on the SIOP-RTSG Coordination Group  Be responsible for the safe and effective conduct of SIOP-RTSG trials and studies in his/her own country; in particular the NC is responsible for receiving and resolving queries from the trial management committees, to monitor and ensure pathology review; and to facilitate arrangements for transport of material for biological studies  Be responsible for the implementation of pharmacovigilance procedures within his/her country  Collaborate with the International Data Centre to regularly validate (see Data Validation process) and update the data  Audit participating centres and do side visits according to regulations in their group or country  Finance a trial and study at a National level or for their group

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Those countries without a NC may wish to maintain existing arrangements with other countries for data submission, pathology review etc. In that case the centres are responsible for ensuring that the necessary processes are in place to facilitate timely and accurate data return. New countries or/and new centres, who wish to participate in any SIOP-RTSG trial or study, will need to demonstrate their ability to meet these requirements.

19.9.1.5 Discipline Panels The following Discipline Panels (DPs) have been identified:  Radiology  Surgery  Pathology  Radiotherapy  Biology  Late effects  Data management & Statistics Panels will coordinate the contribution of their members to provide expert advice and evaluation of specific aspects of trial data or protocol requirements. Each Panel consists of a representative of participating countries. The participating institutions of each individual country nominate their representative. She or he has to have a longstanding experience in the discipline she or he is nominated for. In countries with small numbers of patients cooperation with other countries should be considered, if no person with acknowledged experience is available in that country. Each participating country should build a national review centre including at least two specialists with longstanding experience in the specific discipline. This national review centre provides a rapid review of all study materials of patients entered into the SIOP Nephroblastoma Study in their discipline. Difficult cases should be reviewed by selected members of the Panel immediately to avoid delay in getting an appropriate diagnosis. The chairman of the discipline is nominated by the Panel and has to be approved by the Board of the SIOP Nephroblastoma Study. The chairmanship ends with the end of the study. Every Panel should meet at least once a year to review cases. The Panel should review only selected cases being of special interest in the SIOP Nephroblastoma Study, e.g. all high risk tumours, all low risk tumours, etc. In addition a randomly selected number of cases (e.g. 10 percent of all cases) should be reviewed by the Panel to provide a quality control of submitted data. Each panel must ensure that a representative, normally the Chair, is able to attend meetings of the Coordination Group and that representation is provided to each trial management committee as required / appropriate. Scientific work of members of the Panel using cases of the SIOP Nephroblastoma Study has to be discussed within the Panel. Authorship of possible publications follows the rules as written in the Publication section of this document (see 19.8.4).

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Specific roles of the different Panels Radiology  To review imaging studies before start of treatment  To provide advice for local centres  To analyse the radiological data Surgery  To review all surgical forms  To provide advice for local centres  To analyse the surgical data Pathology  To review all cases before the end of the trial  To provide advice for local pathologists  To analyse the pathology data Radiotherapy  To review treatment plans of cases receiving radiotherapy before the end of the trial  To provide advice for local centres  To analyse the radiotherapeutic data Biology  To establish a tumour and tissue banking  To coordinate molecular and biological research projects  To analyse molecular biology data Late effects  To collect data on late effects  To review late effects and report on them  To analyse the late effects data Data management &  To provide a RDE system (IT infrastructure) for data collection Statistics  To prepare the reports for the different meetings  To clear the data and request missing data

19.9.2 Membership of SIOP-RTSG All countries participating in SIOP-RTSG trials, studies and registries are automatically members of SIOP- RTSG. For more detail see chapter 19.8.14.

19.9.3 International Data Centre The Staff in the International Data Centre (IDC) will be responsible, under the direction of the Head of the IDC, for the day to day management of all studies and for communication within the studies. A Remote Data Entry will be used in the registry. The IDC will liaise with the Chief Investigators of the registry about the conduct of the registry and with the chair of the relevant discipline panel or national coordinator when required.

The IDC will provide an annual progress report to the Board, CG, TMC and IDMC, and will liaise with the coordinators of the safety desk in the provision of an annual safety report, also to be distributed to the Board, CG, TMC and IDMC. This annual safety report has to be forwarded to the legal authorities by the NC in their country. The chair is responsible to send this report to EMA.

