Germline RET Mutations Research Articles

A novel RET gene mutation in a patient with apparently sporadic pheochromocytoma

Pheochromocytoma (Pheo) is a chromaffin tumor arising from the adrenal medulla. The recent discovery of new germline mutations in RET, SDHA, SDHB, SDHC, SDHD, VHL, NF1, TMEM127, MAX genes, increased the rate of genetic disease from 10% to 28% in patients with apparently sporadic tumor. RET germline mutations cause multiple endocrine neoplasia type 2 syndrome (MEN 2A) characterized by complete penetrance of medullary thyroid cancer (MTC), and lower prevalence of Pheo and hyperparathyroidism. We describe the genetic etiology of an apparently sporadic case of monolateral Pheo in a 42-year-old male patient. A new (not previously reported) MEN 2A-associated germline RET mutation located in exon 11 (Glu632Gly, caused by an A>G point mutation at position 1895 of the RET cDNA) was found in the patient but not in his living first-degree relatives. This observation increases the number of possible germline RET mutations. Genotype-phenotype correlation of this new genetic alteration is unknown, but this rare mutation is probably associated with a low risk for MTC (usually the first tumor diagnosed in MEN 2A syndrome) and with the development of Pheo before the onset of MTC. Since we expect MTC to occur in our patient, strict follow-up is mandatory. Our findings emphasize the relevance of genetic testing in patients with Pheo, especially when the clinical presentation (family history, young age at diagnosis, multiple locations, malignant lesions, and bilateralism) is suggestive.

RET and EDNRB mutation screening in patients with Hirschsprung disease: Functional studies and its implications for genetic counseling.

Hirschsprung disease (HSCR) is a major cause of chronic constipation in children. HSCR can be caused by germline mutations in RET and EDNRB. Defining causality of the mutations identified is difficult and almost exclusively based on in silico predictions. Therefore, the reported frequency of pathogenic mutations might be overestimated. We combined mutation analysis with functional assays to determine the frequencies of proven pathogenic RET and EDNRB mutations in HSCR. We sequenced RET and EDNRB in 57 HSCR patients. The identified RET-coding variants were introduced into RET constructs and these were transfected into HEK293 cells to determine RET phosphorylation and activation via ERK. An exon trap experiment was performed to check a possible splice-site mutation. We identified eight rare RET-coding variants, one possible splice-site variant, but no rare EDNRB variants. Western blotting showed that three coding variants p.(Pr270Leu), p.(Ala756Val) and p.(Tyr1062Cys) resulted in lower activation of RET. Moreover, only two RET variants (p.(Ala756Val) and p.(Tyr1062Cys)) resulted in reduced ERK activation. Splice-site assays on c.1880-11A>G could not confirm its pathogenicity. Our data suggest that indeed almost half of the identified rare variants are proven pathogenic and that, hence, functional studies are essential for proper genetic counseling.

The development of rapid and accurate screening test for RET hotspot somatic and germline mutations in MEN2 syndromes

Medullary thyroid carcinoma (MTC) is a rare endocrine malignancy with distinctive features separating it from other thyroid cancers. Cancer may be sporadic or occur as a consequence of the hereditary syndrome called multiple endocrine neoplasia type 2 (MEN2) with three distinct phenotypes in MEN2A, MEN2B and FMTC. Each variant of MEN2 results from different RET gene mutations, with a good genotype–phenotype correlation. The goal of the study was to develop a fast and accurate screening method for a reliable detection of hot-spot RET germline and sporadic tumor mutations. From a cohort of 191 patients with MTC and their relatives, 38 tested positive and 31 tested negative for a germline or somatic tumor RET mutation were selected. A positive HRM mutation pattern was detected in all mutation-positive patients and altogether the method was able to clearly differentiate between twenty different genotypes. A novel germline variant p.Ala639Thr was detected in MTC patient, which was determined to be likely benign. Analytical specificity was determined to be 98.6% and a sensitivity threshold was determined to be 30%. The fast and accurate HRM method reduces the turnaround time providing fast and important information, especially when targeted anti-tyrosine kinase therapy on tumor samples is considered. Overall, we developed a high-throughput, accurate and cost-effective approach for the detection of RET germline and sporadic tumor mutations.

Bilateral Pheochromocytomas in MEN2A Syndrome: A Two-Institution Experience.

