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

in European Journal of Endocrinology
Correspondence should be addressed to V Tiedje; Email: vera.tiedje@uk-essen.de

(V Tiedje and S Ting contributed equally to this work)

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 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.

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 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.

Introduction

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

According to the American Thyroid Association (ATA) risk classification of RET germline mutations, RET918 mutations confer as the highest risk for early MTC manifestation and an aggressive clinical course. In sporadic MTC, the somatic RET918 mutation has been found as the most frequent RET mutation and has been linked to poorer survival (5, 6). Whether there is a correlation of other somatic RET mutations with distinct prognosis in sporadic MTC patients is currently not known (7).

In addition, activation of intracellular tyrosine kinase pathways has been linked to RET mutational status. Thus, Maliszewska et al. (8) reported upregulation of genes involved in the Wnt, Notch, NF-κB, JAK/STAT and MAPK signalling pathways in sporadic MTC harbouring the Met918Thr RET mutation. A RET-associated expression of tyrosine kinases was also described by Rodriguez-Antona et al. (9), whereby MTC with RET wildtype status showed a lower protein expression of PDGFRB than MTC with a somatic RET mutation.

However, whether somatic RET status and tyrosine kinase gene expression pattern are linked and associated with different disease course has not yet been elucidated.

The knowledge of the molecular pathology of MTC has led to development of therapies targeting RET and other tyrosine kinase signalling. Currently, two drugs are approved for treatment of progressive metastatic MTC, that is, vandetanib (European Medicines Agency (EMA) and Food and Drug Administration (FDA) approved), a RET, VEGFR2 (KDR), VEGFR3 (FLT4) and EGFR inhibitor (2) and cabozantinib (FDA approved, conditional EMA approved), a RET, VEGFR2, MET, KIT, AXL and FLT3 inhibitor (2, 10). A subanalysis with regard to progression-free survival (PFS) in RET mutated vs RET wildtype patients was performed in a phase III trial and a benefit for PFS was suggested if a Met918Thr RET mutation was present in the tumour (2).

In this study, we determined RET mutation status by Sanger sequencing and next-generation sequencing (NGS) and performed quantitative expression analysis of a panel of 33 tumour-cell and endothelial-cell tyrosine kinases in sporadic MTC patients with long-term follow-up. Our aim was to delineate prognostic markers for metastasized MTC and to identify factors associated with response or absent response to vandetanib therapy.

Methods

Patients and tumour samples

In this study, 32 patients with sporadic MTC were included, all treated at the University Hospital Essen (Germany), a tertiary centre for endocrine malignancies. The study was approved by the Ethics Committee of the Medical Faculty, University Duisburg-Essen (Germany). Clinical data, including age at diagnosis, sex, TNM stage, postoperative calcitonin levels, and response to tyrosine kinase inhibitor (TKI) therapy, was analysed retrospectively. Ten patients received vandetanib therapy after tumour progress was radiologically demonstrated according to the Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1. (11); response to therapy was classified as partial response (PR), stable disease (SD), (12) or progressive disease (PD) 3months after initiation of therapy. The time point 3 months was chosen to identify MTC patients with a primary vandetanib resistance.

Formalin-fixed paraffin-embedded (FFPE) tissues of the primary tumour were available in all cases of this study; diagnosis of MTC was reviewed by a board-certified, experienced thyroid pathologist (KWS). Hereditary MTC was excluded in all patients by germline RET gene mutation analysis from peripheral blood samples.

Sanger sequencing of RET gene

DNA was isolated from the primary tumour tissues with the QIAamp DNA FFPE tissue kit (Qiagen). For Sanger sequencing, RET exons 10, 11 and 13–16 were amplified by PCR. The PCR reaction was carried out in a total volume of 50µL with AmpliTaq Gold DNA Polymerase (Life Technologies), according to the recommendations of the supplier. Biometra T3000 Thermocycler (Biometra, Germany) was used for amplification. DNA sequences of both strands, forward and reverse, were analysed on an ABI 3500 Genetic Analyzer (Life Technologies), using BigDye Terminator chemistry (Life Technologies).

Next-generation sequencing of RET gene

For NGS, the MiSeq Illumina (Illumina, San Diego, CA, USA) was used following manufacturer’s instructions. DNA concentrations were determined by Qubit 2.0 Fluorometer dsDNA HS assay kit (Life Technologies). Multiplex PCR and purification were performed with the GeneRead Gene Custom Panel, DNAseq Panel PCR Kit (Qiagen) and Agencourt AMPure XP Beads (Beckman Coulter, Brea, CA, USA), followed by measurement of total DNA amount by Qubit 2.0 Fluorometer dsDNA HS assay kit. The library preparation was performed with NEBNext Ultra DNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s recommendations by using 24 different indices per run. The pooled library was sequenced on MiSeq (Illumina) and analysed by the Cancer Research Workbench (CLC Bio, Qiagen). For targeted sequencing, a customized MTC panel was designed containing regions of interest and hotspots of the RET gene. In all runs, an average coverage of approximately 8000× was obtained.

