RET Mutations Research Articles

How clinically useful is comprehensive genomic profiling for patients with non-small cell lung cancer? A systematic review.

Given the lack of a gold standard, the clinical usefulness of Comprehensive Genomic Profiling (CGP) has not been established. This systematic review aimed to evaluate evidence about the clinical benefit of CGP for patients with Non-small cell lung carcinoma (NSCLC). All controlled studies that evaluated the ability of CGP to detect actionable targets (ATs) reported increases in the number of samples with ATs. The frequency of ATs detected in uncontrolled case series ranged from 0.7 % for RET mutations to 45 % for EGFR mutations. The studies that evaluated therapies targeted to EGFR, ALK, ROS-1, MET, and RET mutations documented significant improvement in clinical outcomes. This review suggests that CGP tests may be clinically helpful for treating patients with NSCLC. Although current evidence is associated with a high risk of bias, the significant impact of NSCLC on individuals and society may justify the routine use of CGP testing for this disease.

FP10.01 The Efficacy of Immunotherapy in non-Small Cell Lung Cancer Patients with Uncommon Mutations: a Real World Research from Single Site

Patients with uncommon mutations such as BRAF, RET, MET, ROS1 or HER2-mutated NSCLC are usually treated with targeted therapies, and immune checkpoint inhibitors have become an important treatment option for non-small cell lung cancer (NSCLC)However, efficacy of immune checkpoint drugs (ICIs) in patients harboring uncommon mutations is limited. We aim to evaluate the efficacy of ICIs in those patients in real-world settings. Retrospective data were collected from NSCLC patients who were BRAF，RET, MET, ROS1 or HER2 mutation positive tested by next generation sequencing (NGS) and treated with immunotherapy The clinicopathological characteristics, overall response rate (ORR), disease control rate (DCR), and progression-free survival (PFS)of patients were analyzed. A total of 29 patients enrolled in the study, of which, median age was 59 years old, 34.5% were smokers and 79.3% were adenocarcinoma. 51.7% of the patients were treated with immunotherapy as the first line and 27.6% as second-line. Patients with BRAF, HER2, MET, RET, ROS1 mutation were 7 ,11 ,5 ,3 ,3 cases, respectively. Programmed cell death ligand 1(PD-L1) expression was assessed in 11 patients with 31.9% incidence rate for PD-L1 positive was and 20.7% for PD-L1≥50%. 24% of patients achieved partial responses with BRAF (n=2), HER2 (n=1), MET (n=2) and RET (n=2) and 58.6% (17/29) achieved stable disease (with total ORR 24.1%. The DCR was 82.8% and median PFS was 6.7 months (95% confidence interval,4.4-9.0). Patients with uncommon mutated NSCLC could benefit from ICIs. Since uncommon mutations are rare and efficacy would be different due to diverse mutation type, data from real word study would provide valuable evidence for clinical practice.

Medullary Thyroid Cancer: Case Series Reports and Literature Review

Background: Medullary thyroid carcinoma (MTC) is a rare neuro-endocrine tumor that arises from the C-cells of the thyroid. About 20- 25 % of MTC cases may be associated with hereditary syndromes like MEN 2A, MEN 2B and Familial MTC. The survival rate is related mainly to the age of the patient, stage of the disease and completion of the surgical resection.
 Methods: Retrospective review of 11 patients who were diagnosed with medullary thyroid cancer in our general surgery department during the period from 2011 to 2021. All patients had preoperative assessment including history taking, clinical examination, tumor marker (calcitonin and CEA), thyroid function testing, ultrasonography and FNAC. All patients underwent genetic assessment to exclude any underlying genetic mutation.
 Results: The mean age of diagnosis was 57.73 ± 16.45 years of age. Three patients were males and eight were females. All patients had total thyroidectomy, central and lateral neck dissection except one patient who had prophylactic thyroidectomy due to familial inherited RET mutation. Two patients had recurrence; both of them had high-stage tumor (T3 and T4) with multiple cervical lymph nodes metastasis. The sensitivity of serum calcitonin for the detection of MTC was about 98%. Patients, who had localized disease and underwent complete surgical resection, had good overall survival rates compared with patients with advanced disease.
 Conclusion: MTC represent a heterogeneous group of thyroid cancers. The overall survival is better than that of undifferentiated thyroid cancers. Complete resection of the thyroid tumor and any local or regional metastases provides the only cure for patients with MTC. Further researches are still needed to improve our understanding and management of MTC.

