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Algorithms for molecular testing in solid tumours

A. Hébrant, Ir, PhD1, M. Lammens, MD, PhD2, C. Van den Broecke, MD3, N. D’Haene, MD, PhD4, J. Van den Oord, MD, PhD5, A. Vanderstichele, MD, PhD6, A. Dendooven, MD, PhD7,8, P. Neven, MD, PhD6,9, K. Punie, MD, PhD10, G. Floris, MD, PhD7,11,12, J. Van der Meulen, Ir, PhD13, HA. Poirel, MD, PhD14, C. Dooms, MD, PhD15, S. Rottey, MD, PhD16, T. Boterberg, MD, PhD17, L. Brochez, MD, PhD18, MC. Burlacu, MD19, G. Costante, MD20, D. Creytens, MD, PhD8,21, P. De Paepe, MD, PhD22, R. De Pauwn, MD23, B. Decallonne, MD, PhD24, F. Dedeurwaerdere, MD25, H. Denys, MD, PhD16, L. Ferdinande, MD, PhD8,21, R. Forsyth, MD, PhD26, M. Garmyn, MD, PhD27, T. Gevaert, MD, PhD28,29, J. De Greve, MD, PhD30, E. Govaerts, MD31, E. Hauben, MD, PhD5,11, J. Kerger, MD32, O. Kholmanskikh Van Criekingen, MD, PhD33, V. Kruse, MD, PhD16, Y. Lalami, MD32, L. Lapeire, MD, PhD16,21, P. Lefesvre, MD, PhD34, JP. Machiels, MD, PhD35, B. Maes, MD, PhD36, G. Martens, MD, PhD37, M. Remmelink, MD, PhD4, I. Salmon, MD, PhD4, R. Sciot, MD, PhD11, S. Tejpar, MD, PhD38, K. Van de Vijver, MD, PhD8,21, L. Van de Voorde, MD39, I. Van den Berghe, MD22, A. Van den Bruel, MD40, K. Vandecasteele, MD, PhD17,21, L. Vanwalleghem, MD22, K. Vermaelen, MD, PhD41, R. Salgado, MD, PhD42, E. Wauters, MD, PhD15, B. Weynand, MD, PhD5, E. Van Valckenborgh, PhD1, G. Raicevic, PhD1, M. Van den Bulcke, PhD1, P. Pauwels, MD, PhD2

