Prognostic Markers and Response to Vandetanib Therapy in Sporadic Medullary Thyroid Cancer Patients

175:3

V Tiedje, S Ting and others Prognosis and therapy 175:3 173–180 Clinical Study response in sporadic MTC

Prognostic markers and response to vandetanib therapy in sporadic medullary thyroid cancer patients

Vera Tiedje1,*, Saskia Ting2,*, Robert Fred Walter2,3, Thomas Herold2,4, Karl Worm2, Julia Badziong1, Denise Zwanziger1, Kurt Werner Schmid2 and Dagmar Führer1

1Department of Endocrinology and Metabolism, 2Institute of Pathology, 3Department of Interventional Pneumology, Ruhrlandklinik, West German Lung Centre, University Hospital Essen, University of Duisburg-Essen, Essen, Germany, and 4German Cancer Consortium (DKTK), German Cancer Research Correspondence Center (DKFZ), Heidelberg, Germany should be addressed *(V Tiedje and S Ting contributed equally to this work) to V Tiedje Email [email protected]

Abstract

Objective: Medullary thyroid carcinoma (MTC) occurs sporadically in 75% of patients. Metastatic disease is associated with significantly poorer survival. The aim of this study was to identify prognostic markers for progressive MTC and oncogenic factors associated with response to vandetanib therapy. Design and methods: Clinical courses of 32 patients with sporadic MTC (n = 10 pN0cM0, n = 8 pN1cM0, n = 14 pN1cM1) were compared with genetic profiles of the patients’ primary tumour tissue. Analysis for RET proto-oncogene mutations was performed by Sanger sequencing and next-generation sequencing (NGS). The mRNA expression (mRNA count) of 33 targets was measured by nCounter NanoString analysis. Results: Somatic RET mutations occurred in 21/32 patients. The RET918 mutation was found in 8/14 pN1cM1 patients. BRAF (P = 0.019), FGFR2 (P = 0.007), FGFR3 (P = 0.044) and VEGFC (P = 0.042) mRNA expression was significantly lower

European Journal European of Endocrinology in pN1cM0/pN1cM1 compared with pN0cM0 patients, whereas PDGFRA (P = 0.026) mRNA expression was significantly higher in pN1cM0/pN1cM1 when compared with pN0cM0 patients. Among the 10/32 vandetanib-treated patients, 5 showed partial response (PR), all harbouring the RET918 mutation. mRNA expression of FLT1 (P = 0.039), FLT4 (P = 0.025) and VEGFB (P = 0.042) was significantly higher in therapy responders. Conclusions: In this study, we identified molecular markers in primary tumour tissue of sporadic MTC associated with the development of metastasis (both lymph node and organ metastasis) as well as response to vandetanib therapy.

European Journal of Endocrinology (2016) 175, 173–180

Introduction

Medullary thyroid carcinoma (MTC) is a rare thyroid is known between the type of RET mutation and onset cancer and accounts for approximately 5% of all thyroid of MTC (2). However, in 75% of patients sporadic MTC is malignancies. Curation is only possible by surgery. diagnosed. In these MTC somatic RET and RAS mutations, Advanced, metastasized disease results in poorer prognosis which occur mutually exclusive, are apparently the driver with a 10-year survival of 40% or less (1). In approximately mutations (3, 4). 25% of cases MTC occurs hereditary, due to a germline According to the American Thyroid Association (ATA) RET proto-oncogene mutation. In these patients with risk classification of RET germline mutations, RET918 multiple endocrine neoplasia type 2, a close correlation mutations confer as the highest risk for early MTC

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10.1530/EJE-16-0252 Clinical Study V Tiedje, S Ting and others Prognosis and therapy 175:3 174 response in sporadic MTC