Comprehensively Exploring the Mutational Landscape and Patterns of Genomic Evolution in Hypermutated Cancers.

Simple SummaryTo identify potential genetic markers for evaluating hypermutated cancers, we investigated driver mutations, mutational signatures, tumor-associated neoantigens, and molecular cancer evolution in the genetic variants of 533 cancer patients with six different cancer types. Driver mutations, including RET, CBL, and DDR2 gene mutations, were identified in the hypermutated cancers. Cancer driver mutations and mutational signatures are associated with sensitivity or resistance to immunotherapy, representing potential genetic markers in hypermutated cancers. Using computational predictions, we identified two tumor-associated neoantigens. Sequential mutations were used in a logistic model to predict hypermutated cancers according to genomic evolution. The sequential mutation order and coexisting genetic mutations were found to influence the hypermutation phenotype. Based on our observations, we developed a new concept for hypermutated cancers, whereby sequential mutations are significant for hypermutated cancers, which are mutationally heterogeneous. Through the comprehensive assessments of cancer gene panels, mutational pattern analysis was conducted as a basis for providing recommendations regarding therapeutic strategies for hypermutated cancer patients.Tumor heterogeneity results in more than 50% of hypermutated cancers failing to respond to standard immunotherapy. There are numerous challenges in terms of drug resistance, therapeutic strategies, and biomarkers in immunotherapy. In this study, we analyzed primary tumor samples from 533 cancer patients with six different cancer types using deep targeted sequencing and gene expression data from 78 colorectal cancer patients, whereby driver mutations, mutational signatures, tumor-associated neoantigens, and molecular cancer evolution were investigated. Driver mutations, including RET, CBL, and DDR2 gene mutations, were identified in the hypermutated cancers. Most hypermutated endometrial and pancreatic cancer patients carry genetic mutations in EGFR, FBXW7, and PIK3CA that are linked to immunotherapy resistance, while hypermutated head and neck cancer patients carry genetic mutations associated with better treatment responses, such as ATM and BRRCA2 mutations. APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) and DNA repair defects are mutational drivers that are signatures for hypermutated cancer. Cancer driver mutations and other mutational signatures are associated with sensitivity or resistance to immunotherapy, representing potential genetic markers in hypermutated cancers. Using computational prediction, we identified NF1 p.T700I and NOTCH1 p.V2153M as tumor-associated neoantigens, representing potential therapeutic targets for immunotherapy. Sequential mutations were used to predict hypermutated cancers based on genomic evolution. Using a logistic model, we achieved an area under the curve (AUC) = 0.93, accuracy = 0.93, and sensitivity = 0.81 in the testing set. The sequential patterns were distinct among the six cancer types, and the sequential mutation order of MSH2 and the coexisting BRAF genetic mutations influenced the hypermutated phenotype. The TP53~MLH1 and NOTCH1~TET2 sequential mutations impacted colorectal cancer survival (p-value = 0.027 and 0.0001, respectively) by reducing the expression of PTPRCAP (p-value = 1.06 × 10−6) and NOS2 (p-value = 7.57 × 10−7) in immunity. Sequential mutations are significant for hypermutated cancers, which are characterized by mutational heterogeneity. In addition to driver mutations and mutational signatures, sequential mutations in cancer evolution can impact hypermutated cancers. They characterize potential responses or predictive markers for hypermutated cancers. These data can also be used to develop hypermutation-associated drug targets and elucidate the evolutionary biology of cancer survival. In this study, we conducted a comprehensive analysis of mutational patterns, including sequential mutations, and identified useful markers and therapeutic targets in hypermutated cancer patients.