Nanostring nCounter preparation and prestatistical analysis

RNA was isolated from the primary tumour tissues with miRNAeasy FFPE kit (Qiagen) and total RNA concentrations were determined by using a fluorescent dye-based quantification method (Qubit, Life Technologies). Each sample was adjusted to a total RNA concentration of 200ng/µL for the nCounter preparation following established protocols (13). For gene expression analysis, the General Purpose Reagent nCounter Elements TagSet-48 and reagents according to the supplier were used (NanoString Technologies, Seattle, WA, USA). Probe Pools A (5nM per oligo) and B (25nM per oligo) were supplied by Integrated DNA Technologies (Coralville, IA, USA). The sample preparation was performed according to the nCounter Elements manual. The final hybridization was performed in an Eppendorf Mastercycler nexus (Hamburg, Germany) for 1.300min at 67°C with a heated lid (72°C). After hybridization, samples were directly loaded into the nCounter Prep Station (NanoString Technologies, Seattle, WA, USA) or cooled to 4°C until further proceeding within the next 4h. Samples were analysed on the nCounter Digital Analyzer (NanoString Technologies, Seattle, WA, USA) at 555 fields of view (FOV).

The raw data were analysed according to the following automated protocol: a background correction was performed, calculating the average counts for the negative controls plus two times the standard deviation. These values were subtracted from all sample counts. The geNorm algorithm (14) was used to identify the three most stable reference genes out of a set of nine reference genes (ACTB, GAPDH, HPRT1, GUSB, CLTC, NEDD8, PGK1, TTC1, TUBB). HPRT1, NEDD8 and TTC1 were identified to be the most stable reference genes. A normalization factor was calculated by using the geometric mean of the mRNA counts of the three most stable reference genes. Each sample was normalized using the sample-specific normalization factor. The normalized data were used for further statistical analysis (15).

Statistical analysis

Statistical analysis was performed by IBM SPSS Statistics Version 21. For comparison of mRNA count between MTC subgroups, Student’s t-test was performed. Correlation of clinicopathological parameters with presence of sporadic RET mutations was calculated by Chi square. Statistical significance was set at a P-value <0.05.

Results

The clinical data of the 32 sporadic MTC included in this study are listed in Table 1. The median age at diagnosis was 56 years. The patients were observed until December 2014 with a median observation period of 42 (3–159) months. Until then, 10 patients had disease-free status (pN0cM0), 8 patients had lymph node metastases (pN1cM0) and 14 patients developed distant metastatic disease (pN1cM1). Disease-free status was defined as biochemical and structural cure (calcitonin and CEA not detectable) during follow-up. Nine patients died of MTC during follow-up. Among the 32 patients, 14 received TKI treatment. Ten out of these 14 patients were started on vandetanib due to PD according to RECIST criteria 1.1. Characteristics of the vandetanib-treated patients are listed in Table 2. Among these 10 patients, 6 were treated as part of a clinical trial (ClinicalTrials.gov Nbib1298323).

Table 1

Patient characteristics, somatic RET mutation status and ATA risk level.

SexAge at diagnosisTNM at the end of follow-upPostoperative calcitonin (pg/mL)Sanger sequencingNext-generation sequencingExon
MTC1F58pT1pN0cM0<2c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC3M46pT2pN1cM17486c.1858T>C / p.Cys620Argc.1858T>C / p.Cys620ArgExon 10
MTC4F50pT3pN1cM064c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC5F29pT4pN1cM0xc.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC8M32pT2pN1bcM12363c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC12F45pT1pN0cM0<2c.1858T>A / p.Cys620Serc.1858T>A / p.Cys620SerExon 10
MTC13M53pT1bpN0cM0<2c.1900T>C / p.Cys634Argc.1900T>C / p.Cys634ArgExon 11
MTC14M58pT4pN1cM013
MTC15M55pT3pN1cM0830
MTC16F56pT1pN1cM012c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
c.1900T>C / p.Cys634ArgExon 11
c.1946C>T / p.Ser649LeuExon 11
MTC18M55pT4pN1cM0797c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC19F45pT3pN1cM031.866c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC21M44pT1pN1cM0<2c.1900T>C / p.Cys634Argc.1900T>C / p.Cys634ArgExon 11
c.1946C>T / p.Ser649Leuc.1946C>T / p.Ser649Leu
MTC23F43pT3pN1cM142
MTC24F50pT3bpN1cM15c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC27M85pT2pN1cM0<2c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC31F39pT2pN0cM0<2
MTC45M79pT1apN0cM0<2
MTC48F75pT1pN0cM0<2
MTC51F54pT1pN1cM1135c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC54F64pT3pN0cM196
MTC56M80pT2pN1bcM0119c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC58F60pT1apN0cM0<2
MTC63F49pT2apN0cM0<2
MTC65F63pT1bpN1cM052c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC70F55pT1pN1cM013
MTC71F72pT4pN1cM03c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC75F54pT3pN1bcM0248c.2753T>C / p.Met918Thrc.2753T>C / p.Met918ThrExon 16
MTC84F86pT3pN1acM01314
MTC85F30pT1ap N1bcM07
MTC91M65pT2pN0cM0<2c.1888T>C / p.Cys630Argc.1888T>C / p.Cys630ArgExon 11
MTC95F70pT4apN1bcM03c.1902 C>G / Cys634TrpExon 11

F, female; M, male.