Bilateral pheochromocytoma (PHEO) is more frequently found in patients with multiple endocrine neoplasia 2A carrying a RET germline mutation located in codon 634 (C634). However, it is unclear whether different amino acid substitutions within C634 cause differences in bilateral PHEOs expression. We aimed to answer this by pooling data from two Asian institutions. Sixty-seven patients had confirmed C634 germline mutation. Age-dependent penetrance of bilateral PHEO was calculated from date of birth to the date when bilateral PHEO was first diagnosed or when the contralateral gland became a PHEO (if the patient already had one adrenal gland removed). Age-dependent penetrance was estimated by the Kaplan-Meier method and compared by log-rank test. The 4 different amino acid substitutions included C634R (arginine) (n = 19, 28.4 %), C634Y (tyrosine) (n = 36, 38.8 %), C634G (glycine) (n = 4, 6.0 %), and C634W (tryptophan) (n = 8, 11.9 %). The age-related penetrance of PHEO was similar between C634R, C634Y, C634G, and C634W (by age 40, 69.8, 55.2, 25.0, and 56.2 %, respectively) (p = 0.529). However, the age-related penetrance of bilateral PHEO in C634R was significantly higher than C634Y (by age of 40, 59.3 % vs. 25.2 %, p = 0.046) or C634Y, C634G, and C634W combined (59.3 % vs. 21.5 %, p = 0.024). Nevertheless, the accumulative risk of bilateral PHEOs across all four C634 mutations almost approached 100 % over time. The accumulative risk of bilateral PHEOs almost reached 100 % but its onset was significantly earlier in C634R mutation. These findings implied that those with C634R mutation might benefit from earlier screening of contralateral PHEO than other C634 mutations after an unilateral adrenalectomy.

Molecular pathogenesis of thyroid tumors

The molecular pathogenesis of thyroid tumors has been an evolving field in the past years. The constitutive activation of intracellular tyrosine kinases has been identified as a hallmark of thyroid cancer. The activation of MAPK and PI3K pathways through somatic gene mutations or gene rearrangements seem to play a pivotal role in the pathogenesis of follicular-cell-derived tumors. In poorly differentiated tumors and anaplastic tumors often an accumulation of genetic alterations from differentiated thyroid cancer but also novel gene mutations can be observed. The C-cell-derived medullary thyroid cancer evolves through the constitutive activation of the RET kinase, either through germline RET mutations or somatic RET and RAS mutations. The better knowledge of the molecular pathogenesis allowed the development of targeted therapies in thyroid cancer patients. The identification of molecular response markers to tyrosine kinase inhibitor therapy is desirable.

Reference range of serum calcitonin in pediatric population.

Children belonging to the multiple endocrine neoplasia type 2 (MEN 2) pedigree and carrying germline RET mutations are candidates for prophylactic thyroidectomy, the timing of which is based on the mutation-associated risk and the calcitonin (CT) levels. The aim of this study was to establish the reference range for serum CT in a pediatric population. The study included 2740 subjects (1339 females and 1401 males) ranging in age from 1 day to 16 years and undergoing blood testing for any medical condition not affecting serum CT. Overall, serum CT was undetectable in 61.5% of the samples and detectable in 38.5%. Detectable samples were more frequent in the first 2 years of life. Thereafter, undetectable samples became more frequent, particularly in females. Mean serum CT concentrations were higher in the first year of life (9.81 ± 8.8 pg/mL; range, 2.0-48.9 pg/mL) and the second year of life (4.56 ± 2.64 pg/mL; range, 2.0-14.7 pg/mL). A significant decrease of serum CT levels was observed thereafter (P < .001), and starting from the third year of life serum CT levels were similar to those found in adults. No gender difference was found in any age group. Based on these results, age-specific CT reference ranges are needed in the pediatric population, and especially in the first 2 years of life. This is the first study defining the reference range for serum CT in the pediatric population and large enough to be statistically meaningful. Our proposal may facilitate the process of decision making when dealing with gene carriers of MEN 2.

Twenty years of lesson learning: how does the RET genetic screening test impact the clinical management of medullary thyroid cancer?

