Objective: The aim of this study was to determine the genomic alterations of cancer-related genes in advanced medullary thyroid carcinoma during the course of clinical care. Methods: Hybrid-capture-based comprehensive genomic profiling was performed on 34 consecutive medullary thyroid carcinoma cases to identify all four classes of genomic alterations, and outcome for an index patient was collected. Results:RET was mutated in 88% (30/34) of cases, with RET M918T being responsible for 70% (21/30) of the RET alterations. The other RET alterations were RET E632_L633del, C634R, C620R, C618G/R/S, V804M, and RET amplification. Two of the four RET wild-type patients harbored mutations in KRAS or HRAS (1/34 each). The next most frequent genomic alterations were amplifications of CCND1, FGF3, and FGF19 and alterations in CDKN2A (3/34 each). One case with a RET M918T mutation developed acquired resistance to progressively dose-escalated vandetanib. When the mTOR inhibitor everolimus was added to continued vandetanib treatment, the patient achieved a second 25% reduction of tumor volume (RECIST 1.1) for 8 months. Conclusions: Comprehensive genomic profiling identified the full breadth of RET alterations in metastatic medullary thyroid carcinoma and possible cooperating oncogenic driver alterations. This approach may refine the use of targeted therapy for these patients.

Patients with early-stage medullary thyroid carcinoma (MTC), a tumor derived from the neuroendocrine cells of the thyroid, have a favorable prognosis, as surgery is effective in this setting. For patients with metastatic or recurrent disease, however, the 10-year overall survival (OS) rate is only 40% regardless of whether the MTC arose in a familial setting or as a sporadic malignancy [1]. Chemotherapy and external beam radiotherapy have shown very little efficacy in patients with advanced MTC [2,3]. Two targeted therapies are approved in the US and Europe to treat unresectable, locally advanced, or metastatic MTC, but they have not been well linked to efficacy in the context of a particular genomic alteration (GA). Vandetanib (Caprelsa® 300 mg once daily) significantly prolonged median progression-free survival (PFS) compared with placebo (30.5 vs. 19.3 months) in a phase 3 study of patients with advanced MTC [4]. A retrospective subgroup analysis of patients with sporadic MTC suggested a higher objective response rate (ORR) in RET M918T mutation-positive versus -negative patients (54.5 vs. 30.2%), although RET status was unknown for a high fraction (45%) of patients. The approval of cabozantinib (Cometriq®) is based on a phase 3 trial of patients with progressive metastatic MTC, in which cabozantinib (140 mg once daily) significantly improved median PFS compared with placebo (11.2 vs. 4.0 months) and achieved an ORR of 28% (0% for placebo) [5]. Responses were observed in patients both with and without any detectable RET mutations (ORR of 32 and 25%, respectively), while sporadic MTC with RET M918T had a numerically lower hazard ratio (HR) than RET M918T-negative cases in this study [5]. Median PFS on cabozantinib was longer for patients with a RET mutation than for patients with wild-type RET (60 vs. 25 weeks, p = 0.0001), and RET M918T was associated with longer PFS than any other RET mutation (61 vs. 36 weeks, p = 0.009), suggesting that RET mutation, which correlates with worse outcome [6], and RET M918T in particular confer a survival benefit from cabozantinib treatment [7]. The secondary endpoint of this phase 3 study was improved OS, and it was not met with a median OS of 26.6 months for cabozantinib and 21.1 months for placebo (HR = 0.85, p = 0.241); however, patients with RET M918T mutations experienced a significantly longer median OS with cabozantinib (44.3 vs. 18.9 months with placebo, HR = 0.60, p = 0.026) [8].

Examining the results of these trials, the heterogeneity of responses suggests the possibility of de novo resistance to vandetanib or cabozantinib. The gatekeeper RET V804M/L mutation, present in 1-2% of MTC patients, has been characterized as resistant to vandetanib and less sensitive to cabozantinib [9,10,11,12]. Moreover, a phase 2 study of vandetanib in multiple endocrine neoplasia type 2B (MEN2B)-associated MTC reported 3 patients who experienced disease progression after an initial decrease in tumor size, indicative of acquired resistance, despite a high prevalence of the RET M918T driver mutation (>95%) in this population [13].

In this study, comprehensive genomic profiling (CGP) was performed in the course of clinical care on 34 advanced MTC cases to assess opportunities for benefit from targeted therapy. An index case (patient 1) with acquired resistance to vandetanib monotherapy derived clinical benefit from the addition of everolimus to the regimen.

