Rearranged During Transfection Variants Research Articles

Genotype/phenotype correlations in multiple endocrine neoplasia type 2.

Multiple endocrine neoplasia type 2 (MEN 2) is a rare hereditary endocrine tumour syndrome caused by mutations in the rearranged during transfection (RET) gene. MEN 2 is divided into two main entities, MEN 2A and MEN2B, both of which present with medullary thyroid cancer (MTC) in approximately 100% of cases and pheochromocytoma in 50% of cases. Specific RET mutations are associated with a risk of early onset of MTC, from 1 year of age (highest risk) to 5 years of age (high risk). This risk defines the optimal timing for thyroidectomy, ideally at an age when the disease has not spread. This is the most important genotype-phenotype correlation observed in MEN 2. Specific RET mutations also define the penetrance of pheochromocytoma. However, despite the presence of these highest/high risk variants, some patients unexpectedly present with non-aggressive MTC or never present with pheochromocytoma, suggesting that factors other than the major RET variant may modify the natural history and genotype-phenotype correlations. Improving our understanding of the genotype-phenotype correlations would allow individualizing the management and follow-up of patients with MEN 2. The aim of this brief review is to discuss the main genotype-phenotype correlations in MEN 2 and the potential factors that might influence these correlations.

Maternal Vitamin A Status as a Risk Factor of Hirschsprung Disease in the Child.

The gene-environment interaction of the REarranged during Transfection ( RET ) gene with vitamin A in the etiopathogenesis of Hirschsprung disease (HSCR) has been suggested in rodents. The aim of this study was to evaluate vitamin A status in mothers of children with HSCR and to assess its association with pathogenic variants of the RET gene in affected children. This was a case-control study of stable isotope-based vitamin A measurement stores of mothers of children diagnosed with HSCR (within 8 months from birth, n = 7) and age-matched mothers of normal children (n = 6). Next-generation sequencing of RET exons, along with their upstream promoter region, was performed in the 7 HSCR proband-parent triads to evaluate pathogenic variants. Maternal vitamin A stores in the HSCR group was almost 50% that of those in controls, tending toward significance (0.50 ± 0.17 vs 0.89 ± 0.51 μmol/g respectively, P = 0.079). Two novel pathogenic de novo mutations were identified in 2 cases, and a rare single-nucleotide deletion was detected in the 3.5-kb RET upstream region, in a heterozygous state, in all 7 proband-parent triads. Low-penetrance RET haplotypes associated with HSCR were detected in 5 cases. Mothers with children with HSCR had lower vitamin A liver stores than mothers with normal children, and the children who were affected had HSCR despite having no established pathogenic RET variants. Lower maternal vitamin A status may increase the penetrance of genetic mutations in RET , and vitamin-A mediated gene-environment interactions may underpin some of the etiology of HSCR.

Interaction of Wild Type and V804l and V804m-Mutated Ret Protein Kinase with Emodin: In Silico Approach

Medullary thyroid carcinoma (MTC) is a malignant endocrine tumor originating from parafollicular calcitonin-producing C cells. The proto-oncogene RET (REarranged during Transfection) is known as the main actor in the development and outbreak of MTC. Gain of function mutations of RET constitutively activate the receptor which are found to be responsible for the high percentage of MTC cases. The two FDA-approved drugs used for MTC, vandetanib and cabozantinib, are resistant to two mutant variants of RET which are V804L and V804M. In this study, the interactions of emodin, a natural molecule found in plants, with wild-type as well as V804L and V804M-mutated RET kinase were investigated via molecular docking. Pymol was used to create point mutations on wild type RET. Vandetanib and cabozantinib were used as the reference drugs. The binding free energy of vandetanib with wild-type RET, V804L and V804M variants were found to be -9.3, -9.1 and -8.6 kcal/mol. Similarly, the binding free energy of cabozantinib with wild-type RET, V804L and V804M variants were found to be -10.6, -10.4, and -9.5 kcal/mol, respectively. Clearly, the binding affinity of vandetanib and cabozantinib to RET kinase was found to be reduced in mutated variants as compared to wild type. In the meantime, the binding energy between emodin and wild-type RET was shown to be -9.3 kcal/mol. Interestingly, the binding affinity of emodin to V804L and V804M variants was determined to be increased (-9.9 and -9.8 kcal/mol, respectively) compared to wild type. Furthermore, many H-bonds and hydrophobic interactions between emodin and mutated RET variants were shown. Therefore, strong binding affinity of emodin to wild-type and the mutated variants of RET was suggested in this study. In conclusion, emodin was found to be a potential molecule to inhibit RET kinase activity and could be used as a therapeutical agent against medullary thyroid carcinoma.