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19.9.4 Publication Policy and Authorship Each Cooperative Group within SIOP-RTSG agrees that the results of SIOP-RTSG trials and studies will not be published separately by any of the groups. Participating centres or national groups may publish details of their own cases but will agree to allow the trial management committee the exclusive right to publish the results of each study under its jurisdiction, in part or in total. All publications using data from the SIOP-RTSG trials and studies are considered to be official SIOP-RTSG papers. Authorship and content should be agreed within the Trial Management Committee and proposed to the Board for approval before commencing work on a manuscript. Panels will discuss their participation in different projects and they decide who is going to get involved in a paper. People from National groups/Panels can only get involved in the whole material if invited by relevant chairs. Every author should have participated sufficiently in the work to take public responsibility for the content of the final manuscript.

Fig. 19.8.4: Schema of publication policy

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All manuscripts will clearly state that the publication is offered on behalf of SIOP-RTSG and will acknowledge the contribution of the participating centres by names. At an appropriate place in the article one or more statements should specify contributions that need acknowledging for general support, technical help, financial and material support, etc.

Request for Publication

Name: Country

Centre: Phone: Fax:

Email:

Title or question of work :

Initiators :

Methodology:

Retrospective work: ☐ Prospective work: ☐

From database: SIOP 93-01 ☐ SIOP 2001 ☐ UMBRELLA ☐ Planned time for the first draft:

Accepted by the SIOP-RTSG Coordination Group Date _____/_____/______

Names of colleagues who should be involved in this work:

______

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All manuscripts and abstracts (including abstracts for presentation at meetings) and other documents that contain data from the central SIOP-RTSG data bank must be submitted to the Board for approval prior to submission for publication (in the case of abstract submission this should be no later than 21 days prior to the deadline for conference submission). A template for approval is shown above. The publication policy and instructions to authors are in accordance with the Uniform Requirements of the International Committee of Medical Journal Editors (ICMJE)7.

19.9.5 Biobanking and tissue sharing For biobanking and tissue sharing formal agreements need to be signed between the biomaterial provider and the researcher requesting the material. The researcher has to demonstrate that he/she is able to undergo the research including financial issues. The research needs to be approved by the RTSG board if it enrolls material from all countries and not only a sub-cohort of specimen. The International data centre will provide corresponding clinical data. All regulations regarding data safety and security will be respected. Informed consent needs to be given by parents or patients. Results of the research project need to be published. Publication policy and authorship applies as written in 19.8.4 without any change. A template of an agreement between the biomaterial provider and the researcher is given below.

7 www.ICMJE.org

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Agreement for tissue sharing and analysis between a tissue provider and the principle investigator (PI) of a research using biomaterial

The SIOP-RTSG agrees allowing the following research project between the biomaterial provider: …………………………………………………………………………………………………………. and the following PI:

………………………………………………………………………………………………………….

Title of project: …………………………………………………………………………………………………………………………………….

Short summary of the project:

Request of needed biomaterial to undertake the research (type and number):

………………………………………………………………………………………………………………………………………………………………….

The research is done in the following laboratory:

………………………………………………………………………………………………………………………………………………………………….

The research is financed by:

………………………………………………………………………………………………………………………………………………………………….

The research wil be finished at:

………………………………………………………………………………………………………………………………………………………………….

Informed consent for the research is available: yes ☐ no ☐

The biomaterial provider agrees to provide the following material (type and number)

☐ ………………………………………………………………………………………………………………………………………………………….

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The biomaterial provider agrees that:

☐ Samples have been obtained in accordance with national regulations for consent and approval for research use. ☐ Where relevant, samples have been assessed for tumour cell content and histology and these data will be made available with the specimens (applies to extracted nucleic acids). ☐ Material will be coded with reference to the UMBRELLA ID

The principal investigator (PI) of the research agrees that:

☐ Patient material and characterists will be used only for the purpose of this above mentioned study. Additional projects have to be approved separately. For that reason, separate contracts are needed. ☐ Authorship and acknowledgements are handled according to the guidelines provided in 19.8.4 ☐ This agreement will last for the duration of 2 years starting from the date of the signed agreement. Prolongation can be considered thereafter. A yearly progress report will be asked by the SIOP-RTSG. The first report will be send by the PI of the biological study 1 year after the agreement was signed. ☐ When the project will be discontinued preliminary, this needs be reported immediately to the SIOP-RTSG board ☐ No information or material will be given to third parties without permission of the owners of the materials and the SIOP-RTSG board. ☐ Financial agreement: Shipping of material will be paid by the research group that performs the analysis.