Medullary thyroid carcinoma (MTC) is a rare disease that can be inherited or sporadic; its pathogenesis is related to activating mutations in the RET gene. This study describes our 20-year experience regarding RET genetic screening in MTC. We performed RET genetic screening in 1556 subjects, 1007 with an apparently sporadic MTC, 95 with a familial form and 454 relatives of RET-positive patients with MTC. A germline RET mutation was found in 68 of 1007 (6·7%) patients with sporadic MTC, while 939 patients with MTC were negative for germline RET mutations. We then identified a total of 137 gene carriers (GC). These subjects initiated a clinical evaluation for the diagnosis of MEN 2. A total of 139 MEN 2 families have been followed: 94 FMTC, 33 MEN 2A and 12 MEN 2B. Thirty-three different germline RET mutations were identified. Codon 804 was the most frequently altered codon particularly in FMTC (32/94, 34%), while codon 634 was the most frequently altered codon in MEN 2A (31/33, 94%); MEN 2B cases were exclusively associated with an M918T mutation at exon 16. Our 20-year study demonstrated that RET genetic screening is highly specific and sensitive, and it allows the reclassification as hereditary of apparently sporadic cases and the identification of GC who require an adequate follow-up. We confirmed that FMTC is the most prevalent MEN 2 syndrome and that it is strongly correlated with noncysteine RET mutations. According to these findings, a new paradigm of follow-up of hereditary MTC cases might be considered in the next future.

Tandem Germline RET Mutations in a Family Pathogenetic for Multiple Endocrine Neoplasia 2B, Confirmed by a Natural Experiment

A family with germline tandem V804M/Y806C mutations in the RET proto-oncogene was reported. The in vitro study results showing that these mutations were on the same allele and that RET with these mutations had a moderate transforming activity were confirmed by the clinical features of the offspring as a natural experiment. Thus, the tandem double RET mutations are pathogenetic for MEN 2B.

Abstract 1072: TAM and CD44 in medullary thyroid carcinoma

Abstract Background: Tumor-associated macrophages (TAMs) are alternatively activated or M2 macrophages that infiltrate cancer tissues. It has been suggested that TAMs exert several pro-tumor functions by releasing several growth factors and proangiogenic cytokines that favor tumor progression. In fact, TAM density has been associated with poor prognosis. Although it is well known the pro-tumor roles of TAM in several tumor types, its role in medullary thyroid cancer (MTC) is still unknown. We here evaluated the M2 macrophage marker CD163 in MTC and correlated with RET mutational status and clinical and pathological features. Because it has been suggested TAMs could interact with CD44-positive tumor cells to during tumorigenesis process, we also investigated the expression of CD44 in MTC. Patients and Methods: CD163 and CD44 expression we investigated in 78 MTC by immunohistochemistry (IHC). Because hereditary form is caused by germline mutations in the RET gene, all patients were screened for RET mutations (exons 8, 10, 11, 13-16) in the DNA isolated from blood samples. Those MTC that were negative for RET germline mutations, were also screened for somatic mutations in the RET gene (exons 10, 11 e 16). Results: Germline mutation in the RET gene were identified in 29 patients (p.G533C, p.C609Y, p.C611Y, p.C620Y, p.C634R, p.C634Y/Y791F, p.S891A and p.M918V). Approximately 49 cases are sporadic cases in which somatic RET mutations were identified in about 6 cases (p.M918T, p.C634R and p.C634Y). CD163 expression was mainly found in sporadic MTC when compared to hereditary MTC (p=0.021), suggesting that TAM may play a role in the progression of sporadic MTC. On the other hand CD44 expression was mainly found in hereditary MTC than in sporadic cases (p=0.017), particularly in patients with RET germlime mutation at codon C634. Conclusion: TAM seems to be more important for sporadic MTC tumorigenesis when compared to hereditary MTC. On the other hand, CD44 expression correlated with hereditary MTC, particularly in patients with mutation at codon C634. Citation Format: MARTA MIYAZAWA, MARIA JOSE C. P. SANTOS, LARISSA V. BIM, ERIKA S. L. ORTI-RADUAN, FLÁVIA O. F. VALENTE, ADAUTO F. NUNES, ROSANA DELCELO, RUI M. B. MACIEL, JANETE M. CERUTTI. TAM and CD44 in medullary thyroid carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1072. doi:10.1158/1538-7445.AM2014-1072

Prevalence by Age and Predictors of Medullary Thyroid Cancer in Patients with Lower Risk GermlineRETProto-Oncogene Mutations