manifestation and an aggressive clinical course. In sporadic approved by the Ethics Committee of the Medical Faculty, MTC, the somatic RET918 mutation has been found as University Duisburg-Essen (Germany). Clinical data, the most frequent RET mutation and has been linked to including age at diagnosis, sex, TNM stage, postoperative poorer survival (5, 6). Whether there is a correlation of calcitonin levels, and response to tyrosine kinase inhibitor other somatic RET mutations with distinct prognosis in (TKI) therapy, was analysed retrospectively. Ten patients sporadic MTC patients is currently not known (7). received vandetanib therapy after tumour progress was In addition, activation of intracellular tyrosine kinase radiologically demonstrated according to the Response pathways has been linked to RET mutational status. Thus, Evaluation Criteria In Solid Tumors (RECIST) version 1.1. Maliszewska et al. (8) reported upregulation of genes (11); response to therapy was classified as partial response involved in the Wnt, Notch, NF-κB, JAK/STAT and MAPK (PR), stable disease (SD), (12) or progressive disease (PD) signalling pathways in sporadic MTC harbouring the 3 months after initiation of therapy. The time point 3 Met918Thr RET mutation. A RET-associated expression of months was chosen to identify MTC patients with a tyrosine kinases was also described by Rodriguez-Antona primary vandetanib resistance. et al. (9), whereby MTC with RET wildtype status showed Formalin-fixed paraffin-embedded (FFPE) tissues of a lower protein expression of PDGFRB than MTC with a the primary tumour were available in all cases of this somatic RET mutation. study; diagnosis of MTC was reviewed by a board-certified, However, whether somatic RET status and tyrosine experienced thyroid pathologist (KWS). Hereditary kinase gene expression pattern are linked and associated MTC was excluded in all patients by germline RET gene with different disease course has not yet been elucidated. mutation analysis from peripheral blood samples. The knowledge of the molecular pathology of MTC has led to development of therapies targeting RET and Sanger sequencing of RET gene other tyrosine kinase signalling. Currently, two drugs are approved for treatment of progressive metastatic MTC, DNA was isolated from the primary tumour tissues with that is, vandetanib (European Medicines Agency (EMA) the QIAamp DNA FFPE tissue kit (Qiagen). For Sanger and Food and Drug Administration (FDA) approved), a sequencing, RET exons 10, 11 and 13–16 were amplified RET, VEGFR2 (KDR), VEGFR3 (FLT4) and EGFR inhibitor by PCR. The PCR reaction was carried out in a total (2) and cabozantinib (FDA approved, conditional EMA volume of 50 µL with AmpliTaq Gold DNA Polymerase approved), a RET, VEGFR2, MET, KIT, AXL and FLT3 (Life Technologies), according to the recommendations inhibitor (2, 10). A subanalysis with regard to progression- of the supplier. Biometra T3000 Thermocycler (Biometra, European Journal European of Endocrinology free survival (PFS) in RET mutated vs RET wildtype Germany) was used for amplification. DNA sequences patients was performed in a phase III trial and a benefit of both strands, forward and reverse, were analysed on for PFS was suggested if a Met918Thr RET mutation was an ABI 3500 Genetic Analyzer (Life Technologies), using present in the tumour (2). BigDye Terminator chemistry (Life Technologies). In this study, we determined RET mutation status by Sanger sequencing and next-generation sequencing (NGS) and performed quantitative expression analysis of a panel Next-generation sequencing of RET gene of 33 tumour-cell and endothelial-cell tyrosine kinases in For NGS, the MiSeq Illumina (Illumina, San Diego, sporadic MTC patients with long-term follow-up. Our aim CA, USA) was used following manufacturer’s instructions. was to delineate prognostic markers for metastasized MTC DNA concentrations were determined by Qubit 2.0 and to identify factors associated with response or absent Fluorometer dsDNA HS assay kit (Life Technologies). response to vandetanib therapy. Multiplex PCR and purification were performed with the GeneRead Gene Custom Panel, DNAseq Panel PCR Kit (Qiagen) and Agencourt AMPure XP Beads (Beckman Methods Coulter, Brea, CA, USA), followed by measurement of total DNA amount by Qubit 2.0 Fluorometer dsDNA HS Patients and tumour samples assay kit. The library preparation was performed with In this study, 32 patients with sporadic MTC were included, NEBNext Ultra DNA Library Prep Set for Illumina (New all treated at the University Hospital Essen (Germany), a England Biolabs, Ipswich, MA, USA), according to the tertiary centre for endocrine malignancies. The study was manufacturer’s recommendations by using 24 different