Table 2

Characteristics of vandetanib-treated patients.

CharacteristicsNumber
Sex
 Female6
 Male4
Age at diagnosis*58±10
Site of metastasis
 Lung2
 Lung+bone5
 Lung+bone+liver2
 Lung+bone+cerebral1
Patients with PD under therapy7
Median PFS, months7
Deceased patients6
Previous TKI treatment
 Sorafenib3
 Cabozantinib2

Age in years (mean±s.d.).

PD, progressive disease; PFS, progression-free survival.

Sanger sequencing of the RET proto-oncogene revealed somatic RET mutations in the primary tumour tissues of 20 patients with sporadic MTC. The most frequent RET mutation was the Met918Thr mutation (14/20, 70%), followed by the Cys634Arg mutation (2/20, 10%). Further somatic RET mutations, each found in only one case, included RET codons 620 (Cys620Arg and Cys620Ser), codon 630 (Cys630Arg) and codon 649 (Ser649Leu). Through NGS, one additional MTC with a RET mutations was found, which had not been detected by Sanger sequencing. Two further cases with somatic RET mutations already revealed by Sanger sequencing showed additional mutations in NGS. The detailed list of the somatic RET mutations identified in 32 sporadic MTCs is shown in Table 1.

The frequency of RET mutations in MTC in patients with metastatic (pN1cM0 and pN1cM1) disease at the end of follow-up (16/23, 69.57%) differed from the frequency in MTC of patients with pN0cM0-stage (5/10, 50.00%, non significant P=0.411). The Met918Thr mutation was detected with significantly higher frequency in metastatic vs non-metastatic MTC (56.52% vs 10.00%, P=0.035). The Cys634Arg mutation was found in MTC of two patients with pN0cM0 stage, who were cured by surgery and in two patients with metastatic disease.

Furthermore, we asked whether expression pattern of tyrosine kinases differed in tumours of patients with MTC confined to the thyroid (pN0cM0) vs patients showing or developing lymphonodular (pN1cM0) or distant metastatic disease (pN1cM1) at the end of follow-up. For this, quantitative nCounter analysis was employed and mRNA count for a panel of 33 distinct tumour-cell or endothelial-cell-derived tyrosine kinases was determined in the primary tumour tissues. We found that expression of BRAF (P=0.019), FGFR2 (P=0.007), FGFR3 (P=0.044), PDGFRA (P=0.026) and VEGFC (P=0.042) differed significantly between MTC with pN0cM0 stages and metastatic MTC (pN1cM0 and pN1cM1 stages) (Fig. 1). In case of metastatic disease, BRAF, FGFR2, FGFR3 and VEGFC were significantly downregulated, whereas expression of PDGFRA was significantly upregulated in the primary tumour tissues, in particular in patients with lymph node metastases (P=0.007).

Figure 1

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Figure 1

mRNA count in pN0cM0 vs pN1cM0/pN1cM1 patients.

Citation: European Journal of Endocrinology 175, 3; 10.1530/EJE-16-0252

Comparison of RET mutation status with expression pattern of tyrosine kinases showed a proportional upregulation of AKT1 (P=0.045), KRAS (P=0.046) and KDR (P=0.021) in MTC with somatic RET mutations vs RET wildtype status (Fig. 2). Especially MTC harbouring a RET Met918Thr mutation had significantly higher expression of AKT1 (P=0.030), FGFR1 (P=0.038), KDR (P=0.015) and PDGFRA (P=0.032) compared with MTC with RET wildtype status. Within the group of MTC harbouring somatic RET mutations, significant upregulation of FGFR1 (P=0.027) and VEGFB (P=0.049) was found in tumours with the RET Met918Thr mutation (data not shown).

Figure 2

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Figure 2

mRNA count in RET mutant vs RET wildtype MTC.

Citation: European Journal of Endocrinology 175, 3; 10.1530/EJE-16-0252

Ten patients in our series were treated with vandetanib and were included in a subanalysis to determine whether RET mutation status and/or tyrosine kinase expression pattern in primary tumours of these patients were suitable markers to predict response. Response to vandetanib therapy was assessed at the initial staging after 3months by RECIST 1.1 criteria. Five patients showed PR, three patients showed SD and two patients showed PD. All five patients showing PR had a MTC with a somatic Met918Thr RET mutation; however, in the small patient series we found no significant correlation between presence of a RET mutation in the primary tumour and treatment response at 3months. In contrast, significantly higher expression of tyrosine kinases FLT1 (P=0.039), FLT4 (P=0.025) and VEGFB (P=0.042) was found in MTC of patients showing PR to vandetanib, compared with patients with stable or PD at 3 months of vandetanib treatment (Fig. 3).