Treatment of RET-Positive Advanced Medullary Thyroid Cancer with Multi-Tyrosine Kinase Inhibitors-A Retrospective Multi-Center Registry Analysis.

Simple SummaryLately, a more personalized approach in the management of advanced thyroid cancer patients has improved the outcomes, and several novel molecularly guided therapies, including selective RET inhibitors (sRETis), have demonstrated promising efficacy in clinical trials. RET (rearranged during transfection) variants are the most prevalent oncogenic event in medullary thyroid cancer (MTC). We here found RET oncogene variants in 44/48 prospectively collected MTC tumor samples from patients treated with more unselective kinase inhibitors vandetanib and/or cabozantinib. Our study shows that RET variants were highly prevalent in patients with advanced MTC, and the treatment results in RET-positive cases were similar to those reported in unselected cohorts.Background: RET (rearranged during transfection) variants are the most prevalent oncogenic events in medullary thyroid cancer (MTC). In advanced disease, multi-tyrosine kinase inhibitors (MKIs) cabozantinib and vandetanib are the approved standard treatment irrespective of RET status. The actual outcome of patients with RET-positive MTC treated with MKIs is ill described. Methods: We here retrospectively determined the RET oncogene variant status with a targeted DNA Custom Panel in a prospectively collected cohort of 48 patients with advanced MTC treated with vandetanib and/or cabozantinib at four German referral centers. Progression-free survival (PFS) and overall survival (OS) probabilities were estimated using the Kaplan-Meier method. Results: In total, 44/48 (92%) patients had germline or somatic RET variants. The M918T variant was found in 29/44 (66%) cases. In total, 2/32 (6%) patients with a somatic RET variant had further somatic variants, while in 1/32 (3%) patient with a germline RET variant, additional variants were found. Only 1/48 (2%) patient had a pathogenic HRAS variant, and no variants were found in 3 cases. In first-line treatment, the median OS was 53 (95% CI (95% confidence interval), 32–NR (not reached); n = 36), and the median PFS was 21 months (12–39; n = 33) in RET-positive MTC patients. In second-line treatment, the median OS was 18 (13–79; n = 22), and the median PFS was 3.5 months (2–14; n = 22) in RET-positive cases. Conclusions: RET variants were highly prevalent in patients with advanced MTC. The treatment results in RET-positive cases were similar to those reported in unselected cohorts.

Designing Novel Compounds for the Treatment and Management of RET-Positive Non-Small Cell Lung Cancer-Fragment Based Drug Design Strategy.

Rearranged during transfection (RET) is an oncogenic driver receptor that is overexpressed in several cancer types, including non-small cell lung cancer. To date, only multiple kinase inhibitors are widely used to treat RET-positive cancer patients. These inhibitors exhibit high toxicity, less efficacy, and specificity against RET. The development of drug-resistant mutations in RET protein further deteriorates this situation. Hence, in the present study, we aimed to design novel drug-like compounds using a fragment-based drug designing strategy to overcome these issues. About 18 known inhibitors from diverse chemical classes were fragmented and bred to form novel compounds against RET proteins. The inhibitory activity of the resultant 115 hybrid molecules was evaluated using molecular docking and RF-Score analysis. The binding free energy and chemical reactivity of the compounds were computed using MM-GBSA and density functional theory analysis, respectively. The results from our study revealed that the developed hybrid molecules except for LF21 and LF27 showed higher reactivity and stability than Pralsetinib. Ultimately, the process resulted in three hybrid molecules namely LF1, LF2, and LF88 having potent inhibitory activity against RET proteins. The scrutinized molecules were then subjected to molecular dynamics simulation for 200 ns and MM-PBSA analysis to eliminate a false positive design. The results from our analysis hypothesized that the designed compounds exhibited significant inhibitory activity against multiple RET variants. Thus, these could be considered as potential leads for further experimental studies.