1Cancer Centre, Sciensano, Brussels, 2Department of Pathology, Antwerp University Hospital, and Centre for Oncological Research (CORE), University of Antwerp, Antwerp, 3Department of Pathology, AZ Sint-Lucas Ghent, Ghent, 4Department of Pathology, Hôpital Erasme - Université Libre de Bruxelles (ULB), Brussels, 5Department of Pathology, KU Leuven- University Hospitals Leuven, Leuven, Belgium, 6Department of Oncology, KU Leuven, Leuven, 7Department of Pathology, UZ Antwerp; MIPRO/LEMP, University of Antwerp, Antwerp, 8Department of Pathology, UZ Gent, Ghent, 9Department of Gynecology and Obstetrics, University Hospitals Leuven, Leuven, 10Department of General Medical Oncology. University Hospitals Leuven, 11Department of Imaging and Pathology, KU Leuven- University of Leuven, Leuven, 12Laboratory of Translational Cell & Tissue Research, Leuven, 13Molecular Diagnostics Ghent University Hospital (MDG), Ghent University Hospital, Ghent, 14Belgian Cancer Registry, Brussels, 15Department of Respiratory Diseases and Respiratory Oncology Unit, KU Leuven, Leuven, 16Department of Medical Oncology, Ghent University Hospital, Ghent, 17Department of Radiation Oncology, Ghent University Hospital, Ghent, 18Dermatology University Hospital Ghent, Cancer Research Institute Ghent (CRIG), Ghent, 19Department of Endocrinology, Cliniques Universitaires St-Luc, Brussels, 20Endocrinology Clinic, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, 21Cancer Research Institute Ghent (CRIG), Ghent University, Ghent, 22Department of Pathology, AZ Sint-Jan Brugge-Oostende, 23Department of Pulmonology, AZ Sint-Jan Brugge-Oostende, 24Department of Endocrinology, University Hospitals Leuven, 25Department of Pathology, AZ Delta, Roeselare, 26Department of Pathology, University Hospital Brussels and Experimental Pathology, Vrije Universiteit Brussel, Brussels, 27Department of Dermatology, University Hospitals Leuven, Leuven, 28Organs Systems, KU Leuven, Leuven, 29Department of Pathology, AZ Klina, Brasschaat, 30Department of Medical Oncology, Oncologisch Centrum, UZ Brussel, Vrije Universiteit Brussel, Brussels, 31Department of pneumology, AZ Sint-Lucas Hospital, Ghent, 32Medical Oncology, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, 33Federal Agency for Medicines and Health Products, Brussels, 34Department of Anatomic Pathology, UZ Brussel, Brussels, 35Department of medical oncology, Cliniques universitaires Saint-Luc, Brussels, 36Department of Clinical Biology, Jessa Hospital, Hasselt, 37Department of Laboratory Medicine, AZ Delta, Roeselare, 38Molecular Digestive Oncology, Kuleuven, Leuven, 39Department of Radiation Oncology, AZ Sint-Lucas, Ghent, 40Department of Internal Medicine, Endocrinology, AZ Sint Jan Brugge- Oostende, 41Departement of Pulmonary Medicine, Ghent University Hospital, Ghent, 42Department of Pathology GZA-ZNA, Antwerp. Please send all correspondence to: A. Hébrant, Ir, PhD, Rue Juliette Wytsman 14, 1050 Brussels, Belgium, tel: +32 2 642 57 32, email: aline. [email protected]. Conflict of interest: K. Punie declares to receive honoraria for consulting/advisory from Astra Zeneca, Novartis, Pfizer, Roche and Vifor Pharma as well as speaker fees from Novartis and Pfizer. Honoraria and fees were paid to the institution. Keywords: brain cancer, endometrium cancer, head and neck cancer, lung cancer, melanoma, molecular tests, NGS, oncology, ovarian cancer, soft tissue tumours, thyroid tumours. Acknowledgements: The authors would like to acknowledge all the ComPerMed experts.

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SUMMARY In order to advise the Federal Government on the reimbursement of molecular tests related to Personalised Medicine in Oncology, the Commission of Personalised Medicine (ComPerMed), represented by Belgian experts, has developed a methodology to classify molecular testing in oncology. The different molecular tests per cancer type are represented in algorithms and are annotated with a test level reflecting their rele- vance based on current guidelines, drug approvals and clinical data. The molecular tests are documented with recent literature, guidelines and a brief technical description. This methodology was applied on different solid tumours for which molecular testing is a clear clinical need. (BELG J MED ONCOL 2019;13(7):286-95)