Age-related risk of medullary thyroid carcinoma (MTC) development in presymptomatic carriers of lower risk germline RET mutations is uncertain; such data may aid counseling patients regarding timing of thyroidectomy. From an institutional database and an exhaustive literature review, we identified 679 patients with American Thyroid Association (ATA) level A or B mutations who were identified because of family screening (index cases of MTC were excluded to minimize selection bias). We evaluated age at thyroidectomy or last evaluation if no thyroidectomy, preoperative calcitonin level (elevated or not), the mutated codon, and outcome (MTC vs. no MTC after thyroidectomy or no clinical evidence of MTC if thyroid intact). Data were used to estimate the cumulative prevalence of MTC and/or assess likelihood of MTC stratified by codon. After exclusion of cases with missing data or small representation, 503 patients with mutations in codons 533, 609, 611, 618, 620, 791, and 804 were analyzed. 236 patients had MTC. Cumulative prevalence and median time to MTC varied by codon and within ATA risk levels (p<0.0001). Patients with a codon 620 mutation were 2.8-6.9 times more likely to have MTC than other level B mutation carriers, and 5.1-21.7 times more likely than level A mutation carriers included in our focus population. The youngest median time to MTC was 19 years for codon 620 and the oldest was 56 years for codon 611. Cumulative prevalence of MTC by age 20 was 10% or lower for codons 533, 609, 611, 791, and 804. By age 50, it ranged from 18% for codon 791 to 95% for codon 620. An elevated preoperative calcitonin level strongly predicted MTC on final pathology, though false-negative rates varied by codon (p<0.0001). Positive predictive values ranged from 76% to 100% by codon with an overall positive predictive value of 87% across codons. This study offers a better understanding of the age-related development of MTC in lower risk RET mutation carriers, provides evidence of further distinctions between lower risk mutations within ATA subgroups, and clarifies the clinical significance of codon 791 mutations. The data support individualized "codon-based" management approaches coupled with clinical data such as calcitonin levels.

RET gene mutations (genotype and phenotype) of multiple endocrine neoplasia type 2 and familial medullary thyroid carcinoma

The rapid technical advances in molecular biology and accelerating improvements in genomic and proteomic diagnostics have led to increasingly personalized strategies for cancer therapy. Such an approach integrates the genomic, proteomic, and molecular information unique to the individual to provide an accurate genetic diagnosis, molecular risk assessment, informed family counseling, therapeutic profiling, and early preventative management that best fits the particular needs of each patient. The discovery of mutations in the RET proto-oncogene resulting in variable onset and severity of multiple endocrine neoplasia type 2 (MEN2) was the first step in developing direct genetic testing for at-risk individuals. Patients with germline RET mutations may undergo risk assessment and appropriate intervention based on specific mutations. Moreover, family members of affected individuals receive counseling based on understanding of the genetic transmission of the disease. Increasingly, clinicians are able to make therapeutic choices guided by an informative biomarker code. Improvements in detection and management of patients with MEN2 resulting from understanding of the RET proto-oncogene are evidence of the benefits of personalized cancer medicine. This review describes the discovery of the RET proto-oncogene, the association between genotype and phenotype, and the role of mutation analysis on diagnosis and treatment of MEN2.

Cabozantinib: a MET, RET, and VEGFR2 tyrosine kinase inhibitor.

Cabozantinib is a receptor tyrosine kinase inhibitor with activity against MET, VEGFR2, FLT3, c-KIT, and RET. Activity of cabozantinib toward a broad range of tumor models could be detected in several preclinical studies. Of note, cabozantinib decreases metastasis potential and tumor invasiveness when compared with placebo or agents that target VEGFR and have no activity against MET. Clinical phase I and II studies with cabozantinib have been conducted in various malignancies including medullary thyroid cancer (MTC), NSCLC, breast, ovarian, pancreatic, and prostate cancer. In MTC, gain of function mutations of RET are central for tumorigenesis. Hereditary forms of MTC (MEN II) are caused by germline mutations of RET, in sporadic MTC in up to 50% of cases RET mutations occur. Additionally, activating molecular changes in VEGFR and MET pathways have also been implicated in MTC progression. Clinical responses with cabozantinib in MTC could be observed in early clinical trials, and following confirmation of clinical benefit in a randomized phase III trial, cabozantinib gained FDA approval for first-line treatment of advanced MTC in 2012. In prostate cancer models, MET expression increases with androgen ablation and clinical progression of bone and lymph node metastasis. A phase II trial with cabozantinib also showed very promising response rates in patients with metastatic prostate cancer. Therefore, randomized phase III studies are currently ongoing to validate the efficacy of cabozantinib in heavily pretreated prostate cancer patients.