Figure 3

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Figure 3

mRNA counts according to response to vandetanib therapy. Partial response (PR), stable disease (SD), progressive disease (PD).

Citation: European Journal of Endocrinology 175, 3; 10.1530/EJE-16-0252

Discussion

In this study, somatic RET mutation status and expression pattern of tyrosine kinases were analysed in primary tumour tissues of 32 patients with sporadic MTC to determine whether genetic and/or signalling profiles could provide useful information on disease course and response to vandetanib treatment.

Parallel RET mutation analysis in MTC tissues by Sanger sequencing and NGS showed that the latter is more sensitive to detect somatic mutations in tumour tissues (4). In accordance with published literature, somatic RET mutations were detected in 70% of sporadic MTC and Met918Thr was found as the most frequent somatic RET mutation in sporadic MTC (3).

A risk stratification on the basis of the type of somatic RET mutation is so far less certain, mainly because of low patient numbers. In our series, the Met918Thr mutation was associated with distant metastatic disease in seven patients and this was a significant finding.

We further addressed the potential usefulness of tyrosine kinase expression pattern as prognostic markers in sporadic MTC. We found that the tumour-cell receptor tyrosine kinases FGFR2, FGFR3, the VEGFR ligand VEGFC and the intracellular tyrosine kinase BRAF were significantly downregulated in patients with metastatic MTC compared with patients cured by surgery. In contrast, PDGFRA, located at endothelial cells (e.g., blood vessels), was significantly upregulated in metastatic MTC. Previously reported increase of EGFR protein expression in patients with metastatic disease (16) could not be confirmed in our study (data not shown).

The fibroblast growth factor receptor (FGFR) family is essential for proliferation, survival and angiogenesis (17). Its role in cancer has been studied broadly in gastric (18), endometrial (19) and lung cancer (20) and FGFR gene mutations, amplifications and overexpression have been described. Bernard et al. (21) investigated the expression of FGFR1–4 in normal thyroid tissue as well as in papillary, follicular and poorly differentiated thyroid cancer. Interestingly, FGFR2 was the only receptor to be expressed in normal thyroid tissue and was downregulated in thyroid carcinoma, suggesting a protective role of FGFR2 in carcinogenesis. This is in accordance with our results, showing a higher FGFR2 mRNA count in patients with localized vs metastatic MTC.

Since the MAPK pathway is activated through the RET receptor tyrosine kinase in MTC (22), expressions of BRAF and the downstream mitogen-activating kinases (MAP2K1, MAP2K2, MAPK1, MAPK3) were investigated in our study. Interestingly, the mRNA count of BRAF is significantly higher in MTC of patients with disease-free status cured after surgery in comparison to tumours of patients with metastatic disease. In contrast, the mRNA counts of the mitogen-activating kinases are similar in both subgroups (data not shown). The mechanism of MAPK activation differs in follicular-cell-derived tumours from medullary thyroid cancer. In follicular-cell-derived tumours, for example, papillary thyroid cancer, BRAF mutations are often causative (23). In medullary thyroid cancer, MAPK activation is effectuated through the RET mutations (22). The higher BRAF count in cured patient could imply that BRAF is involved in the initial tumour development, but is not essential for tumour progression.

VEGF are specific ligands of the VEGFR family and are secreted by tumour cells, promoting lymph angiogenesis, blood vessel growth and tumour progression (24). In our study, we found that the VEGFC mRNA count is significantly higher in primary tumours of non-metastatic MTC compared with metastatic MTC disease, while VEGFA and VEGFB expression is similar (data not shown). VEGFC is a KDR (VEGFR2) and FLT1 (VEGFR3) ligand. KDR and FLT1 mRNA counts show an opposite expression pattern with KDR upregulation and FLT1 downregulation in the primary tumour tissue of patients with metastatic disease.

Rodriguez-Antona et al. (16) reported a higher protein expression of KDR in metastatic tissues compared with primary tumour tissue of MTC patients. Our data suggest that a higher KDR mRNA count in MTC mirrors a more aggressive tumour biology, while high VEGFC and FLT1 expression are compatible with disease-free status at the end of follow-up.

Platelet-derived growth factor receptors are a family of receptor tyrosine kinases located at the membrane of endothelial cells and are promoting cell survival, growth and proliferation. The overexpression of PDGFRs has been demonstrated in various cancers, such as prostate (25), ovarian (26) and non-small-cell lung cancer (27). In medullary thyroid cancer, this has so far not been investigated. Zhang et al. (28) analysed PDGFRA expression in normal thyroid and papillary thyroid cancer tissue. PDGFRA expression was significantly higher in thyroid cancer tissues, especially in PTC with lymph node metastases. In our MTC series, we also found that the PDGFRA mRNA count was significantly higher in MTC with lymph node or distant metastases in comparison to MTC with N0M0 stage. Therefore, it can be hypothesized that the PDGFRA pathway might play a role in the development of lymphatic and haematogenous metastases. Targeting the PDGFRA pathway is a potential therapeutic approach, and sorafenib, sunitinib and pazopanib are also inhibitors of this tyrosine kinase receptor. For sorafenib (29), sunitinib (30) and pazopanib, (31) phase II trials have shown some treatment response in medullary thyroid cancer. However, so far no phase III studies have been performed.