INTRODUCTION type, the corresponding working group of the ComPerMed The clinical management of patients with cancer has become (Table 1) started with a systematic evaluation of all molecular more challenging with the advent of numerous innovative tests currently performed in clinical routine in Belgium, tak- molecular tests giving the opportunity to the care givers to ing into account their test utility (diagnostic, prognostic or tailor the patient’s management by fine-tuning the diagno- therapeutic), and then assigned, for each of them, a test lev- sis or by predicting the resistance or response to a thera- el. Three test levels (Table 2) were defined, with level 1 rep- py. In addition, test results may also provide information resenting the highest priority of implementation of the test on prognosis. by the policy makers. These test levels are assigned based on In order to advise the Federal Government on the reimburse- their use in clinical guidelines, expert opinions and on reim- ment of molecular tests related to Personalised Medicine in bursement rules for drugs in Belgium. This test level scale al- Oncology, the Belgian Commission of Personalised Medi- lows easy implementation of common biomarker testing into cine (ComPerMed) was created in 2016. This commission clinical practice. Furthermore, it facilitates the development includes representatives of all professional organisations di- of reimbursement criteria. rectly involved in personalised medicine, including experts In this paper, the methodology has been applied for lung, from the College of Genetics, the College of Oncology, the melanoma, brain, endometrium, ovarian, head and neck, soft Commission of Anatomic Pathology, the Commission of tissue, and thyroid tumours. Clinical Biology and different working groups with Belgian oncologists, haematologists, pathologists, clinical biologists, ALGORITHMS geneticists and other scientists. The ComPerMed is organised To design the molecular test algorithms, only tests with a lev- in different working groups according to the tumour type. el of 1 or 2A were retained. However, some tests with level 2B The ComPerMed advises the Platform Companion Diagnos- were also added to the algorithm if the expert group estimat- tics (CDx platform) from the Belgian reimbursement agency ed that they will become a test level 1 or 2A in the near future. National Institute for Health and Disability Insurance (NIH- For lung, melanoma, brain, endometrium, ovarian, head and DI) or RIZIV/INAMI on decision making related to the reim- neck, soft tissue and thyroid tumours, the different molecu- bursement of diagnostic tests in personalised medicine. The lar tests used in Belgium are sequentially represented in the CDx platform, launched in January 2016, aims to evaluate shape of algorithms (Figures 1-8). These algorithms are pub- the reimbursement of a molecular test and the correspond- lished on the ComPerMed website www.compermed.be/ with ing patient management in personalised medicine. additional information, such as tumour incidence (provid- The ComPerMed has set up a methodology in order to sys- ed by the Belgian Cancer Registry), and, for each molecular tematically evaluate and prioritise, for different tumour types, test, its utility (diagnostic, prognostic or therapeutic utility), the molecular tests currently performed in Belgium in clini- its corresponding test level and a brief technical test descrip- cal routine or in research phase.1 tion. In case of next generation sequencing (NGS) testing, the genes and regions that have to be analysed at minimum are METHODOLOGY also described. These algorithms will be reviewed annual- The methodology has been previously described and applied ly. Additional updates of the gene tests are possible when re- for digestive and breast tumours.2,3 Briefly, for each tumour quested by experts.

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TABLE 1. ComPerMed expert groups.

Lung tumour experts Endometrium tumour experts (to be continued) Jacques De greve Sabine Tejpar Rebecca De Pauw Koen Van de Vijver Franceska Dedeurwaerdere Ivo Van den Berghe Nicky D'Haene Katrien Vandecasteele ChristopheDooms Adriaan Vanderstichele Liesbeth Ferdinande Martens Geert Ovarian tumour experts Elke Govaerts Hannelore Denys Brigitte Maes Nicky D'Haene Patrick Pauwels Giuseppe Floris Myriam Remmelink Martens Geert Sabine Tejpar Thomas Gevaert Caroline Van den Broecke Joseph Kerger Lieve Vanwalleghem Patrick Neven Karim Vermaelen Patrick Pauwels Els Wauters Kevin Punie Birgit Weynand Sabine Tejpar Koen Van de Vijver Melanoma tumour experts Ivo Van den Berghe Lieve Brochez Katrien Vandecasteele Nicky D'Haene Adriaan Vanderstichele Marjan Garmyn Martens Geert Head and neck cancer experts Joseph Kerger Sylvie Rottey Vibeke Kruse Nicky D'Haene Brigitte Maes Esther Hauben Patrick Pauwels Yassine Lalami Sabine Tejpar Martin Lammens Ivo Van den Berghe Jean-Pascal Machiels Joost van den Oord Patrick Pauwels Myriam Remmelink Brain tumour experts Sabine Tejpar Tom Boterberg Lieve Vanwalleghem Pascale De Paepe Franceska Dedeurwaerdere Sarcoma experts Amelie Dendooven David Creytens Nicky D'Haene Pascale De Paepe Martens Geert Ramses Forsyth Martin Lammens Lore Lapeire Pierre Lefesvre Pierre Lefesvre Brigitte Maes Patrick Pauwels Patrick Pauwels Raf Sciot Isabelle Salmon Sabine Tejpar Raf Sciot Sabine Tejpar Thyroid tumour experts Lien Van De Voorde Maria-Christina Burlacu Caroline Van den Broecke Giuseppe Costante Brigitte Decallonne Endometrium tumour experts Nicky D'Haene Hannelore Denys Esther Hauben Nicky D'Haene Martin Lammens Giuseppe Floris Patrick Pauwels Martens Geert Isabelle Salmon Thomas Gevaert Sabine Tejpar Joseph Kerger Caroline Van den Broecke Patrick Neven Annick Van den Bruel Patrick Pauwels Lieve Vanwalleghem Kevin Punie

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TABLE 2. Test levels.