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Downloaded from Bioscientifica.com at 09/29/2021 06:01:19PM via free access European Journal of Endocrinology MTC95 MTC91 MTC85 MTC84 MTC75 MTC71 MTC70 MTC65 MTC63 MTC58 MTC56 MTC54 MTC51 MTC48 MTC45 MTC31 concentration of200 Technologies). EachsamplewasadjustedtoatotalRNA dye-based quantificationmethod(Qubit,Life concentrations weredeterminedbyusingafluorescent with miRNAeasyFFPEkit(Qiagen)andtotalRNA tumour tissues RNA was isolated from theprimary prestatistical analysis Nanostring nCounterpreparation and average coverageofapproximately8000×wasobtained. of interest and hotspots of the a customizedMTCpanelwasdesignedcontainingregions Workbench (CLCBio,Qiagen).Fortargetedsequencing, MiSeq (Illumina)andanalysedbytheCancerResearch wassequencedon indices perrun.Thepooledlibrary MTC27 MTC24 MTC23 MTC21 MTC19 MTC18 MTC16 MTC15 MTC14 MTC13 MTC12 MTC8 MTC5 MTC4 MTC3 F, female;M,male. MTC1 Table 1 Clinical Study F M F F F F F F F F M F F F M F M F F M F M F M M M F M F F M F Sex Patient characteristics,somaticRETmutationstatusandATA risklevel. Age atdiagnosis 70 65 30 86 54 72 55 63 49 60 80 64 54 75 79 39 85 50 43 44 45 55 56 55 58 53 45 32 29 50 46 58 ng/µL forthenCounterpreparation pT4apN1bcM0 pT2pN0cM0 pT1ap N1bcM0 pT3pN1acM0 pT3pN1bcM0 pT4pN1cM0 pT1pN1cM0 pT1bpN1cM0 pT2apN0cM0 pT1apN0cM0 pT2pN1bcM0 pT3pN0cM1 pT1pN1cM1 pT1pN0cM0 pT1apN0cM0 pT2pN0cM0 pT2pN1cM0 pT3bpN1cM1 pT3pN1cM1 pT1pN1cM0 pT3pN1cM0 pT4pN1cM0 pT1pN1cM0 pT3pN1cM0 pT4pN1cM0 pT1bpN0cM0 pT1pN0cM0 pT2pN1bcM1 pT4pN1cM0 pT3pN1cM0 pT2pN1cM1 follow-up TNM attheendof pT1pN0cM0 RET gene. In all runs, an V Tiedje, STing andothers Postoperative Postoperative calcitonin 31.866 (pg/mL) 1314 2363 7486 248 119 135 797 830 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 13 52 96 42 12 13 64 3 7 3 5 x c.1888T>C /p.Cys630Arg c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.1946C>T /p.Ser649Leu c.1900T>C /p.Cys634Arg c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.1900T>C /p.Cys634Arg c.1858T>A /p.Cys620Ser c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.1858T>C /p.Cys620Arg c.2753T>C /p.Met918Thr Sanger sequencing USA) at555fieldsofview(FOV). Digital Analyzer(NanoStringTechnologies, Seattle,WA, the next4 WA, USA)orcooledto4°Cuntilfurtherproceedingwithin nCounter PrepStation(NanoStringTechnologies, Seattle, After hybridization,samplesweredirectlyloadedintothe Germany) for1.300 performed inanEppendorfMastercycler nexus(Hamburg, nCounter Elements manual. The final hybridization was The samplepreparationwasperformedaccordingtothe by IntegratedDNATechnologies (Coralville,IA,USA). A (5 (NanoString Technologies, Seattle,WA, USA).ProbePools TagSet-48 andreagentsaccordingtothesupplierwereused analysis, theGeneralPurposeReagentnCounterElements following establishedprotocols(13).Forgeneexpression response insporadicMTC Prognosis andtherapy nM peroligo)andB(25 h. SampleswereanalysedonthenCounter min at67°Cwithaheatedlid(72°C). Downloaded fromBioscientifica.com at09/29/202106:01:19PM c.1902 C>G/Cys634Trp c.1888T>C /p.Cys630Arg c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.1946C>T /p.Ser649Leu c.1900T>C /p.Cys634Arg c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.1946C>T /p.Ser649Leu c.1900T>C /p.Cys634Arg c.2753T>C /p.Met918Thr c.1900T>C /p.Cys634Arg c.1858T>A /p.Cys620Ser c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.2753T>C /p.Met918Thr c.1858T>C /p.Cys620Arg c.2753T>C /p.Met918Thr sequencing Next-generation nM peroligo)weresupplied 175:3 www.eje-online.org Exon 11 Exon 11 Exon 16 Exon 16 Exon 16 Exon 16 Exon 16 Exon 16 Exon 16 Exon 11 Exon 16 Exon 16 Exon 11 Exon 11 Exon 16 Exon 11 Exon 10 Exon 16 Exon 16 Exon 16 Exon 10 Exon 16 Exon 175 via freeaccess Clinical Study V Tiedje, S Ting and others Prognosis and therapy 175:3 176 response in sporadic MTC

The raw data were analysed according to the Table 2 Characteristics of vandetanib-treated patients. following automated protocol: a background correction Characteristics Number was performed, calculating the average counts for the Sex negative controls plus two times the standard deviation. Female 6 These values were subtracted from all sample counts. The Male 4 geNorm algorithm (14) was used to identify the three Age at diagnosis* 58 ± 10 most stable reference genes out of a set of nine reference Site of metastasis Lung 2 genes (ACTB, GAPDH, HPRT1, GUSB, CLTC, NEDD8, PGK1, Lung + bone 5 TTC1, TUBB). HPRT1, NEDD8 and TTC1 were identified Lung + bone + liver 2 to be the most stable reference genes. A normalization Lung + bone + cerebral 1 Patients with PD under therapy 7 factor was calculated by using the geometric mean of the Median PFS, months 7 mRNA counts of the three most stable reference genes. Deceased patients 6 Each sample was normalized using the sample-specific Previous TKI treatment normalization factor. The normalized data were used for Sorafenib 3 Cabozantinib 2 further statistical analysis (15).