Multigene Combined Detection by RT-qPCR Using Cytological Specimens

Objective: The aim of the study was to investigate the mutation status of multiple driver genes by RT-qPCR and their significance in advanced lung adenocarcinoma using cytological specimens. Materials and Methods: 155 cytological specimens that had been diagnosed with lung adenocarcinoma in the Fourth Hospital of Hebei Medical University were selected from April to November 2019. The cytological specimens included serous cavity effusion and fine-needle aspiration biopsies. Among cytological specimens, 108 cases were processed by using the cell block method (CBM), and 47 cases were processed by the disposable membrane cell collector method (MCM) before DNA/RNA extraction. Ten drive genes of EGFR, ALK, ROS1, BRAF, KRAS, NRAS, HER2, RET, PIK3CA, and MET were combined detected at one step by the amplification refractory mutation system and ABI 7500 RT-qPCR. Results: The purity of RNA (p = 0.005) and DNA (p = 0.001) extracted by using the MCM was both significantly higher than that extracted by using the CBM. Forty-seven cases of fresh cell specimens processed by the MCM all succeeded in multigene detections, while of 108 specimens processed by the CBM, 6 cases failed in multigene detections. Among 149 specimens, single-gene mutation rates of EGFR, ALK, ROS1, RET, HER2, MET, KRAS, NRAS, BRAF, and PIK3CA mutations were 57.71%, 6.04%, 3.36%, 2.68%, 2.01%, 2.01%, 1.34%, 0.67%, 0% and 0% respectively, and 6 cases including 2 coexistence mutations. We found that mutation status was correlated with gender (p = 0.047), but not correlated with age (p = 0.141) and smoking status (p = 0.083). We found that the EGFR mutation status was correlated with gender (p = 0.003), age (p = 0.015) and smoking habits (p = 0.007), and ALK mutation status was correlated with age (p = 0.002). Conclusion: Compared with the CBM, the MCM can improve the efficiency of DNA/RNA extraction and PCR amplification by removing impurities and enriching tumor cells. And we speculate that the successful detection rate of fresh cytological specimens was higher than that of paraffin-embedded specimens. EGFR, ALK, and ROS1 mutations were the main driver mutations in patients with advanced lung adenocarcinoma. We speculate that EGFR and ALK are more prone to concomitant mutations, respectively. Targeted therapies for patients with coexisting mutations need further study.

High Prevalence of DICER1 Mutations and Low Frequency of Gene Fusions in Pediatric Follicular-Patterned Tumors of the Thyroid.

Follicular-patterned tumors of the thyroid in the adult population frequently harbor RAS mutations or PAX8-PPARG rearrangement, but little is known about molecular profiles in the pediatric patients with thyroid tumors, which is rare. To identify the molecular profile of pediatric follicular-patterned tumors, we enrolled 41 pediatric patients with follicular-patterned tumors from two institutions. We did next-generation sequencing using a mutation panel targeting 49 thyroid-tumor-related genes and a fusion panel targeting 88 types of thyroid-related gene fusions. We identified nonsynonymous mutations in at least one target gene in most of the tumors (28/41, 68%). Somatic DICER1 mutations (22%, n = 9) were the most common genetic alteration, followed by mutations of NRAS (15%), FGFR3 (15%), PTEN (12%), and STK11 (10%). Infrequent genetic alterations (≤ 5% of all cases) included mutations of HRAS, APC, TSHR, CTNNB1, TP53, EIF1AX, FGFR4, GNAS, RET, and SOS1, and gene fusion of THADA-IGF2BP3. DICER1 and RAS mutations were mutually exclusive. No patients had tumors related to the DICER1 syndrome or the Cowden syndrome. There was no significant difference in total mutation burden or distribution between follicular adenoma and follicular carcinoma. In the literature, the DICER1 mutation has been reported in 20 to 53% of pediatric patients with follicular-patterned tumors. In conclusion, our study reinforces the role of the DICER1 mutation in the development of pediatric thyroid tumors. Gene fusions rarely occur in pediatric follicular-patterned tumors. Mutation or gene fusion alone could not distinguish benign from malignant follicular-patterned tumors in pediatric patients.