Standard of care biomarker for diagnosis and/or prognosis * 1 Biomarker predictive of response or resistance to a reimbursed drug in Belgium for this indication Recommended standard of care biomarker for diagnosis and/or prognosis + 2A Biomarker predictive of response or resistance to an EMA-approved drug for this indication Biomarker predictive of response or resistance to an EMA-approved drug for another indication or to a drug for 2B which a clinical trial is available for this indication Clinical evidence supporting the biomarker for diagnosis and/or prognosis °Biomarker predictive of response or 3 resistance to (1) a drug for which a clinical trial is not available in this indication or to (2) a compassionate use of drug * Standard of care: Included in guidelines (WHO…) AND consensus from ComPerMed experts + Recommended standard of care: Clinical evidence AND consensus from ComPerMed experts ° Preliminary evidence and no consensus from ComPerMed experts

CONCLUSION Other crucial initiatives are also ongoing to ensure Bel- To conclude, a systematic evaluation of new molecular tests gian healthcare quality, such as national quality control in different cancer types and a clear workflow for an optimal assessments of the molecular tests linked with their reim- clinical management of patients with cancer are required in bursements and the development of Belgian guidelines for order to further improve diagnosis, knowledge of progno- homogeneous interpretation and reporting of the molecu- sis and treatment in the era of personalized medicine. Test lar test results. levels have to be used to classify the molecular tests regard- ing their potential clinical utility. Standard-of-care tests (lev- REFERENCES el 1or 2A) are necessary for the proper management of the 1. Hébrant A, Van Valckenborgh E, Salgado R, et al. Opportunities and chal- cancer patient and therefore require appropriate reimburse- lenges in oncology and molecular testing: the Belgian strategy. Belg J Med ment. A regular update of the reimbursement rules is nec- Oncol. 2018;12:16-50. essary to include molecular tests that reached a higher test 2. Hébrant A, Jouret-Mourin A, Froyen G, et al. Molecular test algorithms for level or to cancel reimbursement of tests that are outdated. digestive tumours. Belg J Med Oncol. 2019;13:4-10. This will avoid extra costs for laboratories, patients and in 3. Hébrant A, Punie K, Duhoux FP, et al. Molecular test algorithms for breast the end the community. tumours. Belg J Med Oncol. 2019;13:40-5.

KEY MESSAGES FOR CLINICAL PRACTICE

1. For an optimal and harmonised management of cancer patients in Belgium, a Commission of Personalised Medicine (ComPerMed) has set up workgroups of Belgian experts, to develop and update molecular testing algorithms.

2. This manuscript presents the updated algorithms for the standard-of-care testing of lung, melanoma, brain, endometrium, ovarian, head and neck, soft tissue and thyroid tumour samples.

3. A common test level scale allows easy implementation of biomarker testing into clinical practice and facilitates the development of reimbursement criteria.

4. Molecular testing algorithms are useful tools to link the molecular test and the therapy reimbursement by the INAMI/RIZIV.

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Figure 1.A-C: Lung cancer algorithms. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level > 2A are in a grey rectangle.

A

IF squamous cell carcinoma

IF never-smoker IF smoker

See algorithm B TEST: PDL1 (IHC)

depending on PDL1 status: - Immunotherapy (monotherapy or combination with chemotherapy) - chemotherapy

B IF non squamous cell carcinoma (presence of an ADC component OR poorly differentiated carcinomas where an ADC cannot be excluded)

In parallel or In parallel or TEST: Mutational status of (EGFR, KRAS, In parallel or TEST: PDL1 TEST: rearr. (ALK) (IHC) TEST: rearr. (ROS1) (IHC) sequentially sequentially BRAF, MET 14 exon skipping, HER2) (PCR for sequentially (IHC) EGFR, NGS) IF IHC ALK positive IF IHC ROS1 positive depending on PDL1 status: - Immunotherapy IF EGFR OR BRAF OR IF KRAS TEST: rearr. (ALK) (ISH) TEST: rearr. (ROS1) (ISH) MET 14 exon skipping mut (monotherapy or OR HER2 mut combination with chemotherapy) - chemotherapy IF rearrangement IF rearrangement