On behalf of the SIOP-RTSG:

Date:

Name (chair) Signature:

The principal investigator of the study:

Date:

Name Signature:

The provider of biomaterial:

Date:

Name Signature:

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19.10 Appendix 9: IT infrastructure The leading clinical trial management system (CTMS) to be used is ALEA®. In German speaking countries ObTiMA will be used. Data stored in ObTiMA will be uploaded to the ALEA® CTMS. ALEA® has been developed and deployed over the last two decades in collaboration with European academics. ALEA® is delivered as Software-as-a-Service (SaaS) based on Cloud Computing technologies. ALEA® provides online data management tools for use in the clinical trials of the pharmaceutical industry and the academic clinical research community. The use of ALEA® increases the efficiency at clinical trial sites, reduces the time to market of drugs and reduces the overall cost of data collection. The reduction of administrative overhead makes it possible for investigators to spend more time on research and makes it possible that new drugs find their way to the market quicker. Which improves the lives of many patients. ALEA® is currently used by more than 40 organizations on over 500 clinical trials spanning all therapeutic areas. ObTiMA is an ontology-based clinical trial management system intended to support clinicians in both designing and conducting clinical trials. The design phase is facilitated by the Trial Builder in which all aspects of a clinical trial can be specified: A trial chairman can define the outline and metadata of a trial in a master protocol to describe, e.g., trial goals or administrative data. The ontology-based creation of CRFs is one of ObTiMA’s major functionalities. A graphical user interface allows defining content, navigation, and layout of CRFs to capture all patient data during a trial, e.g., medical findings or diagnostic data. Since many trials collect similar or equal data, it is possible to store components of or complete CRFs in a repository as templates. When setting-up a clinical trial, appropriate CRFs’ template can either be directly reused or can be quickly created by composing them from existing CRF components. This in turn fosters the CRF standardization since CRFs can then readily be compared on the level of single items (through ontological concepts) and also on component level or in their entirety.

Fig. 19.9.1: Infrastructure for ObTiMA

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ObTiMA itself is composed of different modules and fulfils all GCP criteria, including an Audit Trail. Data safety and security are guaranteed as pseudonymization of private data is implemented according to roles and rights assigned to users of ObTiMA.

ObTiMa provides the following features 1. eCRFs 2. Access to biobanking 3. Access to a DICOM server 4. SAE and SUSAR reporting eCRFs for the UMBRELLA, the registries and the trial are available through ALEA and ObTiMA. The ObTiMA data are stored in a central database that is located in a militarized zone at the Saarland University Hospital to ensure data safety and data protection (fig. 12). Via the Internet remote data entry is possible. To get access to ALEA or ObTiMA and the eCRFs, each participating centre needs to register for getting member of the SIOP-RTSG and the SIOP 2016 UMBRELLA study as well as for other studies and trials run by SIOP-RTSG. After registration and signing a contract for participation in the UMBRELLA or any other study or trial credentials to use ALEA or ObTiMA will be provided. The contracts for participation are listed in Appendix 1.

19.10.1 Legal framework All data within ALEA as well as ObTiMA will be encrypted and in addition personal data are pseudonymized. Only treating physicians can see real names and only have access to their patients. The national coordinators will have no access to real names or other private data of patients. Patients are only accessible via their pseudonyms by study centres and reference centres. For that purpose pseudonyms can be printed as barcodes and patients can be searched via scanning of these barcodes. Local centres can provide real names to reference centres, if they have received informed consent from patients/parents for the purpose of a reference consultation. In case of referring biomaterial to samples collection and storage, including reference pathology, the material as well as the request for samples collection and storage should be labelled with the barcode of the patient.

19.10.2 Virtual samples collection and storage ALEA as well as ObTiMA can handle virtual samples collection and storage database, and both databases can be linked to the SIOP 2016 UMBRELLA protocol. Specific eCRFs are developed to store information about biomaterial for each patient. Together with the p-bioSPRE tool existing biomaterial can be queried.