A retrospective analysis of eleven gene mutations, PD-L1 expression and clinicopathological characteristics in non-small cell lung cancer patients

ObjectivesTo investigate the associations among expression of programmed cell death ligand 1 (PD-L1), eleven mutated genes, and clinicopathological characteristics in 273 patients with non-small cell lung cancer (NSCLC). MethodsWe retrospectively examined tumor PD-L1 expression in 247 surgically resected primary and 26 advanced NSCLC patients by immunohistochemistry using SP263 antibody assay. Gene mutations of EGFR, TP53, KRAS, PIK3CA, ERBB2, MET, RET, ALK, BRAF, ROS1, and APC were examined by NGS sequence. Data analysis was carried out using SPSS 22.0. The associations among PD-L1 expression, eleven mutated genes and clinicopathological characteristics were assessed by univariate and multivariate analysis. ResultsAmong the total 273 patients, 68 (24.9%) patients were positive for PD-L1 expression. Data showed that mutated rate of EGFR gene was the highest with 63.0% (172/273), followed by TP53 (11.7%, 32/273) and KRAS (5.5%, 15/273). The female, non-smoker, and patients with adenocarcinoma (ADC) were more likely to have EGFR mutations. Multivariate logistic regression showed that PD-L1 expression was significantly associated with Non-ADC, lymphatic invasion, EGFR wild type and TP53 mutation (p = 0.041, <0.001, 0.004 and 0.014, respectively). Moreover, PD-L1 expression in adenocarcinoma was associated with lymphatic invasion, mutation of TP53 and KRAS gene (p = 0.012, <0.025 and 0.041, respectively). ConclusionsMutations of EGFR, KRAS and TP53 should be routinely detected in clinical practice to better guide the immunotherapy for NSCLC patients. Future investigations are warranted to illustrate the potential mechanisms between driver mutations and PD-L1 expression for guiding immunotherapy in patients with NSCLC.

Abstract 3132: Tumorigenesis of MEN2 pheochromocytoma

Abstract Backgrounds: Pheochromocytomas are catecholamine-secreting neuroendocrine tumors, which occur sporadically or as part of inherited syndromes, such as multiple endocrine neoplasia type2 (MEN2), caused by deleterious mutations within the RET protooncogene. Although the RET mutation is implicated in the development of MEN2-associated pheochromocytoma, there is a long (typically 40 years) latency period before the disease onset, suggesting the need of the acquisition of secondary hits. However, little is known about such secondary alterations involved in the pathogenesis of MEN2. In this study, we compared genetic profiles of MEN2 pheochromocytoma and its precursor lesion, adrenal medullary hyperplasia (AMH). Materials and methods: Samples were collected from surgical specimens in 6 MEN2 pheochromocytoma cases, including a MEN2B case with RET p.M918T and 5 MEN2A cases with RET p.C634F, p.C634F, p.C634Y, p.E768D and p.C620S, and 1 AMH case with MEN2A carrying RET p.C634Y. Spatially separated samples were collected from a tumor slice (“tumor parts”). Multiple microscale sampling was also performed from apparently normal adrenal medulla (“non-tumor parts”). Somatic mutations and copy number alterations (CNAs) were then evaluated by whole exome sequencing. Result: Regarding “tumor parts”, a total of 20 samples from 6 MEN2 pheochromocytomas was analyzed. The number of somatic mutations per sample was ranging from 2 to 10. No somatic mutations commonly implicated in sporadic pheochromocytomas were detected. In a case with MEN2B, 1p, 3q and 21q losses were shared across all 3 samples, while 11p and 11q losses and 5p, 5q, 12p, 12q and 15q gains were private, suggesting that 1p, 3q and 21q losses occurred early in the tumorigenesis. In 5 MEN2A cases, 1p (80%), 3q (100%), 21q (60%) and 22q (60%) losses were frequently shared across all samples in each case. As for “non-tumor parts”, a total of 12 samples from 2 MEN2 pheochromocytoma cases and 1 AMH case were analyzed. The number of somatic mutations per sample ranged from 0 to 15. In 2 MEN2 pheochromocytoma cases, no somatic mutations were shared across the samples or no CNAs were detected, while 12 mutations were shared in two samples derived from an AMH case. Interestingly, these samples harbored 17p loss alone, while 1p, 3q, 21q or 22q losses were not found. Conclusion and perspective: MEN2 pheochromocytoma and AMH had different genetic characteristics, where 1p, 3q, 21q and 22q losses could have a role in early tumorigenesis. Further studies involving an increasing number of patients with MEN2 pheochromocytoma and AMH are warranted. Citation Format: Tatsuki Ogasawara, Yoichi Fujii, Nobuyuki Kakiuchi, Yusuke Shiozawa, Hiromichi Suzuki, Ryuichi Sakamoto, Yusaku Yoshida, Yuichi Shiraishi, Satoru Miyano, Seishi Ogawa. Tumorigenesis of MEN2 pheochromocytoma [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 3132.