Targeted therapy Mutually exclusive with (EGFR, ALK, RET, BRAF, Targeted therapy Targeted therapy ROS1) and stop molecular testing

C

IF progression of any type of lung cancer under targeted therapy à rebiopsy

TEST: Mutational status of EGFR (T790M) (PCR) (on tissue or liquid biopsy) AND/OR of other genes involved in resistance

Treatment

Optional targets

TEST: Clinical trials/compassionate AND/ TEST: MET copy number analysis AND/ TEST: rear. (RET) (NGS, AND TEST: rear. (TRK) (ISH use (NGS) OR (NGS, ISH) OR ISH) /OR and/or IHC, NGS)

FIGURE 1A-C. Lung cancer algorithms. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level >2A are in a grey rectangle.

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Figure 2: Melanoma algorithm. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level > 2A are in a grey rectangle.

IF metastatic OR lymph node metastasis stage III melanoma

TEST: Mutational status of BRAF V600 (PCR) OR TEST: Mutational status of (BRAF, NRAS, c-KIT) (NGS)

IF BRAF WT IF NRAS mut IF NRAS WT IF BRAF mut (V600)

Immunotherapy (in combination or monotherapy)

IF progression

TEST: Mutational status of (NRAS, c-KIT) (if not yet done) (NGS)

immunotherapy OR BRAFi/MEKi IF no mutation IF c-KIT mut

Treatment (c-KIT inhibitors)

IF progression

IF progression after MAPK inhibitors TEST: Clinical trials/compassionate use (NGS)

Clinical trials OR compassionate use / immunotherapy (monotherapy or in combination)

FIGURE 2. Melanoma algorithm. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level >2A are in a grey rectangle.

Figure 3.A-C: Adult brain cancer algorithms. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level > 2A are in a grey rectangle.

A IF morphology of oligodendrogliomas OR diffuse astrocytomas

TEST: IDH1 AND ATRX AND GFAP AND in some cases SYN AND NF (IHC)

IF IDH1 + AND ATRX - IF IDH1 + AND ATRX + IF IDH1 – AND ATRX +/-

TEST: Mutational status of (IDH1, TEST: 1p/19q codeletion (ISH, NGS, LOH, …) IF IDH1 OR IDH2 mut IDH2) (NGS)

IF codeletion - IF codeletion + IF no mutation

Diffuse astrocytoma, IDH-wildtype Anaplastic astrocytoma, IDH-wildtype Diffuse astrocytoma, IDH-mutant Oligodendroglioma, (Reconsider also ‘circumscribed’ astrocytoma’s (pilocytic astrocytoma, pleiomorphic Anaplastic astrocytoma, IDH- IDH-mutant and xanthoastrocytoma, ganglioglioma) on morphological base and IHC (add NeuN, Syn, NF)) mutant 1p/19q-codeleted Midline glioma H3K27 mutated Anaplastic Oligodendroglioma,I DH-mutant 1p/19q- codeleted TERT promoter testing (NGS, PCR …) EGFR high level amplification testing (FISH, If glioma on midline NGS) TEST: mutational status of (H3K27) Chr -7, +10 (FISH) (NGS or IHC) BRAF - Fusion testing (RNA seq) BRAF V600E testing (PCR, NGS, IHC)

If pTERT mut or EGFR amplification or Midline glioma H3K27 mutated CHr+7,-10 Diffuse astrocytoma, IDH-wt, with molecular features of GBM

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B IF morphology of glioblastoma

IF the combination of radio- and chemotherapy is not feasible (depending of the patient’s fitness)

TEST: Methylation MGMT (PCR,…)

IF no hyper methylation

Adapt treatment

TEST: IDH1 AND Ki67 AND GFAP AND CD45 (if GFAP negative) (IHC)

IF IDH1 + IF IDH1 -

IF younger than 55 à TEST: Mutational status of (IDH1, IDH2) (NGS) IF diffuse pediatric gliomas: add HIST1H3B/C, H3F3A, BRAF

TEST: 1p/19q codeletion (ISH OR LOH OR …) IF IDH1 OR IDH2 mut IF BRAF mut IF IDH1 OR IDH2 WT IF H3F3A mut AND on midline