Patient affected by neurofibromatosis type 1 and thyroid C-cell hyperplasia harboring pathogenic germ-line mutations in both NF1 and RET genes

Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease with an estimated incidence of 1 in 3000/3500 live births. NF1 is caused by a mutation in a gene which encodes a protein known as neurofibromin. In up to 5% of cases, NF1 is associated with pheochromocytomas.RET proto-oncogene encodes a member of the receptor tyrosine kinase family involved in the normal development or the neoplastic growth of neural crest cell lineages. Germ-line RET mutations account for cases of Multiple Endocrine Neoplasia type 2 (MEN2), an autosomal dominant genetic syndrome where medullary thyroid carcinoma (MTC) is the major and more clinically severe feature, with nearly complete penetrance. C-cell hyperplasia (CCH) is described in MEN2 patients, and it has been implicated as the precursor of in situ MTC. Patients with RET mutations develop pheochromocytomas in 50% of cases.Rarely, patients with NF1 have been found to present, in addition to the NF1 clinical picture, other lesions, such as parathyroid hyperplasia/adenoma and/or medullary thyroid carcinoma. In spite of the presence of these MEN2 lesions, in none of these patients mutations of gene RET have been found so far.In this report, we describe the first case of a patient affected by a germ-line mutation in both NF1 and RET genes.

Role of Preoperative Basal Calcitonin Levels in the Timing of Prophylactic Thyroidectomy in Patients With Germline RET Mutations

The American Thyroid Association (ATA) published recommendations for the timing of prophylactic surgery for medullary thyroid carcinoma based on the specific mutation, patient age, family history, and serum calcitonin levels. The aim of this study was to assess the role of preoperative basal calcitonin (prebCt) levels in predicting the presence of medullary carcinoma of the thyroid in patients with RET mutations. We conducted a retrospective study in two endocrine surgery departments. Between 1986 and 2012, a total of 32 patients with RET mutations underwent prophylactic thyroidectomy. The patients were stratified into four ATA risk levels: A, B, C, and D. All of the patients were biologically cured. Microcarcinoma was observed in the final pathology report for four of the 20 patients with normal prebCt (25%) and for nine of the 12 patients with elevated prebCt (75%). In the level A group, four patients with normal prebCt and one patient with elevated prebCt presented with microcarcinoma. In the level C group, one patient with normal prebCt and six of the seven patients with elevated prebCt (86%) presented with microcarcinoma. PrebCt can predict the presence of microcarcinoma according to surgical pathological analysis. Patients with microcarcinoma can be biochemically and clinically cured using prophylactic thyroidectomy.

Abstract A64: Synergistic cooperation between sunitinib and cisplatin promotes apoptotic cell death in human medullary thyroid cancer.

Abstract Recent clinical trials with tyrosine kinase inhibitors (TKIs) have demonstrated therapeutic benefit in patients with advanced medullary thyroid cancer (MTC), a disease poorly responsive to conventional chemotherapy. However, new therapeutic strategies are needed to obtain complete durable responses. The RET TK receptor, which is deeply implicated in the pathogenesis of both inherited and sporadic MTCs, is a major target of most TKIs under clinical investigation in MTC. RET germline mutations represent the pathogenic drivers of MEN2 syndromes and somatic oncogenic mutations of RET are present in 30-50% of sporadic MTCs. Since RET has been shown to negatively regulate CD95 death receptor activation in preclinical models of RET-dependent MTC, we investigated the therapeutic potential of the combination of the RET targeting TKI sunitinib with cisplatin, a known activator of the apoptotic extrinsic pathway. Experiments were designed to determine the effects of the drug combination in human MTC cell lines harboring oncogenic RET mutations. RET signaling, cell growth, apoptosis, autophagy, tumor growth in mice, and the mechanisms underlying the drug interaction were investigated using standardized methods. Assessment of the drug interaction was carried out by the Combination Index method. Sunitinib and cisplatin synergistically inhibited the growth of MZ-CRC-1 cells harboring the RET M918T activating mutation typical of the vast majority of MEN2B-associated and sporadic MTCs. The combination enhanced apoptosis activation through CD95-mediated, caspase-8 dependent, pathway. Moreover, sunitinib induced a severe perturbation of the autophagic flux characterized by autophagosome accumulation and a remarkable lysosomal dysfunction which was further enhanced, with lysosomal leakage induction, by cisplatin. Administration of the drug combination to mice xenografted with MZCRC-1 cells significantly improved the antitumor efficacy, as compared to single agent treatments, enhancing tumor growth inhibition and inducing complete responses in 30% of treated mice. In addition, a significant increase in caspase-3 activation (P&amp;lt;0.01 vs cisplatin, P&amp;lt;0.0005 vs sunitinib) and apoptosis was observed in tumor cells. Overall, these findings indicate that addition of cisplatin to sunitinib potentiates drug induced cell death in MTC cells through a mechanism involving both the apoptotic and the autophagic/lysosomal systems, and has promising preclinical activity in MTCs harboring the RET M918T oncogene. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A64. Citation Format: Alessia Lopergolo, Valentina Nicolini, Denis Cominetti, Marco Folini, Paola Perego, Vittoria Castiglioni, Eugenio Scanziani, Maria Grazia Borrello, Nadia Zaffaroni, Giuliana Cassinelli, Cinzia Lanzi. Synergistic cooperation between sunitinib and cisplatin promotes apoptotic cell death in human medullary thyroid cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A64.