Abstract LB002: Mechanisms of acquired resistance to KRAS G12C inhibition in cancer

Abstract Background: KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRASG12C) occur in ~13% of non-small cell lung cancers (NSCLC), ~3% of colorectal cancers (CRC), and less commonly in other cancer types. In early phase clinical trials of patients with KRASG12C-mutant cancers, promising antitumor activity has been reported with drugs such as adagrasib (MRTX849) and sotorasib (AMG510) which are direct inhibitors of KRASG12C. These small molecule irreversible inhibitors bind covalently to cysteine 12 within the switch II pocket which is formed, in part, by residues H95, Y96, and Q99. Mechanisms of acquired resistance to these therapies are currently unknown. Methods: Patients with KRASG12C-mutant NSCLC and CRC who were enrolled on adagrasib clinical trials and developed subsequent disease progression were included in this study if they were also consented to institutional review board-approved correlative studies at participating institutions. Tissue biopsies were analyzed histologically; tumor and/or circulating tumor DNA samples underwent next generation sequencing at the time of disease progression which was compared to that from pre-treatment samples when available. Results: A total of 30 patients were included in this study, 23 with NSCLC and 7 with CRC (25 with ctDNA, 7 with tissue sequencing, 2 with both). At the time of acquired resistance to adagrasib, we observed multiple on-target acquired KRAS alterations: mutations of the covalent-binding C12 residue, including C12W, C12F, C12V; a KRAS G13D mutation; high-level amplification of the KRASG12C allele; and mutation of the switch II binding pocket residues R68S, H95D, H95R, and Y96C. Furthermore, we detected several acquired off-target bypass mechanisms of resistance such as EGFR or MET amplification; activating mutations in NRAS (Q61K), BRAF (V600E), MAP2K1 (K57N, I99_K104del, E102_I103del), and RET (M918T); and oncogenic fusions involving RET, BRAF, RAF1, and FGFR. In two NSCLC cases with repeat tissue biopsies, histologic transformation from lung adenocarcinoma to squamous cell carcinoma was observed with no identifiable genomic mechanism of acquired resistance. In several cases, multiple coincident resistance mechanisms were identified in the same patient. Deep scanning mutagenesis studies were performed in parallel and identified the landscape of resistance mutations to adagrasib and sotorasib. While most resistance mutations confer high-level resistance to both therapies, some second-site mutations display differential sensitivity to distinct KRASG12C inhibitors, suggesting potential therapeutic strategies for overcoming drug resistance to specific mutations. Conclusion: Diverse genomic and histologic mechanisms impart resistance to covalent KRASG12C inhibitors in patients with cancer. Acquired genomic mutations, amplifications, and rearrangements may be potentially targetable by combining KRASG12C inhibition with available kinase inhibitors or SHP2 inhibitors. Citation Format: Mark Awad, Shengwu Liu, Kathryn Arbour, Viola Zhu, Melissa Johnson, Rebecca Heist, Tejas Patil, Gregory Riely, Joseph Jacobson, Julien Dilly, Xiaoping Yang, Nicole Persky, David Root, Lynette Sholl, Lee Lim, Kavita Garg, Mark Li, Lars Engstrom, Laura Waters, J. David Lawson, Peter Olson, James Christensen, Piro Lito, Sai-Hong Inatius Ou, Pasi Janne, Andrew Aguirre. Mechanisms of acquired resistance to KRAS G12C inhibition in cancer [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 LB002.