CGP was performed in a CLIA-certified, CAP-accredited, NYS-regulated reference laboratory (Foundation Medicine, Inc.). At least 50 ng of DNA per specimen was extracted from 34 clinical formalin-fixed paraffin-embedded consecutively submitted MTC tumor samples, and next-generation sequencing was performed on hybridization-captured, adaptor ligation-based libraries to high, uniform coverage (>500×) for all coding exons of 315 cancer-related genes and 28 genes commonly rearranged in cancer (n = 22), 236 cancer-related genes and 19 genes frequently rearranged in cancer (n = 8), or 182 genes and 14 genes frequently rearranged in cancer (n = 4), with the different number of sequenced genes representing different generations of FoundationOne®.

Base substitutions, short insertions and deletions (indels), focal gene amplifications, homozygous deletions, and select rearrangements were determined and reported for each patient sample. To maximize mutation detection sensitivity in heterogeneous tumor biopsies and resections, the test was validated to detect base substitutions at ≥10% mutant allele frequency (MAF) with ≥99% sensitivity and indels at ≥20% MAF with ≥95% sensitivity, with a false discovery rate of <1% [14]. Actionable alterations are defined as those whose effect is targetable using anticancer drugs currently on the market or in registered clinical trials. Local site permissions to use clinical samples were obtained for this study.

A computational method was used to predict somatic versus germline RET variant status for each of the RET-mutant specimens without a matched normal control as previously described [15].

We reviewed the medical records of a patient (case 1) with sporadic MTC who presented to the Department of Investigational Cancer Therapeutics at The University of Texas MD Anderson Cancer Center after failing standard of care therapy. Treatment and consent on investigational trial (NCT01582191) as well as data collection were conducted in accordance with the guidelines of the University of Texas MD Anderson Cancer Center Institutional Review Board. Tumor response was determined using RECIST (version 1.1) by computed tomography (CT) scans obtained about every 6-8 weeks. Clinical evaluation and assessments were performed per protocol.

Thirty-four consecutively submitted MTC cases had CGP performed in the course of clinical care (table 1). The median age of the patients was 53 years (range 21-85); 71% (23/34) of the patients were male, and 29% (10/34) were female. MTC generally shows a slight female preponderance [1,16], and we do not have an explanation for the relatively high frequency of male cases in this unselected dataset. At the time of profiling, 33 MTC were stage IV and 1 MTC was stage II. CGP was performed on tumor specimens from the primary tumor in 47% (16/34) of cases, from lymph node metastases in 21% (7/34) of cases, or from other metastatic sites in 32% (11/34) of cases.

Table 1

Genomic alterations (GAs) identified in 34 cases of MTC

Genomic alterations (GAs) identified in 34 cases of MTC
Genomic alterations (GAs) identified in 34 cases of MTC

All cases harbored at least one GA (fig. 1), with 83 total GAs identified for an average of 2.4 GAs per tumor (range 1-11). There were 44 base substitutions and indels, 23 gene amplifications, 15 truncations, 1 homozygous deletion, and no rearrangements (fig. 1). Sixty-seven percent (56/83) of the GAs were considered clinically relevant GAs and could potentially guide decisions regarding targeted therapy, with an average of 1.6 clinically relevant GAs per tumor (range 1-7).

Fig. 1

Tile plot of genomic alterations identified by comprehensive genomic profiling in 34 cases of advanced MTC.

Fig. 1

Tile plot of genomic alterations identified by comprehensive genomic profiling in 34 cases of advanced MTC.

Close modal

RET was the most frequently altered gene, mutated in 88% (30/34) of cases (table 1; fig. 1). RET M918T was the most common RET alteration, observed in 62% (21/34) of patients [17,18,19]. RET C618G/R/S was detected in 3 cases (9%), RET C634R and E632_L633del each in 2 cases (6%); RET V804M or C620R was each present in 1 tumor (3%) [9,20,21,22,23,24,25]. One case harbored both a RET amplification and RET M918T [26], and no other cases had more than 1 RET alteration. A computational method was used to determine the RET variant status without a matched normal control [15] and predicted the RET GA to be somatic in the majority of the RET-positive cases (73%) (table 1), consistent with the pathology reports that did not indicate any MEN2A or MEN2B cases. Although the 2 patients with predicted germline RET M918T (cases 21 and 25) could be MEN2B, their ages are more consistent with sporadic MTC, and their pathology reports did not mention any known clinical history of MEN2B or pheochromocytoma.

Other clinically relevant alterations occurred in CCND1 (9%), CDKN2A(9%), FGF19(9%), KRAS (6%), VHL (6%), and for 1 case each (3%) in ATM, CCND2, CDK4, CDK6, CDKN2B, CDKN2C, DNMT3A, ERBB3, ERBB4, GLI1, HRAS, MDM2, MYC, NOTCH2, and PTEN(fig. 1).