19.10.3 Imaging tool for reference radiology From every patient, imaging studies at time of diagnosis and after preoperative chemotherapy need to be analysed by reference radiologists in each participating country. For that purpose a tool will be provided allowing the upload of DICOM files to a DICOM server that will automatically pseudonymize the data and link them to the clinical data provided by ObTiMA. After upload of the files the corresponding reference radiologist will be informed that a new imaging study is available. The

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radiologist can download the study to his PACS system and give his results on the corresponding CRF in ObTiMA. After storing the CRF in ObTiMA, the trial chairman and the local treating physician are informed about the result of the reference radiologist via the corresponding Radiology CRF. The trial chairman is able to comment the result of the reference radiologists, i.e. if preoperative chemotherapy can be given, or if the patient is a candidate for the stage IV randomized trial. Reference surgeons, trial chairmen and reference pathologists have access to the imaging studies via the DICOM server in case of consultations. For that purpose, consultation CRFs will be provided in ObTiMA.

19.10.4 Pharmacovigilance (SAE and SUSAR reporting) Definitions of SAEs and SUSARs are provided within the SIOP 2016 UMBRELLA protocol. In case a SAE/SUSAR occurs the local treating physician needs to report this SAE/SUSAR within 48 hours to the national study centre via the SAE/SUSAR CRF provided in ObTiMA. The National coordinator will receive a notification that a new SAE/SUSAR CRF is provided by a participating centre. He/she needs to decide if this is a SAE/SUSAR that needs to be reported to participating centres and regulatory authorities. In that case the data of the CRF will be reported automatically to the corresponding people and regular requests to the local centre for update of the outcome of the SAE/SUSAR will be sent. A yearly document of all SAE/SUSARs will be generated automatically. Any adverse event or adverse reaction that results in death, is life-threatening*, requires hospitalization or prolongation of existing hospitalization, results in persistent or significant disability or incapacity, or is a congenital anomaly or birth defect. All SAEs should be classified according to the Common Terminology Criteria for Adverse Events (CTCAE) v. 4.03 (Published June 14, 2010)8. Grade 4 and 5 SAEs needs to be reported with the exception of grade 4 of blood and lymphatic system disorders if caused by chemotherapy. All adverse reactions that are unexpected -not consistent with the applicable product information- and meet the definition of Serious Adverse Events (SUSAR) need to be reported.

19.10.5 Statistics All data stored in ALEA and ObTiMA can be exported in CDISC-ODM format for further analysis with statistical tools such as SAS®9. Export of data is restricted to the statistical centre in an anonymous way for analysis. National coordinators are allowed to download their national data anonymously. Local participating centres can only download their own data. Each download is recorded in an audit trail.

8 http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf 9 http://www.sas.com/en_us/home.html

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19.11 Appendix 10: Flowcharts and treatment schedules

Fig. 19.10.1: Flowchart overview

Fig. 19.10.2: Treatment flowchart

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For dose reductions see section 14.5.3.

19.11.1 Localized Wilms Tumours (stage I – III) Pre-operative chemotherapy

ACT 45 g/kg   VCR 1.5 mg/m2         Weeks 1 2 3 4

Post-operative chemotherapy

Stage I, Intermediate Risk Histology: Regimen AV1 ACT 45 g/kg  VCR 1,5 mg/m2         Weeks 1 2 3 4

Stage I, High Risk Histology: Regimen AVD ACT 45 µg/kg      VCR 1.5 mg/m²             DOX 50 mg/m²                    Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Stage II/III Low and Intermediate Risk Histology: Regimen AV-2 ACT 45 µg/kg      VCR 1.5 mg/m²                             Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

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Stage II/III High Risk Histology: High Risk Regimen HR-1 VP16 150 mg/m2   CARBO 200 mg/m2   CYCLO 450 mg/m2    DOX 50 mg/m2      RT             

Weeks 1-----2----/---3-----4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 

 = Echocardiography: at start of treatment, before week 19, 31 and at end of treatment  = GFR (measure at every third course, or more frequently if there is evidence of renal dysfunction.