Abstract 1221: The landscape of 2,706 RET alterations from 89,754 adult patients with cancer: Clinical implications

Abstract Background: Activating receptor-tyrosine kinase rearranged during transfection (RET) mutations and fusions have been recognized as potent drivers of oncogenesis in multiple malignancies. Recently, highly potent and selective RET inhibitors, selpercatinib and pralsetinib have been FDA approved for RET fusion+ NSCLC and selpercatinib for RET+ thyroid cancers. Here we present a comprehensive analysis of RET alterations in pan-cancer adult malignancies. Patients and methods: 96,324 samples from 89,754 patients available from AACR Project Genie database version 8 were analyzed for the prevalence of RET fusions, mutations, and copy number alterations in diverse cancer types accessed July 21, 2020. The races of the samples within the cohort were 69,962 (72.6%) white, 5,388 (5.6%) Black, 4,909 (5.1%) Asian, 163 (0.17%) Native American, 48 (0.05%) Pacific Islander, and 15,854 (16.5%) were unknown. Results: There were 56,382 (58.5%) samples from primary tumors, 24,204 (25.1%) samples from unspecified metastasis sites, 4,798 (5.0%) from distant organ metastasis, 1,379 (1.4%) from lymph node metastasis, 1,279 (1.3%) from local recurrence, and 8,282 (8.6%) unknown. In 96,324 tumor samples there were 2,706 (2.81%) RET alterations. This included 223 (0.23%) fusions, 1,689 RET mutations found (1.75%) in 21 tumor histologies, and 794 (0.82%) RET amplifications identified; where the most abundant tissues harboring RET fusions were NSCLC (n = 121), papillary thyroid cancer (n = 53), breast cancer (n = 8) and colorectal cancer (n = 7) and the most abundant tissues harboring RET missense mutations were NSCLC (n = 355), colorectal cancer (n = 292), melanoma (n = 236), and thyroid cancer (n = 127). NSCLC samples with RET fusions had significantly co-altered KRAS, SETD2, PVRL4, EZH1, and RRAGC. Conclusions: RET fusions represent extremely rate events in multiple cancers. RET missense mutations occur in 2% of malignancies. Most RET missense variants are described as variants of unknown significance, limiting the impact of precision oncology for the majority of patients with RET alterations. Further functional characterization of RET variants is warranted because RET is highly targetable with therapeutics. Citation Format: Jacob J. Adashek, Alexander Andreev-Drakhlin, Jason Roszik, Aakash P. Desai, Vivek Subbiah. The landscape of 2,706 RET alterations from 89,754 adult patients with cancer: Clinical implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1221.

Pitfalls in RET Fusion Detection Using Break-Apart FISH Probes in Papillary Thyroid Carcinoma

A standardized procedure of fused REarranged during Transfection (RET) gene detection using fluorescence in situ hybridization (FISH) remains to be established in papillary thyroid carcinoma (PTC). Our purpose was to investigate false-negative and false-positive events and their FISH signal characteristics. A total of 111 PTC cases were analyzed using break-apart FISH probes for RET status evaluation. All FISH results were validated using fusion-induced asymmetric transcription assay (FIATA)-based reverse transcription droplet digital PCR (RT-ddPCR). Then, suspected RET-positive cases were tested using quantitative reverse transcription-PCR (RT-qPCR), followed by next-generation sequencing (NGS) for recognizing fusion variants. Thirty RET+ cases were revealed, including 20 CCDC6 (exon 1)-RET (exon 12), 6 NCOA4 (exon 8)-RET (exon 12), 1 NCOA4 (exon 7)-RET (exon 12), 1 CCDC186 (exon 7)-RET (exon 12), 1 ERC1 (exon 12)-RET (exon 12) and 1 SPECC1L (exon 9)-RET (exon 12) tumors. All RET fusion cases occurred in the BRAF- population, with a prevalence of 41.7% (30/72). Four cases of 8% to 13% (cutoff was 7.6%) dominant isolated 3' green (IG) FISH signals were RET-. One FISH- case with isolated 5' red (IR) signals with 94% abnormal tumor cells was demonstrated to be positive, harboring the NCOA4 (exon 7)-RET (exon 12) variant. Compared with RET fusions characterized by dominant break-apart signals with 29% to 100% aberrant cells, RET + with dominant IG-signal patterns all showed more frequent FISH+ cells (84%-92%). RET+ PTC with a break-apart signal pattern was more frequently found in unifocal lesions than in multifocal/bilateral tumors (P = 0.049). A false-positive or false-negative event may exist for RET status detection in PTCs using the traditional FISH scoring method with break-apart probes.