In this study, we also addressed response markers to vandetanib treatment. First, among the five MTC patients showing PR, all had a tumour with a somatic RET918 mutation. Second, the FLT1, FLT4 and FLT1 ligand VEGFB mRNA expression were significantly higher in vandetanib responders. If these findings can be reproduced in larger patient series, this may suggest the potential for a molecular stratification on the basis of primary tumour tissue profiling to predict vandetanib response in metastatic MTC. RET mutations located in codon 804 and 806 are known to cause in vitro resistance to vandetanib therapy (32, 33). In our cohort, no mutations in codon 804 or 806 were detected.

The limitations of this study are its retrospective nature and the limited patient number. Furthermore, we are aware of a potential bias through negative selection of patients that had developed metastases during follow-up. Thus, due to the storage limitation of FFPE in non-university facilities (no longer than 10years), many MTC patients with pN0cM0 stages were excluded from our study because patients were cured and no tissue was available. Thus except one, the patients in our study had distant metastatic disease diagnosed less than 10years after initial MTC diagnosis, representing a more aggressive tumour behaviour. However, our aim was to identify molecular markers that could serve as prognostic factors indicating a more benign or aggressive nature of sporadic MTC. We think that such a comparison between our patients group is valid and that this approach is useful to generate marker profiles that can be evaluated and validated in a multicentre approach involving larger patient series.

Conclusion

In this study, we have reported for the first time that expression pattern of tumour-cell receptor tyrosine kinases and endothelial receptor tyrosine kinases in the primary tumour tissues of sporadic medullary thyroid cancer are linked with tumour stage and development of metastases during follow-up. Moreover, the presence of a somatic RET Met918Thr mutation and higher PDGFRA and KDR expression indicates a more aggressive tumour type with increased risk for metastatic disease. As regards targeted therapy, we show for the first time that the somatic RET918 mutation and the expression of FLT1, FLT3 and VEGFB in primary tumour tissues are linked with PR to vandetanib.

Declaration of interest

D F and V T have received speaker fees and have served on advisory boards for AstraZeneca. S T, R F W, T H, K W, J B, D Z and K W S have no conflict of interest.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Acknowledgements

We thank the MOLBIZ – Molekular Biologisches Zentrum Ruhrlandklinik, University Hospital Essen, University of Duisburg-Essen, Germany for performing the nCounter preparation and data generation.

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  • 9

    Rodriguez-AntonaCMunoz-RepetoIInglada-PerezLde CubasAAMancikovaVCanameroMMaliszewskaAGomezALetonRLeandro-GarciaLJInfluence of RET mutations on the expression of tyrosine kinases in medullary thyroid carcinoma. Endocrine-Related Cancer201320611619. (doi:10.1530/ERC-12-0316)

  • 10

    EliseiRSchlumbergerMJMullerSPSchoffskiPBroseMSShahMHLicitraLJarzabBMedvedevVKreisslMCCabozantinib in progressive medullary thyroid cancer. Journal of Clinical Oncology20133136393646. (doi:10.1200/JCO.2012.48.4659)

  • 11

    EisenhauerEATherassePBogaertsJSchwartzLHSargentDFordRDanceyJArbuckSGwytherSMooneyMNew response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). European Journal of Cancer200945228247. (doi:10.1016/j.ejca.2008.10.026)

  • 12

    MulliganLMKwokJBHealeyCSElsdonMJEngCGardnerELoveDRMoleSEMooreJKPapiLGerm-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature1993363458460. (doi:10.1038/363458a0)

  • 13

    WalterRFMairingerFDWernerRTingSVollbrechtCTheegartenDChristophDCZarogoulidisKKurt WernerSZarogoulidisPSOX4, SOX11 and PAX6 mRNA expression was identified as a (prognostic) marker for the aggressiveness of neuroendocrine tumours of the lung by using next-generation expression analysis (NanoString). Future Oncology20151110271036. (doi:10.2217/fon.15.18)

  • 14

    HruzTWyssMDocquierMPfafflMWMasanetzSBorghiLVerbrugghePKalaydjievaLBleulerSLauleORefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization. BMC Genomics201112156. (doi:10.1186/1471-2164-12-156)

  • 15

    MairingerFDWalterRFWernerRChristophDCTingSVollbrechtCZarogoulidisKHuangHLiQSchmidKWActivation of angiogenesis differs strongly between pulmonary carcinoids and neuroendocrine carinomas and is crucial for carcinoid tumourgenesis. Journal of Cancer20145465471. (doi:10.7150/jca.9235)

  • 16

    Rodriguez-AntonaCPallaresJMontero-CondeCInglada-PerezLCastelblancoELandaILeskelaSLeandro-GarciaLJLopez-JimenezELetonROverexpression and activation of EGFR and VEGFR2 in medullary thyroid carcinomas is related to metastasis. Endocrine-Related Cancer201017716. (doi:10.1677/ERC-08-0304)

  • 17

    KatohM.Cancer genomics and genetics of FGFR2 (review). International Journal of Oncology200833233237.