IF codeletion - IF codeletion + Anaplastic pleomorphic Glioblastoma IDH WT Diffuse glioma xanthostrocytoma H3K27 mut Glioblastoma IDH Anaplastic Oligodendroglioma IDH OR epitheloid gioblastoma mut mutated and 1p19q codeleted

C IF Medulloblastoma IF Pilocytic Astrocytoma

Describe morphology: Classic/Nodular- TEST: BRAF fusion gene (KIAA1549-BRAF) (ISH, desmoplastic/Extensive nodularity/anaplastic-large cell RNA seq) TEST: BRAF (V600) mutational status (PCR, NGS) TEST: Beta-catenine/CTNNB1 (IHC and NGS) (if positive: WNT-type = better prognosis ) TEST: GAP1 (IHC) if positive: WNT or SHH- type TEST: YAP1 (IHC) if positive: SHH-type IF SHH: test p53 (NGS) SHH + p53 -: better prognosis SHH + p53 mut: worse prognosis If WNT– and SHH - : nonWNT-nonSHH type

In all cases: test amplification MYC and NMYC (FISH or alternative method (NGS)) à if amplification: worse prognosis, not admitted to several studies.

If non WNT non SHH: perform methylation array to distinguish between type 3 and type 4 medulloblastoma.

FIGURE 3A-C. Adult brain cancer algorithm. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level >2A are in a grey rectangle.

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Figure 4: Endometrium cancer algorithm. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level > 2A are in a grey rectangle.

IF metastatic endometrial TEST: ER status AND PgR status (IHC) Other patients carcinoma

TEST: mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, PMS2) (IHC) AND/OR TEST: MSI (PCR)

IF Loss of MLH1 AND PMS2 IF Loss of (MSH2 and/or MSH6) IF No loss of nuclear expression for MMR (MSI-H) OR PMS2 proteins (MSS and/or MSI-L) (MSI-H)

TEST: methylation of MLH1 promoter Family history suspect for Lynch syndrome? Or other familial cancer syndrome? Suspicious Lynch Grade 3 endometrioid or serous If TEST + If TEST - syndrome Age <50

Genetic counseling IF Yes IF No Sporadic tumor IF no mut

TEST: mutational status of POLE Exon 9 and exon 13 (P286R/H/L and V411L) (PCR)

IF POLE mut IF POLE wt

TEST: TP53 expression (IHC) Adapt patient management TEST: Clinical trials/compassionate use (NGS)

Clinical trials OR compassionate use

FIGURE 4. Endometrium cancer algorithm. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level >2A are in a grey rectangle.Figure 5: Ovarian cancer algorithm. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level > 2A are in a grey rectangle.

All non-mucinous and non- borderline epithelial ovarian cancers AND fallopian tube and primary peritoneal cancer

TEST: Germline genetic testing in all cases AND/OR TEST: Mutational status of (BRCA1, BRCA2) (NGS) (on tumor sample)

IF germline BRCA1 OR BRCA2 mut IF somatic BRCA1 OR BRCA2 mut IF somatic BRCA1 OR BRCA2 wt AND relapse

Genetic counselling TEST: HRD

Adapt patient management IF HRD + IF HRD -

TEST: Clinical trials/compassionate use (NGS)

Clinical trials OR compassionate use OR standard treatment

FIGURE 5. Ovarian cancer algorithm. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level >2A are in a grey rectangle.

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Figure 6: Head and neck cancer algorithm. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level > 2A are in a grey rectangle.

Oropharyngeal carcinoma

TEST: HPV (ISH, PCR, …) OR/AND TEST: p16 (IHC,…)

Prognosis

IF progression OR recurrence

TEST: Clinical trials/compassionate use (NGS)

Clinical trials OR compassionate use

FIGURE 6. Head and neck cancer algorithm. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level >2A are in a grey rectangle.

Figure 7: Soft tissue tumor algorithm. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level > 2A are in a grey rectangle.