How to Incorporate New Tyrosine Kinase Inhibitors in the Treatment of Patients With Medullary Thyroid Cancer

Medullary thyroid cancer (MTC), a neuroendocrine tumor arising from the parafollicular C cells of the thyroid gland, is rare and accounts for approximately 5% of all thyroid cancers. The majority of cases of MTC are sporadic, and in roughly 25% of cases, MTC is part of a hereditary cancer syndrome with a well-characterized germline RET mutation: multiple endocrine neoplasia 2A, multiple endocrine neoplasia (MEN) 2B, or familial MTC. Patients with newly diagnosed disease are treated with surgery. Calcitonin and carcinoembryonic antigen (CEA) are useful blood markers and are often used to monitor patients after surgery. Systemic chemotherapy and radiation therapy are not believed to represent important or useful options for most patients with MTC. Patients with metastatic disease are not curable and are often monitored with serial scans and biochemical markers. Metastatic MTC can be quite indolent, and timing to initiate therapy varies greatly among physicians and practices. Until recently, no satisfactory options existed for treating patients with metastatic disease in need of therapy. In 2011, the US Food and Drug Administration approved vandetanib (which targets RET, epidermal growth factor receptor, and vascular endothelial growth factor receptor) for the treatment of patients with symptomatic or progressive, locally advanced, or metastatic MTC. This approval was based on the Zactima Efficacy in Thyroid Cancer Assessment (ZETA) phase III study. In 2012, the US Food and Drug Administration also approved cabozantinib for the same indication on the basis of the Efficacy of XL184 (Cabozantinib) in Advanced Medullary Thyroid Cancer (EXAM) trial reported in the article that accompanies this editorial. The ZETA study was a randomized double-blind phase III trial in which patients were randomly assigned in a 2:1 fashion to receive oral vandetanib 300 mg per day (n 231) or a placebo (n 100) until disease progression. The primary end point was progression-free survival (PFS), and relevant secondary end points were objective response rate, overall survival, biochemical response, safety, and tolerability. Patients who were enrolled onto the ZETA trial were not required to have progressive disease before enrollment but were required to have measurable disease at baseline and a calcitonin level of at least 500 pg/mL. The study showed a significant prolongation of PFS with vandetanib versus the placebo (hazard ratio, 0.46; 95% CI, 0.31 to 0.69; P .001). Statistically significant advantages for vandetanib were also seen for objective response rate, disease control rate, and biochemical response. This study allowed patients receiving the placebo to cross over to vandetanib, and an overall survival advantage was not seen at the time of the initial report. The median duration of treatment in the randomized phase was 90.1 weeks for vandetanib and 39.9 weeks for the placebo. The ZETA trial clearly included many patients with indolent disease, as evidenced by a PFS of 19.3 months in the placebo group (with an estimated PFS of 30 months in the vandetanib group). Adverse events such as diarrhea, rash, nausea, and hypertension occurred in more than 30% of patients receiving vandetanib. More patients required dose reduction of vandetanib compared with the placebo for adverse events or QTc prolongation (35% v 3%). Nineteen patients (8%) developed protocol-defined QTc prolongation, but there were no reports of torsades de pointes. The rate of treatment discontinuation because of toxicity was 12%. Because of this risk of QT prolongation, vandetanib is currently only available through the US Food and Drug Administration Vandetanib Risk Evaluation Mitigation Strategy Program. This program is meant to educate practitioners about the risk, appropriate monitoring, and management of QT prolongation should it occur during vandetanib therapy. The EXAM study that is reported in the article that accompanies this editorial sought to examine the effect of cabozantinib in progressive MTC. Cabozantinib is a tyrosine kinase inhibitor (TKI) that targets three potentially important pathways in MTC: MET, vascular endothelial growth factor receptor 2, and RET. EXAM is a double-blind phase III trial comparing oral cabozantinib at 140 mg per day with a placebo in 330 patients with documented radiographic progression of metastatic MTC. The primary end point is PFS. Other end points are response rate, overall survival, and safety. This study also used a 2:1 randomization schema, but unlike the ZETA trial, the EXAM study did not allow cross over to the active drug. The study met its primary end point of prolongation of PFS: 11.2 months for cabozantinib versus 4 months for the placebo (hazard ratio, 0.28; 95% CI, 0.19 to 0.4; P .001). There is no survival advantage noted so far, and analysis is ongoing for the overall survival end point. Objective tumor response rates and biochemical responses were also significantly improved with cabozantinib. JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 29 OCTOBER 1