  • 18

    JangJHShinKHParkJG.Mutations in fibroblast growth factor receptor 2 and fibroblast growth factor receptor 3 genes associated with human gastric and colorectal cancers. Cancer Research20016135413543.

  • 19

    PollockPMGartsideMGDejezaLCPowellMAMallonMADaviesHMohammadiMFutrealPAStrattonMRTrentJMFrequent activating FGFR2 mutations in endometrial carcinomas parallel germline mutations associated with craniosynostosis and skeletal dysplasia syndromes. Oncogene20072671587162. (doi:10.1038/sj.onc.1210529)

  • 20

    DaviesHHunterCSmithRStephensPGreenmanCBignellGTeagueJButlerAEdkinsSStevensCSomatic mutations of the protein kinase gene family in human lung cancer. Cancer Research20056575917595.

  • 21

    St BernardRZhengLLiuWWinerDAsaSLEzzatS.Fibroblast growth factor receptors as molecular targets in thyroid carcinoma. Endocrinology200514611451153.

  • 22

    DrostenMPutzerBM.Mechanisms of disease: cancer targeting and the impact of oncogenic RET for medullary thyroid carcinoma therapy. Nature Clinical Practice Oncology20063564574. (doi:10.1038/ncponbib610)

  • 23

    RomittiMCeolinLSiqueiraDRFerreiraCVWajnerSMMaiaAL.Signaling pathways in follicular cell-derived thyroid carcinomas (review). International Journal of Oncology2013421928. (doi:10.3892/ijo.2012.1681)

  • 24

    AchenMGStackerSA.Tumor lymphangiogenesis and metastatic spread-new players begin to emerge. International Journal of Cancer200611917551760.

  • 25

    ChottASunZMorgansternDPanJLiTSusaniMMosbergerIUptonMPBubleyGJBalkSP.Tyrosine kinases expressed in vivo by human prostate cancer bone marrow metastases and loss of the type 1 insulin-like growth factor receptor. American Journal of Pathology199915512711279. (doi:10.1016/S0002-9440(10)65229-7)

  • 26

    HenriksenRFunaKWilanderEBackstromTRidderheimMObergK.Expression and prognostic significance of platelet-derived growth factor and its receptors in epithelial ovarian neoplasms. Cancer Research19935345504554.

  • 27

    DonnemTAl-SaadSAl-ShibliKAndersenSBusundLTBremnesRM.Prognostic impact of platelet-derived growth factors in non-small cell lung cancer tumor and stromal cells. Journal of Thoracic Oncology20083963970. (doi:10.1097/JTO.0b013e3181834f52)

  • 28

    ZhangJWangPDykstraMGelebartPWilliamsDInghamRAdewuyiEELaiRMcMullenT.Platelet-derived growth factor receptor-alpha promotes lymphatic metastases in papillary thyroid cancer. Journal of Pathology2012228241250. (doi:10.1002/path.v228.2)

  • 29

    LamETRingelMDKloosRTPriorTWKnoppMVLiangJSammetSHallNCWakelyPEJrVaskoVVPhase II clinical trial of sorafenib in metastatic medullary thyroid cancer. Journal of Clinical Oncology20102823232330. (doi:10.1200/jco.2009.25.0068)

  • 30

    CarrLLMankoffDAGoulartBHEatonKDCapellPTKellEMBaumanJEMartinsRG.Phase II study of daily sunitinib in FDG-PET-positive, iodine-refractory differentiated thyroid cancer and metastatic medullary carcinoma of the thyroid with functional imaging correlation. Clinical Cancer Research20101652605268. (doi:10.1158/1078-0432.ccr-10-0994)

  • 31

    BibleKCSumanVJMolinaJRSmallridgeRCMaplesWJMenefeeMERubinJKarlinNSiderasKMorrisJC3rd et al. A multicenter phase 2 trial of pazopanib in metastatic and progressive medullary thyroid carcinoma: Mbib57H. Journal of Clinical Endocrinology and Metabolism20149916871693. (doi:10.1210/jc.2013-3713)

  • 32

    CarlomagnoFGuidaTAnagantiSVecchioGFuscoARyanAJBillaudMSantoroM.Disease associated mutations at valine 804 in the RET receptor tyrosine kinase confer resistance to selective kinase inhibitors. Oncogene20042360566063. (doi:10.1038/sj.onc.1207810)

  • 33

    CarlomagnoFGuidaTAnagantiSProviteraLKjaerSMcDonaldNQRyanAJSantoroM.Identification of tyrosine 806 as a molecular determinant of RET kinase sensitivity to ZD6474. Endocrine-Related Cancer200916233241. (doi:10.1677/ERC-08-0213)

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    mRNA count in pN0cM0 vs pN1cM0/pN1cM1 patients.