A 42-year-old male (case 1) presented with a neck mass, and metabolic imaging suggested a neoplastic thyroid lesion. The patient underwent total thyroidectomy with lymph node dissection. Surgical pathology revealed multifocal MTC with the largest lesion of 2.2 cm in the right lobe. Metastases were detected in 8 out of 8 central nodes, and multiple foci indicated extranodal disease. Hot-spot testing identified the RET M918T mutation. Five months later, right thoracotomy was performed, and mediastinal metastases were resected. Abdominal CT imaging indicated hepatic metastases, and the patient received external beam radiation to treat right choroidal orbital metastases 8 months after the thyroidectomy.

CGP of the thyroidectomy specimen demonstrated that the tumor harbored RET M918T (32% MAF) and revealed the ATM truncations L804fs*4 (30% MAF) and S978fs*12 (28% MAF). The patient was treated with vandetanib monotherapy, which was dosed progressively (100 mg once daily, 3 weeks; 200 mg once daily, 8.5 months; 300 mg once daily, 2 months). Electrocardiographic QTc interval prolongation from 405 ms at baseline to 442 ms was the main side effect. Circulating calcitonin eventually increased, however, and vandetanib was discontinued due to disease progression after 1 year. The patient presented in the phase 1 clinic at MD Anderson and was enrolled on the clinical trial (NCT01582191) combining vandetanib (300 mg once daily) and everolimus (10 mg once daily) at the defined maximum tolerated dose. After 2 cycles of therapy, the patient had a 25% reduction in the greatest unidimensional tumor measurement per RECIST 1.1 (fig. 2), designated as stable disease, while just under the partial response threshold. In addition, the patient reported that the nodes in the neck were less congested. The main toxicities included grade 2 diarrhea, likely from vandetanib, which was controlled by diphenoxylate and atropine, and grade 2 cough. However, the hepatic metastases in the liver were progressing after an initial mixed response. The patient received particulate transarterial embolization for the hepatic metastases and continued on systemic therapy. The stable disease in the neck area lasted for at least 8 months, but the patient was taken off the protocol because of progressive disease in the liver.

Fig. 2

a CT scan of the index patient (case 1) before treatment shows a right mass measuring 2.2 × 1.9 cm. b CT scan of the same patient taken 2 months after the beginning of treatment with vandetanib plus everolimus shows a mass of 1.5 × 1.5 cm. White arrows point to the target lesion.

Fig. 2

a CT scan of the index patient (case 1) before treatment shows a right mass measuring 2.2 × 1.9 cm. b CT scan of the same patient taken 2 months after the beginning of treatment with vandetanib plus everolimus shows a mass of 1.5 × 1.5 cm. White arrows point to the target lesion.

Close modal

The patient underwent hepatic radiotherapy with transarterial 90Y SIR-Spheres®, which led to a decrease in the volume of the liver disease, and a subsequent bilateral adrenalectomy for refractory ACTH-induced Cushing's syndrome. He was started on cabozantinib (100 mg once daily) and experienced a painful keratotic rash on the feet and intermittent diarrhea, which was managed with dose interruption and reduction. Given the symptomatic progressive disease, the patient underwent extended and complicated right parotidectomy with preservation of the seventh cranial nerve, right neck dissection, wide excision of the entire cervical skin, and reconstruction of the neck and upper chest. Pathology demonstrated MTC, and local hot-spot testing identified only a RET M918T mutation. Cabozantinib was increased to 120 mg daily and then to 140 mg daily. The patient experienced transient symptomatic benefit but had a declining performance status and passed away 20 months after initiation of treatment with vandetanib plus everolimus.

MTC is an aggressive tumor of neuroendocrine origin arising from the calcitonin-producing parafollicular C cells of the thyroid gland and accounts for approximately 5% of thyroid cancers, which translates to an incidence of nearly 3,000 new cases per year in the US. Seventy-five percent of MTC cases are dubbed sporadic, and the remainder are part of the inherited autosomal dominant syndromes MEN2A, MEN2B, or familial. Germline mutations in RET are found in almost all patients with inherited MTC [17,20,21,27], and RET somatic mutations have been observed in 40-65% of patients with sporadic MTC [6,28]. The activating RET M918T mutation is the most frequent somatic and MEN2B germline alteration. Mutations in the RAS pathway have been identified in patients with RET wild-type sporadic MTC [29,30,31]. Retrospective next-generation sequencing studies of the exomes or select cancer genes in 17 and 20 sporadic MTC cases, respectively, confirmed mutant RET, HRAS, and KRAS as principal drivers. These studies reported few additional somatic alterations and recurrent mutations only in the gene MDC1, which was not profiled in this study [32,33].