Abstract 1464: Pre-clinical characterization of potent and selective next-generation RET inhibitors

Abstract In May 2020, selpercatinib became the first FDA-approved selective RET inhibitor, indicated for patients (pts) with RET fusion-positive NSCLC and thyroid cancer as well as RET-mutant medullary thyroid cancer. Despite the durable activity of selpercatinib, pts can eventually develop acquired resistance. Previous studies in pts treated with selpercatinib or pralsetinib have reported the shared emergence of recurrent RET G810 mutations at the solvent front of the ATP pocket, which lead to a steric clash and loss of binding potency for both drugs. In some pts, these RET G810 solvent front mutations have been observed to co-occur with RET V804 gatekeeper mutations, demonstrating the importance of identifying agents that can maintain potency against both solvent front and gatekeeper resistance mutations. While multi-kinase inhibitors with potency against RET G810 mutations have been reported, these agents lack activity against V804 mutations and also carry off-target toxicities that further limit efficacy. We have identified a series of potent and selective next-generation RET inhibitors to address the emerging unmet need of pts who relapse on selective RET inhibitors. To assess potency, we evaluated the molecules in HEK293 cell lines engineered to express an M918T RET mutation or a KIF5B-RET fusion, as well as G810S or V804M resistance mutations. One compound, LOX-18228, exhibited nanomolar potency across the M918T RET, KIF5B-RET wild type, KIF5B-RET G810S, KIF5B-RET V804M, and KIF5B-RET V804M/G810S cell lines, with cellular IC50 values of 1.2, 0.9, 5.8, 31 and 51 nM, respectively. LOX-18228 also demonstrated high selectivity for RET when compared to a broad enzyme panel of off-target kinases, followed by relevant cellular assay screening. LOX-18228 also showed robust selectivity against a diverse panel of receptors, transporters and enzymes at a concentration of 10 μM, and exhibited a hERG IC50 of &amp;gt;30 μM. LOX-18228 demonstrated in vitro ADME properties predictive of good in vivo exposure with low predicted intrinsic clearance (range, 2.1-6.6 µL/min/million cells) in human, mouse, rat, and dog hepatocytes, and a measured high bin permeability of Papp = 15 (10-6 cm/s) in an intestinal MDCKII permeability assay. As predicted, LOX-18228 demonstrated high oral exposures in mouse and rat. In a pt-derived xenograft (PDX) model harboring a CCDC6-RET G810S mutation, LOX-18228 demonstrated complete regression at doses ≥30 mg/kg. Similarly, in a PDX model harboring a CCDC6-RET V804M mutation, LOX-18228 exhibited dose-dependent tumor inhibition with 100% tumor growth inhibition observed at 60 mg/kg. These data suggest that LOX-18228, as well as closely related compounds, represent promising next-generation RET inhibitor candidates that could be used to further extend durable disease control for pts with RET-altered cancers following the development of acquired resistance to current agents. An IND is planned for 2021. Citation Format: Gabrielle R. Kolakowski, Erin D. Anderson, Joshua A. Ballard, Barbara J. Brandhuber, Kevin R. Condroski, Eliana B. Gomez, Thomas C. Irvin, Manoj Kumar, Nisha A. Patel, Faith D. Watson, Steven W. Andrews. Pre-clinical characterization of potent and selective next-generation RET inhibitors [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 1464.