19.11.2 Metastatic Wilms Tumours (stage IV)

Pre-operative chemotherapy

ACT 45 g/kg    VCR 1.5 mg/m2       DOX 50 mg/m2         Weeks 1 2 3 4 5 6

Group A1 Patients – regimen AVD150

ACT 45 µg/kg      VCR 1.5 mg/m²             DOX* 50 mg/m²     RT              Weeks 1 2< ---- 3------4----- >5 6 7 8* 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Groups: A2, B, and C – regimen AVD250

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Groups: C where nodules are viable and incompletely resected or representative resection is not feasible – 4-Drug Regimen (HR-2)

19.11.3 Bilateral WT

Chemotherapy guidelines Doses and administration of all drugs are as recommended in the protocol for unilateral tumours.

In case of Non-response (SD, PD or NSS is not possible)

VP16 150 mg/m2   CARBO 200 mg/m2        Weeks 1 2 3 4 5

19.11.4 WT after primary surgery

Regimen 1 (intensive VCR): Stage I, intermediate risk (excluding focal anaplasia).

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Regimen 2 (AV): Stage I, focal anaplasia and Stage II, intermediate risk

VCR 1.5 mg/m²            ACT-D 45 µg/kg     | | | | | | | | | | | Week 1 2 3 4 5 6 7 8 9 10 11 14 17 20 23 26

Regimen 3 (sequential AVD): Stage III intermediate risk (includes focal anaplasia)

VCR 1.5 mg/m²          ACT-D 22.5 g/kg * DOX 50 mg/m²  | |<-15Gy | RT---> | | | | | | Weeks 1 2 3 4 5 6 7 8 9

VCR 1.5 mg/m²     ACT-D 45 g/kg   DOX 50 mg/m²   | | | | | | | | | | Weeks 10 11 12 13 14 15 16 17 18 19 22 25 28

19.11.5 Relapsed WT Group AA

VP16 150 mg/m2   CARBO 200 mg/m2   CYCLO* 500 mg/m2/bd    DOX 50 mg/m2                   Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

*: CYCLO: 500 mg/m2 given every 12 hours for 2 days (difference to HR-1)

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Group BB

IFO* 2.000 mg/m2   CARBO* 560 mg/m2     VP16* 100 mg/m2   VP16* 100 mg/ m2   CYCLO* 440 mg/m2   Melphalan 200 mg/m2                 Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

*Dosages: Route of Drug Dose Day(s) administration i.v. 66.7 mg/kg/day for infants < 12 months Ifosfamide (I) 1-3 over 2 hours 2,000 mg/m² for children ≥ 12 months Cyclophosphamide i.v. 440mg/m2 (total 2.2 g/m2/course) 1-5 (Cy) over 15 min GFR Dose 560 mg/m2 > 150 ml/min/1.73m2 (18.7 mg/kg for infants) 500 mg/m2 100-150 ml/min/1.73m2 (16.6 mg/kg for infants) 370 mg/m2 Carboplatin i.v. 75-99 ml/min/1.73m2 (12.3 mg/kg for infants) 1 (Carbo) over 1 hour 290 mg/m2 50-74 ml/min/1.73m2 (9.7 mg/kg for infants) 200 mg/m2 30-49 ml/min/1.73m2 (6.7 mg/kg for infants) < 30 ml/min/1.73m2 Hold carboplatin Etoposide (VP16) i.v. 3.3 mg/kg/day for infants < 12 months 1-3 during ICE over 1 hour 100 mg/m2 for children ≥ 12 months Etoposide (VP16) i.v. 3.3 mg/kg/day for infants < 12 months 1-5 during CCE over 1 hour 100 mg/m2 for children ≥ 12 months GCSF 5 µg/kg/day subcutaneously from day 4 through ANC > 3,000/µl i.v. infusion over 3 hours during the administration of ifosfamide, at an equivalent ifosfamide dose (2,000 mg/m2/d for children > 12 months). with ifosfamide An additional Mesna 1,000 mg/m2/d dose is required in a parallel liquid infusion over 24 hours, during all the 3 days of ICE. Mesna Reduced dose for children < 12 months i.v. bolus of 200 mg/m2 immediately prior to first dose of with cyclophos- Cyclophosphamide followed by continuous Mesna at 1.0 gr/m2/day phamide until 12 hours after last dose of cyclophosphamide. Reduced dose for children < 12 months

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Group CC

As a priority these children should be referred to centres that are conducting research trials on novel agents in the treatment of children with solid tumours (see http://www.itcc-consortium.org/ for up-to- date news).