Abstract 2073: Exome sequencing reveals PTPRS, NOTCH3, FGFR1/3 mutations as novel mutational features in medullary thyroid cancer

Abstract Background: Approximately 1,000 patients are diagnosed with medullary thyroid cancer (MTC) in the United States each year; a tumor type that disproportionately causes thyroid cancer-related death. The molecular hallmark of MTC are mutations of the Rearranged During Transfection (RET) proto-oncogene: ~25% of MTC patients harbor germline RET variants and somatic RET mutations can be found in ~ 40% of sporadic medullary thyroid cancers. However, with the exception of RAS mutations relatively little is known of the broad molecular landscape of MTC. Material & Methods: To further explore the mutational features of MTC, we performed whole exome sequencing on paired tumor and normal tissue on a cohort of 14 patients with MTC. For variant calling, we focused on the 468 genes included in the MSK-IMPACT panel. Paired RNA sequencing was available for 7 patients. A variant was included as potential pathogenic based on a combination of SIFT, Polyphen and clinical significance prediction. Only coding variants were considered. Results: Out of the 14 patients, 43% were female (n=6), and 57% (n=8) were male (Table 1). The median age was 55.5 years (range: 21-79 years). At time of diagnosis, 29% had stage I (n=4), 14% stage II (n=2), 21% stage III (n=3), and 36% (n=5) had stage IV disease. The majority of patients (71%, n=10) had multifocal disease. While half of the patients had lymphovascular tumor invasion (50%, n=7), only 14% (n=2) had capsular invasion. Of all patients, 29% (n=4) of patients were germline RET mutation carriers. In our somatic tumor profiling, we detected a total of 23 potential pathogenic variants. The median mutation burden per patient was 2 (range= 0-7). Three patients had no detectable mutations. Expectedly, mutations in the RET gene were also the most frequent somatic mutational events and were detected in 43% (n=6) patients. Interestingly, we identified recurrent mutations in 3 genes that to the best of our knowledge have not been previously described in MTC. Protein- Tyrosine phosphate receptor-type sigma (PTPRS), Notch receptor 3 (NOTCH3), and Fibroblast growth factor receptors1/3 (FGFR1/3). Each of these was mutated in 21% (n=2) MTC patients. In addition, 10 genes were found mutated in a single MTC patient (CHEK2, DNMT1, EP300, GNAS, KMT2A, PIK3C2G, PNRC1, POLE, PRKAR1A, SOX2). In the RET wildtype patients, DNMT1, FGFR1, KMT2A, PIK3C2G, PRKAR1A and PTPRS were the only detected lesions, suggesting a possible pathophysiological importance independent of RET. Conclusions: Our exome sequencing study identified recurrent mutations in PTPRS, NOTCH3, FGFR1/3 as novel recurrent mutational features in MTC. Notably, some of them were detected in RET wildtype patients, suggesting a possible functional role of the genes in carcinogenesis. The functional studies to further define the roles of these gene mutations in MTC are ongoing. Citation Format: Isaiah Boateng, Ayse S. Yilmaz, Pamela Brock, Sandya Liyanarachchi, Ravi Patel, Christopher J. Walker, John Phay, Albert de la Chapelle, Matthew Ringel, Ann-Kathrin Eisfeld. Exome sequencing reveals PTPRS, NOTCH3, FGFR1/3 mutations as novel mutational features in medullary thyroid cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2073.

The landscape of RET alterations from 56,970 adult patients with cancer: Clinical implications.

3106 Background: Activating receptor-tyrosine kinase rearranged during transfection ( RET) mutations and fusions have been recognized as potent drivers of oncogenesis. Recent identification of highly potent and selective RET inhibitors holds great promise in the management of RET-dependent tumors. Here we present a comprehensive analysis of RET alterations in pan-cancer adult malignancies. Methods: We analyzed 59,347 samples from 56,970 patients available from AACR Project GENIE (Cancer Discov. 2017) database for the prevalence of RET fusions, mutations, and copy number alterations in diverse cancer types. Results: A total of 1414 RET alterations were detected, including 91 fusions (6.4%), 1166 missense mutations (82.5%), 136 truncating mutations (9.6%), and 21 in-frame mutations (1.5%). RET fusions were observed in 0.15% of tumor samples and were most commonly identified in non-small cell lung cancer, thyroid cancer, colorectal cancer, prostate cancer, and gastric cancer (62.6%, 18.6%, 5.5%, 4.4%, 3.3% of identified RET fusions, respectively). RET fusions were significantly co-altered with MAPK3/ERK1 (p=0.045), SETD2 (p=1.36E-07 ), and EIF4E (p=0.045), while there was a negative association between RET fusions and EGFR (p=0.009634) , TP53 (p=0.02267), and KRAS (p=2.53E-05) alterations. Most common RET gene upstream partners were KIF5B, CCDC6, and NCOA4 (42.9%, 24.2%, 7.7% of identified RET fusions, respectively). RET missense mutations were found in 2.0% of tumor samples; 136 (11.7%) of identified missense mutations, including 8 RET gatekeeper V804M/L mutations, were characterized as likely oncogenic, 12 (1.0%) as likely benign, and 1018 (87.3%) as variants of unknown significance using OncoKB database. RET amplifications occurred in 1.5% of tested samples. Conclusions: While RET fusions represent extremely rare events in multiple cancers, RET missense mutations occur in 2% of malignancies. Most RET missense variants are described as variants of unknown significance, limiting the impact of precision oncology for the majority of patients with RET alterations. Further functional characterization of RET variants is warranted. MAPK pathway co-alterations in patents with RET fusions may present a strategy for future therapeutic combinations.