Acquired Resistance to KRASG12C Inhibition in Cancer

BackgroundClinical trials of the KRAS inhibitors adagrasib and sotorasib have shown promising activity in cancers harboring KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRASG12C). The mechanisms of acquired resistance to these therapies are currently unknown.MethodsAmong patients with KRASG12C-mutant cancers treated with adagrasib monotherapy, we performed genomic and histologic analyses that compared pretreatment samples with those obtained after the development of resistance. Cell-based experiments were conducted to study mutations that confer resistance to KRASG12C inhibitors.ResultsA total of 38 patients were included in this study: 27 with non–small-cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer. Putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired KRAS alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the KRASG12C allele. Acquired bypass mechanisms of resistance included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN. In two of nine patients with lung adenocarcinoma for whom paired tissue-biopsy samples were available, histologic transformation to squamous-cell carcinoma was observed without identification of any other resistance mechanisms. Using an in vitro deep mutational scanning screen, we systematically defined the landscape of KRAS mutations that confer resistance to KRASG12C inhibitors.ConclusionsDiverse genomic and histologic mechanisms impart resistance to covalent KRASG12C inhibitors, and new therapeutic strategies are required to delay and overcome this drug resistance in patients with cancer. (Funded by Mirati Therapeutics and others; ClinicalTrials.gov number, NCT03785249.)

Evaluation of grade in a genotyped cohort of sporadic medullary thyroid carcinomas

Tumour grade and RET mutation status, especially the presence of high-risk exon 15 and 16 RET mutations, have been reported to be prognostic in patients with sporadic medullary thyroid carcinoma (MTC). The aims of our study were to evaluate the performance of two recently proposed grading systems and to assess the association between grade and genotype in a cohort of sporadic MTCs. We identified 44 sporadic MTCs. All available tumour slides were examined, and cases were assigned a grade on the basis of either mitotic count and tumour necrosis, or mitotic count, tumour necrosis, and Ki-67 proliferative index, as described in two recent studies. Additional clinicopathological features and outcome information were obtained from the pathology reports and electronic medical records. The presence of RET and RAS mutations was determined either with direct sequencing or with massively parallel sequencing. Both grading systems were prognostic for progression-free survival and disease-specific survival on univariate analysis. There was no correlation between grade and mutation status. Specifically, neither RET nor high-risk RET mutations were enriched in high-grade tumours, as assessed by either grading scheme. Our findings suggest that grade is not correlated with RET/RAS mutation status, indicating that grade and genotype may give independent prognostic information.

A pooled analysis of response to selective RET inhibitors among patients with medullary thyroid cancer with M918T versus non-M918T RET mutations.

6078 Background: Medullary thyroid cancer (MTC) accounts for 1 to 2% of thyroid cancers in the United States; RET alterations occur in &gt;95% of hereditary and 50% of sporadic forms. Up to 80% of patients with sporadic MTC have somatic M918T RET mutations, which is associated with poor prognosis (1). The tyrosine kinase inhibitors (TKIs) cabozantinib and vandetanib are approved to treat patients with MTC regardless of RET status; however, retrospective analyses have suggested that there may be greater benefit in patients with M918T mutations (1,2). Newly approved therapies selpercatinib and pralsetinib, developed for patients with RET mutations, have demonstrated higher response rates than previous first line therapies. In this analysis, we examine the differences in overall response rate (ORR) between patients with MTC with RET M918T non- RET M918T mutations. Methods: An analysis of ORR in patients with MTC with RET M918T mutations with non-M918T mutations was conducted using the efficacy populations used to support the approvals of pralsetinib and selpercatinib using the following groups: Patients who received prior cabozantinib or vandetanib (referred to as “previously treated”). Patients with no prior cabozantinib or vandetanib (“TKI naïve”). All patients regardless of prior therapy. Results: Exploratory analysis of ORR of pooled population of Selpercatinib and Pralsetinib in patients with MTC with RET M918T mutations and non-M918T mutations. 1 Prior vandetanib or cabozantinib. 2 No prior vandetanib or cabozantinib. Two groups of patients were analyzed ( RET M918T mutation and RET non-M918T mutation), with subgroups with respect to prior treatment. Among all patients regardless of prior therapy, the ORR was similar between M918T non-M918T groups. Among previously treated patients, the ORR was lower in the M918T group vs. the non-M918T group, while in the TKI naïve group the ORR was higher in the M918T groups vs the non-M918T group although the 95% CIs overlap in both comparisons. Conclusions: There were no major differences in ORR among mutational subtypes in patients with MTC treated with RET inhibitors, regardless of prior therapy. ORR was similar between patients with M918T and non-M918T mutations. Additional experience in ongoing clinical studies may provide additional data regarding responses across specific mutation types.