*Age in years (mean ± s.d.). PD, progressive disease; PFS, progression-free survival. Statistical analysis PD according to RECIST criteria 1.1. Characteristics of the Statistical analysis was performed by IBM SPSS Statistics vandetanib-treated patients are listed in Table 2. Among Version 21. For comparison of mRNA count between MTC these 10 patients, 6 were treated as part of a clinical trial subgroups, Student’s t-test was performed. Correlation of (ClinicalTrials.gov NCT01298323). clinicopathological parameters with presence of sporadic Sanger sequencing of the RET proto-oncogene RET mutations was calculated by Chi square. Statistical revealed somatic RET mutations in the primary tumour significance was set at aP -value <0.05. tissues of 20 patients with sporadic MTC. The most frequent RET mutation was the Met918Thr mutation Results (14/20, 70%), followed by the Cys634Arg mutation (2/20, 10%). Further somatic RET mutations, each found The clinical data of the 32 sporadic MTC included in this in only one case, included RET codons 620 (Cys620Arg

European Journal European of Endocrinology study are listed in Table 1. The median age at diagnosis was and Cys620Ser), codon 630 (Cys630Arg) and codon 649 56 years. The patients were observed until December 2014 (Ser649Leu). Through NGS, one additional MTC with a with a median observation period of 42 (3–159) months. RET mutations was found, which had not been detected Until then, 10 patients had disease-free status (pN0cM0), by Sanger sequencing. Two further cases with somatic RET 8 patients had lymph node metastases (pN1cM0) and 14 mutations already revealed by Sanger sequencing showed patients developed distant metastatic disease (pN1cM1). additional mutations in NGS. The detailed list of the Disease-free status was defined as biochemical and somatic RET mutations identified in 32 sporadic MTCs is structural cure (calcitonin and CEA not detectable) during shown in Table 1. follow-up. Nine patients died of MTC during follow-up. The frequency of RET mutations in MTC in patients Among the 32 patients, 14 received TKI treatment. Ten with metastatic (pN1cM0 and pN1cM1) disease at the end out of these 14 patients were started on vandetanib due to of follow-up (16/23, 69.57%) differed from the frequency

Figure 1 mRNA count in pN0cM0 vs pN1cM0/pN1cM1 patients.

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Downloaded from Bioscientifica.com at 09/29/2021 06:01:19PM via free access European Journal of Endocrinology of tumour tissues. Wein the primary found that expression endothelial-cell-derived tyrosinekinaseswasdetermined mRNA count for a panel of 33 distinct tumour-cell or this, quantitativenCounteranalysiswasemployedand metastatic disease(pN1cM1)attheendoffollow-up.For or developinglymphonodular(pN1cM0) or distant confined to thethyroid (pN0cM0) vs patients showing tyrosine kinasesdiffered in tumoursofpatientswithMTC two patientswithmetastaticdisease. andin with pN0cM0stage,whowerecuredbysurgery Cys634Arg mutationwasfoundinMTCoftwopatients vs non-metastaticMTC(56.52%10.00%,P detected withsignificantlyhigherfrequencyinmetastatic non significantP in MTCofpatientswithpN0cM0-stage(5/10,50.00%, of Met918Thr mutationhadsignificantlyhigherexpression ). EspeciallyMTCharbouringaRET wildtype status(Fig. 2 (P upregulation ofAKT1(P pattern oftyrosinekinasesshowedaproportional metastases (P tumour tissues,inparticularpatientswithlymphnode of were significantlydownregulated,whereasexpression case ofmetastaticdisease,BRAF, metastatic MTC(pN1cM0andpN1cM1stages)(Fig. 1 ). In significantly betweenMTCwithpN0cM0stagesand PDGFRA (P somatic RETmutations,significantupregulationofFGFR1 wildtype status.Within thegroupofMTCharbouring and Clinical Study = was significantly upregulated in the primary PDGFRA wassignificantlyupregulatedintheprimary BRAF (P AKT1 (P 0.021) in MTC with somatic RET mutations vs RET Furthermore, weaskedwhetherexpressionpatternof Comparison ofRETmutationstatuswithexpression PDGFRA (P = 0.019), = 0.030), .2) and = 0.026) = 0.007). = = 0.032) comparedwithMTCRET 0.411). TheMet918Thrmutationwas FGFR2 (P FGFR1 (P = 0.045), VEGFC (P = 0.007), FGFR2, = 0.038), KRAS (P V Tiedje, STing andothers FGFR3 andVEGFC FGFR3 (P .4) differed = 0.042) 0.046) and = 0.046) KDR (P 0.035). The = 0.035). = 0.044), = 0.015) KDR mutation insporadicMTC(3). Met918Thr wasfoundasthemostfrequentsomaticRET mutations weredetectedin70%ofsporadicMTCand (4). Inaccordancewithpublishedliterature,somaticRET sensitive todetectsomaticmutationsintumourtissues Sanger sequencingandNGSshowedthatthelatterismore response tovandetanibtreatment. could provideusefulinformationondiseasecourseand determine whethergeneticand/orsignallingprofiles tumour tissuesof 32 patientswithsporadic MTC to pattern oftyrosinekinaseswereanalysedinprimary In thisstudy, somaticRETmutationstatusandexpression Discussion of tyrosinekinasesFLT1 (P at 3 tumourandtreatmentresponse mutation intheprimary found nosignificantcorrelationbetweenpresenceofaRET RET mutation; however, in the small patient series we patients showingPRhadaMTCwithsomaticMet918Thr patients showedSDandtwoPD.Allfive by RECIST1.1criteria.FivepatientsshowedPR,three therapy was assessed at the initial staging after 3 markers topredictresponse.Responsevandetanib tumours of these patients were suitable pattern in primary RET mutation status and/or tyrosine kinase expression and wereincludedinasubanalysistodeterminewhether with theRETMet918Thrmutation(datanotshown). PD at3monthsofvandetanibtreatment(Fig. 3). PR tovandetanib,comparedwithpatientsstableor VEGFB (P (P response insporadicMTC Prognosis andtherapy .2) and = 0.027) Parallel Ten patientsinourseriesweretreatedwithvandetanib months. Incontrast,significantlyhigherexpression = 0.042) was found in MTC of patients showing RET mutationanalysisinMTCtissuesby VEGFB (P wildtype MTC. mRNA countinRETmutantvs Figure 2 stable disease(SD),progressive disease (PD). vandetanib therapy. Partialresponse(PR), mRNA countsaccordingtoresponse to Figure 3 Downloaded fromBioscientifica.com at09/29/202106:01:19PM = 0.049) wasfoundintumours = 0.039), FLT4 (P 175:3 www.eje-online.org .2) and = 0.025) months 177 via freeaccess Clinical Study V Tiedje, S Ting and others Prognosis and therapy 175:3 178 response in sporadic MTC