19.11.6 Adults with WT Stage I non-anaplastic (favourable) histology (Regimen 1)

ACT 1.5 mg/m2      VCR 1.5 mg/m2                       Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Stage II/III non-anaplastic (favourable) histology (Regimen 2) ACT 1.5 mg/m2      VCR 1.5 mg/m2         Dox 30 mg/m2       < ------RT------>            Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Stage IV, non-anaplastic histology Start with Regimen 2 as for stage II/III non-anaplastic. Further treatment is according to metastatic response.

Anaplastic histology, any stage and slowly responding stage IV non-anaplastic histology tumours (Regimen 3 = HR-1) VP16 150 mg/m2   CARBO 200 mg/m2   CYCLO 450 mg/m2    DOX 50 mg/m2      RT             

Weeks 1-----2------3-----4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 

 = Echocardiography: at start of treatment, before week 19, 31 and at end of treatment  = GFR (measure at every third course, or more frequently if there is evidence of renal dysfunction.

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19.12 Appendix 11: List of members of the different Subgroups

19.12.1 Biology Chair: Kathy Pritchard-Jones

Name Country Email

Peter Ambros Austria [email protected] Aurore Coulomb France [email protected] Jarno Drost The Netherlands [email protected] Manfred Gessler Germany [email protected] David Gisselsson Sweden [email protected] Ivo Leuschner Germany [email protected] Mariana Maschietto Brazil [email protected] Eckart Meese Germany [email protected] William Mifsud UK [email protected] Maureen O'Sullivan Ireland [email protected] Daniela Perotti Italy [email protected] Kathy Pritchard-Jones UK [email protected] Paolo Radice Italy [email protected] Marry van den Heuvel-Eibrink The Netherlands m.m.vandenheuvel- [email protected] Harm van Tinteren The Netherlands [email protected] Gordan Vujanic UK [email protected] Richard Williams UK [email protected]

19.12.2 Radiology Chair: Anne Smets

Name Country Email

Karoly Lakatos Austria [email protected] Ana Rizzi Argentina [email protected] Luc Breysem Belgium [email protected] Marleen Smet Belgium [email protected] Henrique Lederman Brazil [email protected] Ayda Aly Youssef Egypt [email protected] Hervé Brisse France [email protected] Jens-Peter Schenk Germany [email protected] Tse Kin Sun Hong Kong [email protected] Gábor Rudas Hungary [email protected] Carlo Morosi Italy [email protected] Dorota Sosnowska Poland [email protected] Lena Gordon (Sweden) Scandinavia [email protected] Mojca Glusic Slovenia [email protected] Ana Coma Spain [email protected]

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Christian Kellenberger Switzerland [email protected] Anne Smets The Netherlands [email protected] Eline Deurloo The Netherlands [email protected] Oystein Olsen United Kingdom [email protected] Anne Smets + NN All other [email protected] countries

19.12.3 Surgery Chair: Jan Godzinski

Name Country Email

Daniel C. Aronson UK [email protected] Georges Audry France [email protected] Torbjörn Backman Sweden [email protected] Davide Biasoni Italy [email protected] Gabriella Guillén Burrieza Spain [email protected] João Luís Castro Portugal [email protected] Giovanni Cechetto Italy [email protected] Tiago Henriques Coelho Portugal [email protected] Henrique Sa Couto Portugal [email protected] Virginie Fouguet France [email protected] Jörg Fuchs Germany [email protected] Frederic Gauthier France [email protected] Jan Godzinski Poland [email protected] Hugo Heij The Netherlands [email protected] Sabine Irtan France [email protected] Lars Johansen Denmark [email protected] Rosa Cabello Laureano Spain [email protected] Marc-David Leclair France [email protected] Bruce Okoye UK [email protected] Mark Powis UK [email protected] Guido Seitz Germany [email protected] Kees van de Ven The Netherlands [email protected] Dietrich von Schweinitz Germany [email protected] Maximilian Stehr Germany [email protected] Raimund Stein Germany [email protected] Steven Warmann Germany [email protected] Jim Wilde The Netherlands [email protected]