TEST: 8 - 15 IHC

IF morphology of desmoid tumor Definitive diagnosis STS Inconclusive diagnosis STS

TEST: beta-catenin (IHC) TEST: 2-6 ISH

IF Typical clinic IF Atypical clinic Definitive diagnosis STS And/or Typical morphology and/or AND IHC beta-catenin positive Atypical morphology and/or Treatment IHC beta-catenin inconclusive

TEST: Mutational status of IF progression AND/OR metastasis CTNNB1 (PCR)

TEST: Clinical trials/compassionate use (NGS) IF CTNNB1 mut NTRK fusion (IHC then RNA seq)

Desmoid Desmoid Clinical trials OR compassionate use

FIGURE 7. Soft tissue tumour algorithm. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level >2A are in a grey rectangle.

ABBREVIATIONS: BRAFi: BRAF inhibitor, FISH: Fluorescent In Situ Hybridization, FNA: Fine Needle Aspiration, GBM: Glioblastoma Multiforme, IHC: Immunohistochemistry, ISH: in situ hybridization, LOH: Loss Of Heterozygosity, MEKi: MEK inhibitor, MSI-H: Microsatellite instability - High, MSI-L: Microsatellite instability - Low, MSS: Microsatellite Stable, Mut: Mutated, NGS: Next Generation Sequencing, PCR: Polymerase Chain Reaction, Rearr: Rearrangement, RNA seq: RNA sequencing, SHH: Sonic Hedgehog, STS: Soft Tissue Sarcoma, WT: Wild Type, WNT: Wingless.

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Figure 8.A-C: Thyroid tumor algorithms. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level > 2A are in a grey rectangle.

On FNA or biopsy, IF follicular neoplasm cytology (Bethesda class 3 or 4) A AND clinical/ ultrasound risk LOW

TEST: Mutational status of (BRAF, KRAS, HRAS, NRAS) (NGS)

IF mutation IF no mutation

TEST: Fusion gene detection of (RET/PTC1, RET/PTC3, PAX8/PPARg) (PCR, ISH, RNA seq)

IF rearrangement IF no rearrangement

Lobectomy or total Lobectomy or total Follow-up rather than thyroidectomy * thyroidectomy * lobectomy *

* Also taking into account other clinical/imaging characteristics (e.g. nodule diameter, multinodularity) and the patient’s preference

B On FNA or biopsy, IF follicular neoplasm cytology (Bethesda class 3 or 4) AND clinical/ ultrasound risk HIGH B On FNA or biopsy, IF follicular neoplasm cytology (Bethesda class 3 or 4) AND clinical/ ultrasound risk HIGH TEST: Mutational status of (BRAF, KRAS, HRAS, NRAS) (NGS)

TEST: Mutational status of (BRAF, KRAS, HRAS, NRAS) (NGS) IF mutation IF no mutation

IF mutation IF no mutation TEST: Fusion gene detection of (RET/PTC1, RET/PTC3, PAX8/PPARg) (PCR, ISH, RNA seq) TEST: Fusion gene detection of (RET/PTC1, RET/PTC3, PAX8/PPARg) (PCR, ISH, RNA seq) IF rearrangement IF no rearrangement

IF rearrangement IF no rearrangement Total thyroidectomy rather than Total thyroidectomy rather Lobectomy lobectomy * than lobectomy * rather than follow-up * Total thyroidectomy rather than Total thyroidectomy rather Lobectomy lobectomy * than lobectomy * rather than follow-up *

* Also taking into account other clinical/imaging characteristics (e.g. nodule diameter, multinodularity) and the patient’s preference * Also taking into account other clinical/imaging characteristics (e.g. nodule diameter, multinodularity) and the patient’s preference C On FNA or biopsy IF Bethesda class 5 or 6 C On FNA or biopsy IF Bethesda class 5 or 6

TEST: Mutational status of (BRAF) (PCR) TEST: Mutational status of (BRAF) (PCR)

IF mutation IF mutation

Consider more aggressive management* Consider more aggressive management*

* Also taking into account other clinical/imaging characteristics (e.g. nodule diameter, * Also taking into accountmultinodularity other clinical) and/imagingthe patient’s characteristicspreference(e.g. nodule diameter, multinodularity) and the patient’s preference

FIGURE 8A-C. Thyroid tumour algorithms. Molecular tests with level 1 or 2A are represented in a red rectangle, molecular tests with a test level >2A are in a grey rectangle.

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