Outcome of Patients with Different MEN2A Germline Mutations

Objectives: Medullary thyroid carcinoma (MTC) behavior in MEN2 present variations, ranging from indolent MTC, in patients harboring intra-cellular “weak mutations, to highly aggressive disease, with RET mutation in codons 634 or 918. We compared the outcomes among patients with different germline mutations. Methods: Retrospective chart review. Between January 2002 and December 2011, 67 MTC patients were operated at our institution: 26 (38.8%) sporadic and 41 (61.2%) carriers of MEN2A germline RET mutations. All patients were tested for RET hot spot mutations (exons 5, 8, 10, 11, 13, 16), pre- and postoperative calcitonin (Ct), carcinoembryonic antigen (CEA), parathyroid hormone, and calcium. They were also submitted to imaging studies (ultrasound, MRI, CT and scintigraphy). Follow-up varied from 1 to 65 months (average 10.9 months). Patients were classified as cured when serum Ct concentration &lt;2pg/mL in the last evaluation (Group A). Patients not cured were divided in two groups: Group B) Ct &gt;2pg/mL and &gt;40pg/mL and Group C) Ct&gt;40pg/mL. Results: Nineteen patients (46.3%) were cured, most of them treated by prophylactic surgery. There was no difference according to the RET mutation. Preoperative calcitonin level ranged from 4.5 to 1367pg/mL (average 124.5) in group A from 9 to 180149 (average 124.5) in group B and from 98 to 8358 (average 2587) in group C. Fifteen patients (36.6%) presented postoperative hypoparathyroidism, not related to cure, although related to extended surgery. Younger patients who underwent prophylactic surgery had better prognosis ( P = 0.02). Conclusions: Genetic test is the most important exam to perform in MTC patients. Outcome was better in younger patients treated by prophylactic surgery.

Comprehensive analysis of RET gene should be performed in patients with multiple endocrine neoplasia type 2 (MEN 2) syndrome and no apparent genotype-phenotype correlation: an appraisal of p.Y791F and p.C634Y RET mutations in five unrelated Brazilian families.

We previously identified a four-generation family with medullary thyroid cancer (MTC) and a germline p.Y791F RET mutation whose cancer lacked a strong genotype-phenotype correlation. The entire gene coding region of the RET gene should be sequenced when genotype-phenotype discrepancies are observed in patients with multiple endocrine neoplasia type 2 (MEN 2), even if a RET hotspot mutation has been identified. A new genetic test was performed in the index case of this family with the p.Y791F RET germline mutation. The entire coding region of the RET gene was investigated by direct sequencing of PCR products. Once a mutation was identified, the target exon was sequenced in all at-risk relatives. An additional p.C634Y germline mutation in the RET gene was identified in the reported family. The double mutation occurred in cis and segregated with the phenotype. Through the Brazilian Genetic Screening Program developed at our institution, we additionally report the combination of these two mutations (p.C634Y/p.Y791F) in the RET gene in four other unrelated families. The overall penetrance of MTC and pheochromocytoma in patients with the p.C634Y/p.Y791F mutations was 79% and 13%, respectively. Our data emphasises that a comprehensive analysis of the RET gene may reveal multiple germline mutations in MEN 2 patients who exhibit an atypical clinical course of the disease.

Efficacy of cabozantinib (Cabo) in medullary thyroid cancer (MTC) patients with RAS or RET mutations: Results from a phase III study.