19.12.4 Pathology Chairs: Ivo Leuschner and Gordan Vujanic

Name Country Email

Isabela Werneck Cuhna Brazil [email protected] Aurore Coulomb France [email protected]

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Ivo Leuschner Germany [email protected] Paola Collini Italy [email protected] Ivo Leuschner Scandinavia [email protected] Enrique de Alava Spain [email protected] Christina Hulsbergen-van de Kaa The Netherlands [email protected] Gordan Vujanic United Kingdom [email protected] Gordan Vujanic / Ivo Leuschner All other countries [email protected] / [email protected]

19.12.5 Radiotherapy Chair: Christian Rübe

Name Country Email

Lorenza Gandola Italy [email protected] Aymeri Huchet France [email protected] Geert Janssens The Netherlands [email protected] Foppe Oldenburger The Netherlands [email protected] Christian Rübe Germany [email protected] Daniel Saunders UK [email protected] Stéphane Supiot France [email protected] Patrick Melchior Germany [email protected] Farida Alam UK [email protected]

19.12.6 Late effects Chair: Annelies Mavinkurve-Groothuis

Name Country Email

Monica Cypriano Brazil [email protected] Leo Kager Austria [email protected] Annelies Mavinkurve-Groothuis The Netherlands A.M.C.Mavinkurve- [email protected] Dennis Ku China [email protected] Patrick Melchior Germany [email protected] m.m.vandenheuvel- Marry van den Heuvel-Eibrink The Netherlands [email protected] Monica Terenziani Italy [email protected] Catriona Duncan UK [email protected] Helene Sudour France [email protected] Carlota Calvo Spain [email protected]

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19.12.7 Data management, Statistics and IT Chair: Harm van Tinteren

Name Country Email

Norbert Graf Germany [email protected] NN

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19.13 Appendix 12: Abbreviations

ABV Absolute blastemal volume ADC Apparent diffusion coefficient AIEP Italian Pediatric Oncology Group ALEA Remote Data Entry System CCSK Clear cell sarcoma CCLG Children’s Cancer and Leukaemia Group (UK) CG Coordination Group CMN Congenital mesoblastic nephroma COG Children’s Oncology Group CPR Central pathology review CRF Case Report Form CRR Central radiology review CT Computed tomography CI Confidence interval DCOG Dutch Childhood Oncology Group DHPLNR Diffuse Hyperplastic Periloar Nephrogenic Rests DICOM Digital Imaging and Communications in Medicine DWI Diffusion weighted imaging EFS Event free survival FFPE Formalin fixed paraffin embedded GALOP Latin American Group Pediatric Oncology GBTR Grupo Brazileiro de Tumores Renais GPOH German Society for Paediatric Oncology and Haematology IDC International Data Centre IMPORT Improving Population Outcomes for Renal Tumours of Childhood

LCMSMS Liquid chromatography mass spectrometry2 LOH Loss of heterozygosity MPLA Multiplex ligation-dependent probe amplification MRI Magnetic resonance imaging MRTK Malignant rhabdoid tumour of the kidney NB Nephroblastomatosis NC National coordinator

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NOPHO Scandinavian Pediatric Oncology Group NR Nephrogenic rest NGS Next generation sequencing NWTSG National Wilms Tumor Study Group ObTiMA Ontology-based Clinical Trial Management Application OS Overall survival p-medicine From data sharing and integration via VPH models to personalized medicine (EU project) PBV Percentage of blastema in Vital Tumour Volume (VTV) PI Principal Investigator PRT Percentage of the regressive tumour part RCC Renal cell carcinoma RTSG Renal Tumour Study Group SEHOP Spanish Pediatric Oncology Group SFCE French Pediatric Oncology Group SIOP International Society of Paediatric Oncology SNP Single nucleotide polymorphism SOP Standard operating procedure TMC Trial Management Committee US Ultrasound VPH Virtual physiological human (EU Institute) VTV Viable tumour volume WT Wilms Tumour WTV Whole tumour volume

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