A risk stratification on the basis of the type of somatic VEGF are specific ligands of the VEGFR family and are RET mutation is so far less certain, mainly because of low secreted by tumour cells, promoting lymph angiogenesis, patient numbers. In our series, the Met918Thr mutation blood vessel growth and tumour progression (24). In was associated with distant metastatic disease in seven our study, we found that the VEGFC mRNA count is patients and this was a significant finding. significantly higher in primary tumours of non-metastatic We further addressed the potential usefulness of MTC compared with metastatic MTC disease, while VEGFA tyrosine kinase expression pattern as prognostic markers and VEGFB expression is similar (data not shown). VEGFC in sporadic MTC. We found that the tumour-cell receptor is a KDR (VEGFR2) and FLT1 (VEGFR3) ligand. KDR and tyrosine kinases FGFR2, FGFR3, the VEGFR ligand FLT1 mRNA counts show an opposite expression pattern VEGFC and the intracellular tyrosine kinase BRAF were with KDR upregulation and FLT1 downregulation in the significantly downregulated in patients with metastatic primary tumour tissue of patients with metastatic disease. MTC compared with patients cured by surgery. In Rodriguez-Antona et al. (16) reported a higher protein contrast, PDGFRA, located at endothelial cells (e.g., blood expression of KDR in metastatic tissues compared with vessels), was significantly upregulated in metastatic MTC. primary tumour tissue of MTC patients. Our data suggest Previously reported increase of EGFR protein expression that a higher KDR mRNA count in MTC mirrors a more in patients with metastatic disease (16) could not be aggressive tumour biology, while high VEGFC and FLT1 confirmed in our study (data not shown). expression are compatible with disease-free status at the The fibroblast growth factor receptor (FGFR) family is end of follow-up. essential for proliferation, survival and angiogenesis (17). Platelet-derived growth factor receptors are a family Its role in cancer has been studied broadly in gastric (18), of receptor tyrosine kinases located at the membrane of endometrial (19) and lung cancer (20) and FGFR gene endothelial cells and are promoting cell survival, growth mutations, amplifications and overexpression have been and proliferation. The overexpression of PDGFRs has described. Bernard et al. (21) investigated the expression been demonstrated in various cancers, such as prostate of FGFR1–4 in normal thyroid tissue as well as in papillary, (25), ovarian (26) and non-small-cell lung cancer follicular and poorly differentiated thyroid cancer. (27). In medullary thyroid cancer, this has so far not Interestingly, FGFR2 was the only receptor to be expressed been investigated. Zhang et al. (28) analysed PDGFRA in normal thyroid tissue and was downregulated in expression in normal thyroid and papillary thyroid thyroid carcinoma, suggesting a protective role of FGFR2 cancer tissue. PDGFRA expression was significantly in carcinogenesis. This is in accordance with our results, higher in thyroid cancer tissues, especially in PTC with European Journal European of Endocrinology showing a higher FGFR2 mRNA count in patients with lymph node metastases. In our MTC series, we also localized vs metastatic MTC. found that the PDGFRA mRNA count was significantly Since the MAPK pathway is activated through the higher in MTC with lymph node or distant metastases in RET receptor tyrosine kinase in MTC (22), expressions comparison to MTC with N0M0 stage. Therefore, it can be of BRAF and the downstream mitogen-activating kinases hypothesized that the PDGFRA pathway might play a role (MAP2K1, MAP2K2, MAPK1, MAPK3) were investigated in the development of lymphatic and haematogenous in our study. Interestingly, the mRNA count of BRAF is metastases. Targeting the PDGFRA pathway is a potential significantly higher in MTC of patients with disease-free therapeutic approach, and sorafenib, sunitinib and status cured after surgery in comparison to tumours of pazopanib are also inhibitors of this tyrosine kinase patients with metastatic disease. In contrast, the mRNA receptor. For sorafenib (29), sunitinib (30) and pazopanib, counts of the mitogen-activating kinases are similar (31) phase II trials have shown some treatment response in both subgroups (data not shown). The mechanism in medullary thyroid cancer. However, so far no phase III of MAPK activation differs in follicular-cell-derived studies have been performed. tumours from medullary thyroid cancer. In follicular- In this study, we also addressed response markers to cell-derived tumours, for example, papillary thyroid vandetanib treatment. First, among the five MTC patients cancer, BRAF mutations are often causative (23). In showing PR, all had a tumour with a somatic RET918 medullary thyroid cancer, MAPK activation is effectuated mutation. Second, the FLT1, FLT4 and FLT1 ligand VEGFB through the RET mutations (22). The higher BRAF count mRNA expression were significantly higher in vandetanib in cured patient could imply that BRAF is involved in responders. If these findings can be reproduced in the initial tumour development, but is not essential for larger patient series, this may suggest the potential tumour progression. for a molecular stratification on the basis of primary