6000 Background: Cabo extends progression-free survival (PFS) in patients (pts) with progressive, metastatic MTC (Schöffski, J Clin Oncol 30, 2012). Mutations in the RET oncogene are associated with most hereditary cases and ~half of sporadic cases of MTC. RAS gene mutations have recently been identified in subsets of RET wild type (wt) cases. Therefore, we investigated the association of RET (a prospectively defined endpoint) and RAS mutations (a post hoc analysis) with efficacy outcomes in the phase 3 study of cabo in MTC. Methods: Pts enrolled into the double-blind, placebo-controlled phase III trial were evaluated for the presence of somatic and germline RET mutations using Sanger and next generation methods. A subset of pts determined to be RET wt (44 pts) or RET unknown (41 pts) were then evaluated for tumor-associated mutations in KRAS, NRAS, and HRAS in codons 12, 13, and 61 by next generation sequencing. Impact of RET and RAS gene mutation status was evaluated with respect to PFS and tumor response rate (RR) according to RECIST. Results: RET status was determined in 65% of the study pts (215/330), of which 79% harbored an activating mutation, and 21% were RET wt. All RET mutational subgroups (RET mutated, RET wt, and RET unknown) showed hazard ratios indicating PFS benefit from cabo treatment, and demonstrated RRs between 22% and 32%. However pts harboring a RET mutation had longer median PFS on cabo (60 wks) than pts with wt RET (25 wks, PFS difference p=0.0001). Also, pts with the poor prognosis mutation RET M918T showed a longer median PFS on cabo treatment (61 wks) than pts with any other RET mutation (36 wks, PFS difference p=0.009). Patients with hereditary MTC had similar PFS to those with sporadic disease, and the presence of the common RET polymorphism G691S had no effect on either PFS or RR. Sixteen of 85 tested pts (5% of total study pts) with wt or unknown RET status were found to harbor a RAS gene mutation. The RAS-mutated pts showed a similar RR (31%) and PFS (47 wks) as RET mutated pts (32% and 60 wks). Conclusions: While hazard ratios indicate PFS improvement for all RET subgroups on cabo, the extent of benefit may depend in part on RET genotype. Cabo treatment benefit is also seen in pts harboring a RAS mutation. Clinical trial information: NCT00704730.

Abstract 5139: Molecular docking exploration of potential RET tyrosine kinase inhibitors at non ATP-binding sites.

Abstract RET (REarranged during Transfection) tyrosine kinase receptor is a transmembrane protein required for the development of neural-crest-derived cells, the urogenital system, and the central and peripheral nervous systems, notably the enteric nervous system. The RET protein has an extracellular domain with a cysteine-rich region and four cadherin-like domains, a transmembrane domain, and an intracellular tyrosine kinase domain, required for RET phosphorylation and downstream signaling. The structure of the RET kinase shares with other tyrosine kinases many conserved functional motifs and regulatory residues that are important for the kinase enzyme function. RET activation requires binding of a glial cell-line-derived neurotrophic factor (GDNF) and a co-receptor of the GDNF family receptors α. Germline mutations of RET, leading to uncontrolled activation, are associated with thyroid cancer, and recently with colorectal and lung cancers, and chronic myelomonocytic leukemia. RET mutations that result in decreased receptor function have been linked to developmental defects, such as Hirschsprung disease and kidney anomalies. The design of receptor tyrosine kinase (RTK) inhibitors has traditionally targeted the enzymes’ highly conserved ATP binding pocket. This approach resulted in the discovery of potent small molecule inhibitors, but with relatively low selectivity. To date there are no RET-specific inhibitors available for therapy, although few small molecule inhibitors are undergoing clinical evaluations as potential RET inhibitors. Since there are important critical regions within the RET molecule (e.g., the substrate binding pocket and activation loop) that contain unique amino acids and structural features, we used the method of molecular docking to virtually screen a diverse library of compounds that potentially target non-ATP binding sites of RET. Known ligands of RET were used to test the success of docking before the library of compounds was screened, docking solutions were inspected and ranked, and best compounds selected for novel structural features and future testing. Multiple comparisons with other known structures of RTK-ligand complexes have been performed in order to identify molecular features that would characterize the discovery of potential RET-specific inhibitors. Citation Format: Adrian C. Nicolescu, Taranjit S. Gujral, Jordan DS Zelt, Lois M. Mulligan. Molecular docking exploration of potential RET tyrosine kinase inhibitors at non ATP-binding sites. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5139. doi:10.1158/1538-7445.AM2013-5139 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.