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    mRNA count in RET mutant vs RET wildtype MTC.

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    mRNA counts according to response to vandetanib therapy. Partial response (PR), stable disease (SD), progressive disease (PD).

References

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RomanSLinRSosaJA.Prognosis of medullary thyroid carcinoma: demographic, clinical, and pathologic predictors of survival in 1252 cases. Cancer200610721342142. (doi:10.1002/(ISSN)1097-0142)

2

WellsSAJrRobinsonBGGagelRFDralleHFaginJASantoroMBaudinEEliseiRJarzabBVasselliJRVandetanib in patients with locally advanced or metastatic medullary thyroid cancer: a randomized, double-blind phase III trial. Journal of Clinical Oncology201230134141. (doi:10.1200/JCO.2011.35.5040)

3

AgrawalNJiaoYSausenMLearyRBettegowdaCRobertsNJBhanSHoASKhanZBishopJExomic sequencing of medullary thyroid cancer reveals dominant and mutually exclusive oncogenic mutations in RET and RAS. Journal of Clinical Endocrinology and Metabolism201398E364E369. (doi:10.1210/jc.2012-2703)

4

SimboloMMianCBarolloSFassanMMafficiniANevesDScardoniMPennelliGRuggeMPelizzoMRHigh-throughput mutation profiling improves diagnostic stratification of sporadic medullary thyroid carcinomas. Virchows Archiv20144657378. (doi:10.1007/s00428-014-1589-3)

5

SchillingTBurckJSinnHPClemensAOttoHFHoppnerWHerfarthCZieglerRSchwabMRaueF.Prognostic value of codon 918 (ATG-->ACG) RET proto-oncogene mutations in sporadic medullary thyroid carcinoma. International Journal of Cancer2001956266. (doi:10.1002/(ISSN)1097-0215)

6

EliseiRCosciBRomeiCBotticiVRenziniGMolinaroEAgateLVivaldiAFavianaPBasoloFPrognostic significance of somatic RET oncogene mutations in sporadic medullary thyroid cancer: a 10-year follow-up study. Journal of Clinical Endocrinology and Metabolism200893682687. (doi:10.1210/jc.2007-1714)

7

MouraMMCavacoBMPintoAEDominguesRSantosJRCidMOBugalhoMJLeiteV.Correlation of RET somatic mutations with clinicopathological features in sporadic medullary thyroid carcinomas. British Journal of Cancer200910017771783. (doi:10.1038/sj.bjc.6605056)

8

MaliszewskaALeandro-GarciaLJCastelblancoEMaciaAde CubasAGomez-LopezGInglada-PerezLAlvarez-EscolaCDe la VegaLLetonRDifferential gene expression of medullary thyroid carcinoma reveals specific markers associated with genetic conditions. American Journal of Pathology2013182350362. (doi:10.1016/j.ajpath.2012.10.025)

9

Rodriguez-AntonaCMunoz-RepetoIInglada-PerezLde CubasAAMancikovaVCanameroMMaliszewskaAGomezALetonRLeandro-GarciaLJInfluence of RET mutations on the expression of tyrosine kinases in medullary thyroid carcinoma. Endocrine-Related Cancer201320611619. (doi:10.1530/ERC-12-0316)

10

EliseiRSchlumbergerMJMullerSPSchoffskiPBroseMSShahMHLicitraLJarzabBMedvedevVKreisslMCCabozantinib in progressive medullary thyroid cancer. Journal of Clinical Oncology20133136393646. (doi:10.1200/JCO.2012.48.4659)

11

EisenhauerEATherassePBogaertsJSchwartzLHSargentDFordRDanceyJArbuckSGwytherSMooneyMNew response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). European Journal of Cancer200945228247. (doi:10.1016/j.ejca.2008.10.026)

12

MulliganLMKwokJBHealeyCSElsdonMJEngCGardnerELoveDRMoleSEMooreJKPapiLGerm-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature1993363458460. (doi:10.1038/363458a0)

13

WalterRFMairingerFDWernerRTingSVollbrechtCTheegartenDChristophDCZarogoulidisKKurt WernerSZarogoulidisPSOX4, SOX11 and PAX6 mRNA expression was identified as a (prognostic) marker for the aggressiveness of neuroendocrine tumours of the lung by using next-generation expression analysis (NanoString). Future Oncology20151110271036. (doi:10.2217/fon.15.18)

14

HruzTWyssMDocquierMPfafflMWMasanetzSBorghiLVerbrugghePKalaydjievaLBleulerSLauleORefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization. BMC Genomics201112156. (doi:10.1186/1471-2164-12-156)

15

MairingerFDWalterRFWernerRChristophDCTingSVollbrechtCZarogoulidisKHuangHLiQSchmidKWActivation of angiogenesis differs strongly between pulmonary carcinoids and neuroendocrine carinomas and is crucial for carcinoid tumourgenesis. Journal of Cancer20145465471. (doi:10.7150/jca.9235)

16

Rodriguez-AntonaCPallaresJMontero-CondeCInglada-PerezLCastelblancoELandaILeskelaSLeandro-GarciaLJLopez-JimenezELetonROverexpression and activation of EGFR and VEGFR2 in medullary thyroid carcinomas is related to metastasis. Endocrine-Related Cancer201017716. (doi:10.1677/ERC-08-0304)

17

KatohM.Cancer genomics and genetics of FGFR2 (review). International Journal of Oncology200833233237.