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Downloaded from Bioscientifica.com at 09/29/2021 06:01:19PM via free access European Journal of Endocrinology the public,commercialornot-for-profit sector. This researchdidnotreceiveanyspecific grantfromanyfundingagencyin Funding of interest. have no conflict and K W S D Z for AstraZeneca. S T, K W, J B, T H, R F W, D FandVThavereceivedspeakerfeesservedonadvisoryboards Declaration ofinterest with PRtovandetanib. FLT3 tumourtissuesarelinked and VEGFBinprimary somatic RET918mutationandtheexpressionofFLT1, targeted therapy, weshowforthefirsttime thatthe type withincreasedriskformetastaticdisease.Asregards and a somaticRETMet918ThrmutationandhigherPDGFRA metastases during follow-up. Moreover, the presence of cancer are linked with tumour stage and development of thyroid tumourtissuesofsporadicmedullary primary kinases andendothelialreceptortyrosineinthe expression patternoftumour-cellreceptortyrosine In thisstudy, wehavereportedforthefirsttimethat Conclusion approach involvinglargerpatientseries. that canbeevaluatedandvalidatedinamulticentre that thisapproachisusefultogeneratemarkerprofiles a comparisonbetweenourpatientsgroupisvalidand aggressive nature of sporadic MTC. We think that such asprognosticfactorsindicatingamorebenignorserve aim wastoidentifymolecularmarkersthatcould a moreaggressivetumourbehaviour. However, our than 10 our studyhaddistantmetastaticdiseasediagnosedless tissue was available. Thus except one, the patients in from ourstudybecausepatientswerecured and no many MTCpatientswithpN0cM0stageswereexcluded in non-universityfacilities(nolongerthan10 follow-up. Thus,duetothestoragelimitationofFFPE of patientsthathaddevelopedmetastasesduring are awareofapotentialbiasthroughnegativeselection nature andthelimited patient number. Furthermore, we 804 or806weredetected. therapy (32,33).Inourcohort,nomutationsincodon 806 areknowntocauseinvitro resistancetovandetanib metastatic MTC.RETmutationslocatedincodon804and tumour tissueprofilingtopredictvandetanibresponsein Clinical Study The limitationsofthisstudyareitsretrospective KDR expressionindicatesamoreaggressivetumour years afterinitialMTCdiagnosis,representing V Tiedje, STing andothers years),

References performing thenCounterpreparationanddatageneration. University Hospital Essen, University of Duisburg-Essen, Germany for We thanktheMOLBIZ–MolekularBiologischesZentrumRuhrlandklinik, Acknowledgements