Precision Targeted Therapy with BLU-667 for RET-Driven Cancers.

The receptor tyrosine kinase rearranged during transfection (RET) is an oncogenic driver activated in multiple cancers, including non-small cell lung cancer (NSCLC), medullary thyroid cancer (MTC), and papillary thyroid cancer. No approved therapies have been designed to target RET; treatment has been limited to multikinase inhibitors (MKI), which can have significant off-target toxicities and limited efficacy. BLU-667 is a highly potent and selective RET inhibitor designed to overcome these limitations. In vitro, BLU-667 demonstrated ≥10-fold increased potency over approved MKIs against oncogenic RET variants and resistance mutants. In vivo, BLU-667 potently inhibited growth of NSCLC and thyroid cancer xenografts driven by various RET mutations and fusions without inhibiting VEGFR2. In first-in-human testing, BLU-667 significantly inhibited RET signaling and induced durable clinical responses in patients with RET-altered NSCLC and MTC without notable off-target toxicity, providing clinical validation for selective RET targeting.Significance: Patients with RET-driven cancers derive limited benefit from available MKIs. BLU-667 is a potent and selective RET inhibitor that induces tumor regression in cancer models with RET mutations and fusions. BLU-667 attenuated RET signaling and produced durable clinical responses in patients with RET-altered tumors, clinically validating selective RET targeting. Cancer Discov; 8(7); 836-49. ©2018 AACR.See related commentary by Iams and Lovly, p. 797This article is highlighted in the In This Issue feature, p. 781.

Challenging clinically unresponsive medullary thyroid cancer: Discovery and pharmacological activity of novel RET inhibitors

It is now known that “gain of function” mutations of RET (REarranged during Transfection) kinase are specific and key oncogenic events in the onset of thyroid gland cancers such as the Medullary Thyroid Carcinoma (MTC). Although a number of RET inhibitors exist and are capable of inhibiting RET variants, in which mutations are outside the enzyme active site, the majority becomes dramatically ineffective when mutations are within the protein active site (V804L and V804M). Pursuing a receptor-based virtual screening against the kinase domain of RET, we found that compound 5 is able to inhibit efficiently both wild type and V804L mutant RET. Compound 5 was able to significantly reduce proliferation of both commercially available TT cell lines and surgical thyroid tissues obtained from patients with MTC and displayed a suitable drug-like profile, thus standing out as a promising candidate for further development towards the treatment of clinically unresponsive MTC.

Detection of RET (rearranged during transfection) variants and their downstream signal molecules in RET rearranged lung adenocarcinoma patients

BackgroundWe screened resected tumor tissues from patients with lung cancer for EGFR mutations, ALK rearrangements, and rearranged during transfection (RET) gene variants (including RET rearrangements and the Kinesin Family Member 5B (KIF5B)-RET fusion gene) using various methods including reverse transcription polymerase chain reaction (RT-PCR), transcript assays, fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). We also examined the protein expression of associated downstream signaling molecules to assess the effect of these variants on patient outcome. MethodWe constructed a tissue microarray (TMA) comprising 581 resected tumor tissues from patients with lung adenocarcinoma and analyzed the microarray by both FISH (using RET break-apart and KIF5B-RET SY translocation probes) and a commercial RET transcript assay. We evaluated the expression of RET and RET-related signaling molecules, including p-AKT and p-ERK, by TMA -based IHC staining. ResultsAmong the 581 specimens, 51 (8.8%) specimens harbored RET rearrangements, including 12 cases (2.1%) carrying a KIF5B-RET fusion gene. Surprisingly, RET expression was lower in KIF5B-RET fusion gene-positive than in RET wild-type specimens. We detected activating EGFR mutations in 11 (21.6%) of the 51 RET variant-positive specimens. Among the KIF5B-RET fusion gene-positive specimens, p-ERK expression was significantly lower in the EGFR mutation subgroup showing RET expression than in the EGFR mutation subgroup that did not express RET. Similarly, the RET rearrangement group showed significant variation in the expression level of p-AKT (P = 0.028) and p-ERK, whose expression remarkably increased in specimens not expressing RET. The expression of p-ERK markedly increased in the RET rearrangement group regardless of RET expression. ConclusionThis result suggests that a combination of RET and ERK inhibitors may be an effective treatment strategy for lung adenocarcinoma patients harboring RET variants.