18

JangJHShinKHParkJG.Mutations in fibroblast growth factor receptor 2 and fibroblast growth factor receptor 3 genes associated with human gastric and colorectal cancers. Cancer Research20016135413543.

19

PollockPMGartsideMGDejezaLCPowellMAMallonMADaviesHMohammadiMFutrealPAStrattonMRTrentJMFrequent activating FGFR2 mutations in endometrial carcinomas parallel germline mutations associated with craniosynostosis and skeletal dysplasia syndromes. Oncogene20072671587162. (doi:10.1038/sj.onc.1210529)

20

DaviesHHunterCSmithRStephensPGreenmanCBignellGTeagueJButlerAEdkinsSStevensCSomatic mutations of the protein kinase gene family in human lung cancer. Cancer Research20056575917595.

21

St BernardRZhengLLiuWWinerDAsaSLEzzatS.Fibroblast growth factor receptors as molecular targets in thyroid carcinoma. Endocrinology200514611451153.

22

DrostenMPutzerBM.Mechanisms of disease: cancer targeting and the impact of oncogenic RET for medullary thyroid carcinoma therapy. Nature Clinical Practice Oncology20063564574. (doi:10.1038/ncponbib610)

23

RomittiMCeolinLSiqueiraDRFerreiraCVWajnerSMMaiaAL.Signaling pathways in follicular cell-derived thyroid carcinomas (review). International Journal of Oncology2013421928. (doi:10.3892/ijo.2012.1681)

24

AchenMGStackerSA.Tumor lymphangiogenesis and metastatic spread-new players begin to emerge. International Journal of Cancer200611917551760.

25

ChottASunZMorgansternDPanJLiTSusaniMMosbergerIUptonMPBubleyGJBalkSP.Tyrosine kinases expressed in vivo by human prostate cancer bone marrow metastases and loss of the type 1 insulin-like growth factor receptor. American Journal of Pathology199915512711279. (doi:10.1016/S0002-9440(10)65229-7)

26

HenriksenRFunaKWilanderEBackstromTRidderheimMObergK.Expression and prognostic significance of platelet-derived growth factor and its receptors in epithelial ovarian neoplasms. Cancer Research19935345504554.

27

DonnemTAl-SaadSAl-ShibliKAndersenSBusundLTBremnesRM.Prognostic impact of platelet-derived growth factors in non-small cell lung cancer tumor and stromal cells. Journal of Thoracic Oncology20083963970. (doi:10.1097/JTO.0b013e3181834f52)

28

ZhangJWangPDykstraMGelebartPWilliamsDInghamRAdewuyiEELaiRMcMullenT.Platelet-derived growth factor receptor-alpha promotes lymphatic metastases in papillary thyroid cancer. Journal of Pathology2012228241250. (doi:10.1002/path.v228.2)

29

LamETRingelMDKloosRTPriorTWKnoppMVLiangJSammetSHallNCWakelyPEJrVaskoVVPhase II clinical trial of sorafenib in metastatic medullary thyroid cancer. Journal of Clinical Oncology20102823232330. (doi:10.1200/jco.2009.25.0068)

30

CarrLLMankoffDAGoulartBHEatonKDCapellPTKellEMBaumanJEMartinsRG.Phase II study of daily sunitinib in FDG-PET-positive, iodine-refractory differentiated thyroid cancer and metastatic medullary carcinoma of the thyroid with functional imaging correlation. Clinical Cancer Research20101652605268. (doi:10.1158/1078-0432.ccr-10-0994)

31

BibleKCSumanVJMolinaJRSmallridgeRCMaplesWJMenefeeMERubinJKarlinNSiderasKMorrisJC3rd et al. A multicenter phase 2 trial of pazopanib in metastatic and progressive medullary thyroid carcinoma: Mbib57H. Journal of Clinical Endocrinology and Metabolism20149916871693. (doi:10.1210/jc.2013-3713)

32

CarlomagnoFGuidaTAnagantiSVecchioGFuscoARyanAJBillaudMSantoroM.Disease associated mutations at valine 804 in the RET receptor tyrosine kinase confer resistance to selective kinase inhibitors. Oncogene20042360566063. (doi:10.1038/sj.onc.1207810)

33

CarlomagnoFGuidaTAnagantiSProviteraLKjaerSMcDonaldNQRyanAJSantoroM.Identification of tyrosine 806 as a molecular determinant of RET kinase sensitivity to ZD6474. Endocrine-Related Cancer200916233241. (doi:10.1677/ERC-08-0213)

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