12 11 10 response insporadicMTC Prognosis andtherapy 3 1 2 4 5 6 7 8 9 cases. Cancer20061072134–2142.(doi:10.1002/(ISSN)1097-0142) in1252 demographic, clinical,andpathologicpredictorsofsurvival thyroidcarcinoma:Roman S, Lin R&Sosa JA.Prognosisofmedullary 2012 30134–141.(doi:10.1200/JCO.2011.35.5040) ofClinicalOncology randomized, double-blindphaseIIItrial.Journal thyroidcancer:a with locallyadvancedormetastaticmedullary Baudin E, Elisei R,Jarzab B,Vasselli JR et al.Vandetanib inpatients Wells SA Jr, Robinson BG, Gagel RF, Dralle H,Fagin JA,Santoro M, jc.2012-2703) Endocrinology andMetabolism201398E364–E369.(doi:10.1210/ ofClinical exclusive oncogenicmutationsinRETandRAS.Journal thyroidcancerrevealsdominantandmutually of medullary Bhan S,Ho AS,Khan Z,Bishop Jet al.Exomicsequencing Agrawal N, Jiao Y, Bettegowda C,Roberts NJ, Sausen M,Leary R, (doi:10.1007/s00428-014-1589-3) thyroidcarcinomas.medullary Virchows Archiv 201446573–78. mutation profilingimprovesdiagnosticstratificationofsporadic Scardoni M, Pennelli G,Rugge M,Pelizzo MRet al.High-throughput Simbolo M, Mian C,Barollo S,Fassan M,Mafficini A,Neves D, 62–66. (doi:10.1002/(ISSN)1097-0215) 200195 ofCancer thyroidcarcinoma. Journal medullary International codon 918(ATG-->ACG) RETproto-oncogenemutationsinsporadic Herfarth C, Ziegler R,Schwab M&Raue F. Prognosticvalueof Schilling T, Burck J, Sinn HP, Clemens A,Otto HF, Hoppner W, Metabolism 200893682–687.(doi:10.1210/jc.2007-1714) a 10-yearfollow-upstudy. ofClinicalEndocrinologyand Journal thyroidcancer: RET oncogenemutationsinsporadicmedullary Vivaldi A, Faviana P, Basolo Fet al.Prognosticsignificanceofsomatic Elisei R, Cosci B,Romei C,Bottici V, Renzini G,Molinaro E, Agate L, (doi:10.1038/sj.bjc.6605056) thyroid carcinomas. ofCancer20091001777–1783. BritishJournal mutations withclinicopathologicalfeaturesinsporadicmedullary Cid MO, Bugalho MJ&Leite V. CorrelationofRETsomatic Moura MM, Cavaco BM,Pinto AE,Domingues R,Santos JR, (doi:10.1016/j.ajpath.2012.10.025) ofPathology2013182350–362. conditions. AmericanJournal thyroid carcinoma reveals specificmarkersassociatedwithgenetic la Vega L, Leton Ret al.Differentialgeneexpressionofmedullary Cubas A, Gomez-Lopez G,Inglada-Perez L,Alvarez-Escola C,De Maliszewska A, Leandro-Garcia LJ, Castelblanco E,Macia A,de 1993 363458–460.(doi:10.1038/363458a0) RET proto-oncogeneinmultipleendocrine neoplasiatype2A.Nature Love DR, Mole SE,Moore JK,Papi L et al. Germ-linemutationsofthe Mulligan LM, Kwok JB,Healey CS,Elsdon MJ, Eng C,Gardner E, ofCancer200945228–247.(doi:10.1016/j.ejca.2008.10.026) Journal criteria insolidtumours:revisedRECIST guideline(version1.1).European Dancey J, Arbuck S, Gwyther S, Mooney M Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH,Sargent D,Ford R, 31 3639–3646.(doi:10.1200/JCO.2012.48.4659) thyroidcancer.progressive medullary ofClinicalOncology2013 Journal Licitra L, Jarzab B,Medvedev V, Kreissl MCet al.Cabozantinibin Elisei R, Schlumberger MJ,Muller SP, Schoffski P, Brose MS,Shah MH, Cancer 201320611–619.(doi:10.1530/ERC-12-0316) thyroidcarcinoma.of tyrosinekinasesinmedullary Endocrine-Related Leandro-Garcia LJ et al.InfluenceofRETmutationsontheexpression Mancikova V, Canamero M,Maliszewska A,Gomez A,Leton R, Rodriguez-Antona C, Munoz-Repeto I,Inglada-Perez L,deCubas AA, Downloaded fromBioscientifica.com at09/29/202106:01:19PM New response evaluation et al. New response evaluation 175:3 www.eje-online.org 179 via freeaccess Clinical Study V Tiedje, S Ting and others Prognosis and therapy 175:3 180 response in sporadic MTC