Nationwide French Study of RET Variants Detected from 2003 to 2013 Suggests a Possible Influence of Polymorphisms as Modifiers.

The presence of single nucleotide polymorphisms (SNPs) in the REarranged during Transfection (RET) gene has been investigated with regard to their potential role in the development or progression of medullary thyroid cancer or pheochromocytomas (PHEO) in patients with the multiple endocrine neoplasia type 2 (MEN2) syndrome. The aim of this study was to evaluate the spectrum of RET variants in France between 2003 and 2013, and to evaluate the impact of SNPs on the MEN2 A phenotype. In this retrospective cohort study, RET variants were screened in 5109 index cases, and RET pathogenic variants were screened in 2214 relatives. Exons 5, 8, 10, 11, 13, 14, 15, and 16 were characterized by Sanger sequencing. RET pathogenic variants, RET variants with unknown functional significance (VUS), and four RET SNP variants-G691S (rs1799939), L769L (rs1800861), S836S (rs1800862), and S904S (rs1800863)-were characterized and are reported in index cases. In silico analysis and classification following the recommendation of the American College of Medical Genetics and Genomics was performed for RET VUS. Each patient's age at the time of diagnosis, sex, and the endocrine neoplasias present at molecular diagnosis were recorded. Twenty-six single VUS in RET without any well-defined risk profiles were found in 33 patients. Nine of these were considered probably pathogenic, 11 of uncertain significance, and six as probably benign. Three double pathogenic variants found in three patients were classified as pathogenic. A study of the entire cohort showed that patients carrying pathogenic variants or VUS in RET together with PHEO were diagnosed earlier than the others. The presence of the G691S SNP, or a combination of SNPs, increased the risk of developing PHEO but did not modify the date of the diagnosis. No association was found between SNPs and medullary thyroid cancer or hyperparathyroidism. The findings propose a classification of 15 of the 26 VUS in RET without any well-defined risk profiles and suggest that the G691S SNP, or a combination of SNPs, may be associated with the development of PHEO.

How to Assess the Clinical Relevance of Novel RET Missense Variants in the Absence of Functional Studies?

Introduction and Background: Familial medullary thyroid cancer (FMTC) is caused by gain of function mutations in the proto-oncogene RET (rearranged during transfection). Missense mutations within exon 14 including p.Val804Met are known to cause FMTC and multiple endocrine neoplasia type 2a/b. The clinical significance of other novel missense variants within this hotspot region of exon 14 is not delineated. Case Description: A three-generation pedigree of FMTC is presented with the co-occurrence of two missense variants within exon 14 of the RET gene, the known variant p.Val804Met and the novel variant p.Val826Met. The female index patient developed medullary thyroid cancer at the age of 42 years and was heterozygous for both missense variants. Her younger sister was also tested to be compound heterozygous for both mutations, and five further relatives were heterozygous for only one of both sequence variants. Prophylactic thyroidectomy was recommended for the two carriers of the RET mutation p.Val804Met, revealing a C-cell hyperplasia for one of them at the age of 19 years. Medical surveillance of 6 heterozygous carriers including repeated neck ultrasound examination as well as basal and calcium (pentagastrin)-stimulated calcitonin levels were recommended. Conclusion: Our data emphasize the importance of an interdisciplinary approach to assess the functional and clinical significance of novel RET variants. In the absence of functional studies, the plausibility of the pathologic significance of a detected endocrine genetic variant can be estimated by in silico methods such as computational analysis of protein structure and biophysical differences or comparative database search for evolutionary conservation.

The involvement of the RET variant G691S in medullary thyroid carcinoma enlightened by a meta‐analysis study