The recent clinical trials for MTC with targeted therapy demonstrated clinical benefit for only some MTC cases, suggesting the other cases may be de novo resistant to targeted therapy. Several observations from the genomic profiles in this series may explain de novo resistance. We identified a range of RET mutations that occurred mutually exclusively to RET M918T, specifically RET C618G/S/R, C634R, E632_L633del, C620R, and RET V804M. Various RET mutations may be differentially sensitive to targeted therapy, as preclinical data suggest for RET V804M, but this remains a hypothesis for now, as clinical evidence linking specific RET alterations other than RET M918T to therapeutic efficacy is lacking. The potential value of RET mutations as predictive markers of therapy response hinges on the ability of the deployed assay to identify RET alterations with a high sensitivity. Both large phase 3 studies of MTC with targeted therapy reported unknown RET mutation status for 30-40% of cases [4,5], increasing the sample size required to correlate RET mutation status with treatment outcome, although many cases with unknown RET status in the cabozantinib trial had no tumor sample available at the time of analysis [5]. Furthermore, both vandetanib and cabozantinib are anti-angiogenic and inhibit other tyrosine kinases in addition to RET, with vandetanib also targeting VEGFR2 and EGFR and cabozantinib also targeting VEGFR2 and MET [4,5]. Predicting responses to these therapies solely on the basis of RET status thus likely misses other mechanisms of benefit and may be improved by correlating responses with comprehensive genomic profiles of MTC.

De novo resistance may also be linked to alterations in genes other than RET. Activated RAS acts downstream of RET and is associated with resistance to therapies targeting other receptor protein tyrosine kinases; however, in the cabozantinib phase 3 trial, patients with a RAS mutation experienced a similar response rate and PFS as patients with a RET mutation (31 vs. 32% and 47 vs. 60 weeks, respectively) [7]. Alterations in CCND1, CCND2, CDK4, CDK6, CDKN2A/B, or CDKN2C occurred in 21% of specimens and may lead to hyperactivation of the cyclin D-dependent kinases CDK4 and CDK6. Future clinical studies need to address whether CCND1 or CDK4 amplification may associate with sensitivity to CDK4/6 inhibitors, as preliminary clinical data suggest for other tumor types [34,35]. CDKN2C has previously been observed to be inactivated in MTC, and CDKN2C loss cooperated with oncogenic RET to promote MTC development in mice [36,37]. Alterations in cell cycle genes coexistent with RET mutations may therefore contribute to resistance to RET targeting. The current study sensitively assesses alterations throughout the entire RET and RAS genes as well as most known cancer genes, and thus is a better foundation for illuminating potential mechanisms of resistance to the approved therapies.

Everolimus demonstrated modest activity against metastatic MTC, as a phase 2 study of everolimus reported durable stable disease (at least 24 weeks) for 7 out of 9 cases, with 5 patients experiencing tumor shrinkage [38]. Case studies describe 1 patient who achieved stable disease for at least 1 year on everolimus [39] and 2 patients with biochemical responses on everolimus plus octreotide [40]. These clinical data are supported by preclinical studies that validate mTOR signaling as a potential target in MTC [41,42]. However, as the efficacy of single-agent everolimus in MTC has been limited and may result in even less clinical benefit in the second-line setting [43], mTOR inhibitors may be most useful in targeted therapy combinations to overcome resistance to RET targeting. A case report describes systemic activity of vandetanib plus everolimus against RET-rearranged lung cancer [44]. Addition of everolimus to HER2-targeted therapy has shown efficacy against trastuzumab-resistant breast cancer [45], and persistent mTOR activation correlates with resistance to other targeted therapies [46]. Although a specimen of the progressive vandetanib-resistant disease was not available for CGP in the case described here, CGP can potentially identify GAs in the mTOR pathway and point to benefit from added everolimus [45]. Vandetanib combined with everolimus may hold merit in the second-line setting for metastatic MTC and could be most rationally deployed with the use of genomic profiling.

In conclusion, CGP of MTC has the potential to better delineate potential markers of response to approved targeted therapies, while suggesting routes to overcome resistance to these therapies. Genomic profiles of MTC need to be further investigated in the course of clinical care for patients with advanced MTC to inform the optimal management of these patients.

We thank Dr. Jon Chung and Dr. Jennifer Spangle for helpful discussions. The University of Texas MD Anderson Cancer Center is supported by theNational Institutes of Health through Cancer Center Support Grant CA016672. V.S. would like to thank the Dennis Lawrence and Joyce Lawrence research funds.

A.M.H., J.X.S., J.A.E., J.C., J.-A.V., R.L.E., D.L., J.S.R., V.A.M., S.M.A., and P.J.S. are employed by and have equity interests in Foundation Medicine, Inc. K.W. and R.Y. are former employees of Foundation Medicine, Inc. S.I.S. has a consultant/advisory role for Eisai. N.L.B. has a consulting/advisory relationship with Eisai and Bayer, and she has received research funding from Bayer. M.H.S. has received research funding from Bayer, Eisai, and Exelixis.

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A.M.H. and V.S. contributed equally to this work.

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