13 Walter RF, Mairinger FD, Werner R, Ting S, Vollbrecht C, carcinomas (review). International Journal of Oncology 2013 42 19–28. Theegarten D, Christoph DC, Zarogoulidis K, Kurt Werner S, (doi:10.3892/ijo.2012.1681) Zarogoulidis P et al. SOX4, SOX11 and PAX6 mRNA expression 24 Achen MG & Stacker SA. Tumor lymphangiogenesis and metastatic was identified as a (prognostic) marker for the aggressiveness of spread-new players begin to emerge. International Journal of Cancer neuroendocrine tumours of the lung by using next-generation 2006 119 1755–1760. expression analysis (NanoString). Future Oncology 2015 11 1027–1036. 25 Chott A, Sun Z, Morganstern D, Pan J, Li T, Susani M, Mosberger I, (doi:10.2217/fon.15.18) Upton MP, Bubley GJ & Balk SP. Tyrosine kinases expressed in vivo by 14 Hruz T, Wyss M, Docquier M, Pfaffl MW, Masanetz S, Borghi L, human prostate cancer bone marrow metastases and loss of the type Verbrugghe P, Kalaydjieva L, Bleuler S, Laule O et al. RefGenes: 1 insulin-like growth factor receptor. American Journal of Pathology identification of reliable and condition specific reference genes 1999 155 1271–1279. (doi:10.1016/S0002-9440(10)65229-7) for RT-qPCR data normalization. BMC Genomics 2011 12 156. 26 Henriksen R, Funa K, Wilander E, Backstrom T, Ridderheim M & (doi:10.1186/1471-2164-12-156) Oberg K. Expression and prognostic significance of platelet-derived 15 Mairinger FD, Walter RF, Werner R, Christoph DC, Ting S, Vollbrecht C, growth factor and its receptors in epithelial ovarian neoplasms. Zarogoulidis K, Huang H, Li Q, Schmid KW et al. Activation of Cancer Research 1993 53 4550–4554. angiogenesis differs strongly between pulmonary carcinoids and 27 Donnem T, Al-Saad S, Al-Shibli K, Andersen S, Busund LT & neuroendocrine carinomas and is crucial for carcinoid tumourgenesis. Bremnes RM. Prognostic impact of platelet-derived growth factors in Journal of Cancer 2014 5 465–471. (doi:10.7150/jca.9235) non-small cell lung cancer tumor and stromal cells. Journal of Thoracic 16 Rodriguez-Antona C, Pallares J, Montero-Conde C, Inglada-Perez L, Oncology 2008 3 963–970. (doi:10.1097/JTO.0b013e3181834f52) Castelblanco E, Landa I, Leskela S, Leandro-Garcia LJ, Lopez- 28 Zhang J, Wang P, Dykstra M, Gelebart P, Williams D, Ingham R, Jimenez E, Leton R et al. Overexpression and activation of EGFR and Adewuyi EE, Lai R & McMullen T. Platelet-derived growth factor receptor- VEGFR2 in medullary thyroid carcinomas is related to metastasis. alpha promotes lymphatic metastases in papillary thyroid cancer. Journal Endocrine-Related Cancer 2010 17 7–16. (doi:10.1677/ERC-08-0304) of Pathology 2012 228 241–250. (doi:10.1002/path.v228.2) 17 Katoh M. Cancer genomics and genetics of FGFR2 (review). 29 Lam ET, Ringel MD, Kloos RT, Prior TW, Knopp MV, Liang J, International Journal of Oncology 2008 33 233–237. Sammet S, Hall NC, Wakely PE Jr, Vasko VV et al. Phase II clinical trial 18 Jang JH, Shin KH & Park JG. Mutations in fibroblast growth factor of sorafenib in metastatic medullary thyroid cancer. Journal of Clinical receptor 2 and fibroblast growth factor receptor 3 genes associated Oncology 2010 28 2323–2330. (doi:10.1200/jco.2009.25.0068) with human gastric and colorectal cancers. Cancer Research 2001 61 30 Carr LL, Mankoff DA, Goulart BH, Eaton KD, Capell PT, Kell EM, 3541–3543. Bauman JE & Martins RG. Phase II study of daily sunitinib in FDG- 19 Pollock PM, Gartside MG, Dejeza LC, Powell MA, Mallon MA, PET-positive, iodine-refractory differentiated thyroid cancer and Davies H, Mohammadi M, Futreal PA, Stratton MR, Trent JM et al. metastatic medullary carcinoma of the thyroid with functional Frequent activating FGFR2 mutations in endometrial carcinomas imaging correlation. Clinical Cancer Research 2010 16 5260–5268. parallel germline mutations associated with craniosynostosis (doi:10.1158/1078-0432.ccr-10-0994) and skeletal dysplasia syndromes. Oncogene 2007 26 7158–7162. 31 Bible KC, Suman VJ, Molina JR, Smallridge RC, Maples WJ, (doi:10.1038/sj.onc.1210529) Menefee ME, Rubin J, Karlin N, Sideras K & Morris JC 3rd et al. A 20 Davies H, Hunter C, Smith R, Stephens P, Greenman C, Bignell G, multicenter phase 2 trial of pazopanib in metastatic and progressive Teague J, Butler A, Edkins S, Stevens C et al. Somatic mutations of the medullary thyroid carcinoma: MC057H. Journal of Clinical protein kinase gene family in human lung cancer. Cancer Research Endocrinology and Metabolism 2014 99 1687–1693. (doi:10.1210/ 2005 65 7591–7595. jc.2013-3713) 21 St Bernard R, Zheng L, Liu W, Winer D, Asa SL & Ezzat S. Fibroblast 32 Carlomagno F, Guida T, Anaganti S, Vecchio G, Fusco A, Ryan AJ, European Journal European of Endocrinology growth factor receptors as molecular targets in thyroid carcinoma. Billaud M & Santoro M. Disease associated mutations at valine 804 Endocrinology 2005 146 1145–1153. in the RET receptor tyrosine kinase confer resistance to selective 22 Drosten M & Putzer BM. Mechanisms of disease: cancer targeting kinase inhibitors. Oncogene 2004 23 6056–6063. (doi:10.1038/ and the impact of oncogenic RET for medullary thyroid carcinoma sj.onc.1207810) therapy. Nature Clinical Practice Oncology 2006 3 564–574. 33 Carlomagno F, Guida T, Anaganti S, Provitera L, Kjaer S, (doi:10.1038/ncponc0610) McDonald NQ, Ryan AJ & Santoro M. Identification of tyrosine 806 23 Romitti M, Ceolin L, Siqueira DR, Ferreira CV, Wajner SM & as a molecular determinant of RET kinase sensitivity to ZD6474. Maia AL. Signaling pathways in follicular cell-derived thyroid Endocrine-Related Cancer 2009 16 233–241. (doi:10.1677/ERC-08-0213)

Received 20 March 2016 Revised version received 1 June 2016 Accepted 8 June 2016

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