Medullary thyroid carcinoma (MTC) is a rare tumor, partially explained by mutations in the rearranged during transfection (RET) proto-oncogene. The nonsynonymous RET polymorphism G691S has been reported as associated with MTC, but findings are discordant. We sought to clarify the role of G691S in MTCs through in silico analysis, genetic association in our patients and a meta-analysis with extensive literature revision. Ninety-three Italian patients were compared to 85 healthy individuals. Results were included in a meta-analysis together with 11 case-control association studies identified through PubMed, EMBASE and Web of Science, with a combined sample of 968 cases and 2,115 controls. No association of G691S with MTC was found in our sample; however, we observed an excess of homozygotes for the variant, significantly higher among females. The overall allelic association in the meta-analysis was significant under the fixed-effect model (odds ratio [OR] = 1.22 [95% confidence intervals: 1.06-1.39], p = 0.0049), but borderline under the random effect model (OR = 1.21 [0.99-1.46], p = 0.0575), with a moderate/high heterogeneity (I(2) = 44.6%, p = 0.047). Under the recessive model of transmission, applied to the eight studies with available genotype frequencies, results were significant under both effect models (OR = 2.016 and OR = 2.022, p = 0.0004). No heterogeneity was anymore detectable. In silico analyses on G691S confirmed a change of the phosphorylation pattern that might account for the enhanced signaling transduction previously reported for G691S in several cancers, thus also explaining its overrepresentation in MTCs. The G691S variant allele does increase the risk for MTC, with a recessive mechanism of action, apparently more evident among females.

Clinical relevance of RET variants G691S, L769L, S836S and S904S to sporadic medullary thyroid cancer

Based on reports of higher frequencies among patients with sporadic medullary thyroid cancer (MTC) relative to external controls, the RET (REarranged during Transfection) variants G691S, L767L, S836S and S904S have been considered disease modifiers, suggesting greater lifetime risks of MTC. Other studies, employing different external controls, failed to confirm this association. Using a complementary approach, this study aimed at exploring differences in clinico-pathological characteristics among patients with sporadic MTC carrying no (wildtype), one (heterozygotes) or both (homozygotes) homologue RET variants in the germline, with wildtype cases acting as internal controls. Included in this investigation were 150 patients with complete genetic information on G691S, L769L, S836S and S904S RET alleles operated on for sporadic MTC at a tertiary referral centre. Not one statistically significant dose-response relationship was identified between any RET variant (wildtype vs RET heterozygotes vs homologue RET homozygotes) and patient age at MTC diagnosis, gender, primary tumour size, extrathyroidal extension, numbers of involved and removed lymph nodes, or distant metastasis. L769L and S836S homozygotes, unlike G691S and S904S homozygotes, were either rare or absent, limiting the analyses to comparisons of heterozygosity versus wildtype. On time-to-event analysis, G691S, L769L, S836S or S904S carriers and noncarriers developed MTC at similar rates. In carriers and noncarriers of the RET variants G691S, L767L, S836S and S904S, sporadic MTC appeared clinically and pathologically indistinguishable. This observation, along with the inconclusive evidence of previous association studies, calls for larger longitudinal association studies with age- and sex-matched external controls and additional functional studies of RET biology.

The RET p.G533C Mutation Confers Predisposition to Multiple Endocrine Neoplasia Type 2A in a Brazilian Kindred and Is Able to Induce a Malignant Phenotype In Vitro and In Vivo

We have previously described a p.G533C substitution in the rearranged during transfection (RET) oncogene in a large family with medullary thyroid carcinoma. Here, we explore the functional transforming potential of RET p.G533C mutation. Plasmids expressing RET mutants (p.G533C and p.C634Y) and RET wild type were stable transfected into a rat thyroid cell line (PCCL3). Biological and biochemical effects of RET p.G533C were investigated both in vitro and in vivo. Moreover, we report the first case of pheochromocytoma among the RET p.G533C-carriers in this Brazilian family and explore the RET mutational status in DNA isolated from pheochromocytoma. Ectopic expression of RET p.G533C and p.C634Y activates RET/MAPK/ERK pathway at similar levels and significantly increased cell proliferation, compared with RET wild type. We additionally show that p.G533C increased cell viability, anchorage-independent growth, and micronuclei formation while reducing apoptosis, hallmarks of the malignant phenotype. RET p.G533C down-regulates the expression of thyroid specific genes in PCCL3. Moreover, RET p.G533C-expressing cells were able to induce liver metastasis in nude mice. Finally, we described two novel RET variants (G548V and S556T) in the DNA isolated from pheochromocytoma while they were absent in the DNA isolated from blood. Our in vitro and in vivo analysis indicates that this mutation confers a malignant phenotype to PCCL3 cells. These findings, in association with the report of first case of pheochromocytoma in the Brazilian kindred, suggest that this noncysteine mutation may be more aggressive than was initially considered.

In vitro transforming potential, intracellular signaling properties, and sensitivity to a kinase inhibitor (sorafenib) of RET proto-oncogene variants Glu511Lys, Ser649Leu, and Arg886Trp

Multiple endocrine neoplasia type 2 and a subset of apparently sporadic medullary thyroid carcinoma (AS-MTC) are caused by germ line activating point mutations of the rearranged during transfection (RET) proto-oncogene. RET encodes a receptor with tyrosine kinase activity that targets several intracellular signaling cascades, such as RAS-RAF-ERK1/2, PIK3-AKT, and STAT transcription factors. The objective of this study was to assess the function of three germ line RET variants Arg886Trp, Ser649Leu, and Glu511Lys of undetermined pathogenic significance, which were found in three kindreds of isolated AS-MTC. For this purpose, we employed vectors expressing each of the RET variants and measured the number of NIH3T3 transformation foci and soft agar colonies, the degree of activation of known RET intracellular signaling targets (ERK1/2, STAT1, STAT3, and TCF4), and the extent of ERK1/2 inhibition on sorafenib treatment. We found that RET variants Arg886Trp and Glu511Lys have shown increased in vitro transforming potential in a glial-derived neurotrophic factor-dependent manner. In contrast, the Ser649Leu variant did not significantly increased the number of foci and agar colonies relative to wild-type RET (RET-WT). The variants Glu511Lys and Arg886Trp showed 10- and 12.5-fold ERK1/2 activation respectively, that was significantly higher than that observed for RET-WT (fivefold). Increased levels of STAT1 and TCF4 activation were only observed for RET Arg886Trp (2.5- and 3-fold versus 1.2- and 2-fold in RET-WT respectively). The three RET variants analyzed here were sensitive to treatment with sorafenib. In conclusion, our results allow to classify previously uncharacterized RET genotypes, which may be of use to define follow-up and therapeutic regimens.

Does the RET variant G691S influence the features of sporadic medullary thyroid carcinoma?

The RET (rearranged during transfection) proto-oncogene G691S variant is over-represented in the germline of patients with sporadic medullary thyroid carcinoma (sMTC) vs. normal controls but so far is not associated with any medical or pathological features of the tumour. The aim of our study was to assess the influence of this variant on the age of onset, clinical, biological and pathological features of sMTC. One hundred patients with histologically proven MTC, for whom the germline genetic analysis of RET was negative and medical records were available, were included in the study. Patients with the heterozygous GS variant or the homozygous SS variant (n = 36) were on average 8.0 years younger than patients with the wild-type GG variant (n = 64, mean age 43.9 vs. 51.9 years, P < 0.01). The former group did not differ from the wild-type group in terms of MTC size, prevalence of C-cell hyperplasia (CCH) or papillary thyroid carcinoma (PTC). However, the prevalence of an increased preoperative basal calcitonin (bCT) level (> 1000 pg/ml) was 2.75-fold higher in the patients with the GS or SS variant than in those with the wild-type variant (P < 0.001). The proportion of patients with lymph node metastases was also higher in the former group (P < 0.05). Multivariate analysis confirmed that the presence of the RET variant is independently associated with higher preoperative bCT values (P = 0.011). Our data demonstrate that the RET G691S variant could modulate the age of onset of sMTC as demonstrated previously for familial tumours. Moreover, this variant is an independent predictor of a higher basal calcitonin synthesis rate in patients with sMTC.

RET(MEN 2B) is active in the endoplasmic reticulum before reaching the cell surface

MEN 2B (multiple endocrine neoplasia type 2B) is an autosomal dominant cancer syndrome caused by an oncogenic form of the receptor tyrosine kinase REarranged during transfection (RET). The MEN 2B syndrome is associated with an abnormal autophosphorylation of the mutated receptor even without ligand-stimulation. Here, we characterize the activation of a RET(MEN 2B) variant carrying the point mutation Met918Thr, and show that the 150 kDa precursor of RET(MEN 2B) becomes phosphorylated already during synthesis in the endoplasmic reticulum (ER). At least three different tyrosine residues (Tyr905, Tyr1062, Tyr1096) of the RET(MEN 2B) precursor are phosphorylated before the oncogenic receptor reaches the cell surface. We also demonstrate that the precursor of RET(MEN 2B) interacts with both growth factor receptor-bound protein and Src homology 2 domain-containing already in the ER, and that this interaction is dependent on the kinase activity of RET. With the aid of two RET mutants (RET(MEN 2B/S32L) and RET(MEN 2B/F393L)), which accumulate in the ER, we show that the oncogenic precursor of the receptor has the capacity to activate AKT, extracellular signal-regulated kinase and signal transducer and activator of transcription 3 from the ER. Taken together, our data demonstrate that the oncogenic precursor of RET(MEN 2B) is phosphorylated, interacts with adapter proteins and induces downstream signalling from the ER.