NTRK, ALK and RET rearrangements in salivary gland cancer.

e18023 Background: We investigated the prevalence of rare genes rearrangements in salivary gland adenocarcinomas and the impact of these fusions on overall survival (OS) and progression free survival (PFS). Methods: For 51/60 (85.0%) patients with different types salivary gland adenocarcinomas (stage I-IVA), sufficient tumor tissue was available for molecular characterization by polymerase chain reaction (PCR) and has been tested for NTRK, ALK, ROS1, RET and MET deletion. OS and PFS curves were estimated with the Kaplan-Meier method and compared using the log-rank test. Results: Two cases (3.92%) of NTRK translocation were found, both are a classic rearrangement of ETV6ex5; NTRK3ex15. Of the other alterations, 2 cases (3.92%) of unbalanced ALK expression. However, it was not possible to establish the option of rebuilding. In addition, were found 2 cases (3.92%) of highly balanced expression of NTRK2 and ALK, and one case (1.96%) of high expression of ROS1. RET mRNA expression was detected in 13 of 48 cases (27.08%). NTRK, RET, ALK and ROS1 fusions were equally common in both men and women (p = 0.89), not depending on age (0.76). All patients bearing ALK, RET and NTRK fusions had shorter overall survival - 13.2 months, 95% confidence interval [CI] 13.0 to 32.0 months in multivariable model 105.0 moth (95% CI 105.0-105.0) in patient's group without mutations (p = 0.1817); and shorter PFS: 5.0 month (95% CI 5.0-16.0) vs not reached PFC in group (mean of PFS 63.15±5.58; 95% CI 52.20-74.09) without mutations (p = 0.0467). ALK fusion is an independent poor prognosis factor affecting on OS: p = 0.0168, hazard ratio (HR) = 8.65 (95% CI 1.47-50.77); and poor prognosis factor affecting on PFS: p = 0.0170, HR = 7.13 (95% CI 1.42-35.80). Conclusions: ALK, NTRK, ROS1 and RET mutations define a new rare subtype of salivary gland adenocarcinomas with enormously poor prognosis. The presence ALK-mutation is an independent poor prognosis factorб negatively affecting both overall survival and progression-free survival. It’s advisable to consider the possibilities of using ALK, NTRK and RET inhibitors in this cohort of patients.

Zebra Case of Papillary Thyroid Cancer in MEN-2 Syndrome

Introduction: The occurrence of papillary thyroid cancer in patients with familial medullary thyroid cancer (FMTC) is extremely rare and underreported. Case Presentation: A 51-year-old caucasian female with no past medical history who presented to primary care physician for her annual visit. She denied any complaints. Vital signs were unremarkable. Physical examination was remarkable for large left thyroid nodule without cervical lymph nodes enlargements. Family history was significant for metastatic MTC of her two sisters (at the age of 55, 60) and her niece at age of 21. She previously declined genetic testing. Labs were unremarkable including serum calcium of 8.3 mg/dL. Thyroid function panel revealed a TSH of 2.1 μU/mL, Free T4 of 1.4 ng/dL, Thyroid peroxidase (TPO) antibody of <6 IU/mL. Neck ultrasonography revealed a mid-left thyroid hypoechoic nodule of 1.8 ×1.5 cm, without cervical lymph nodes enlargements. Fine needle biopsy revealed a papillary thyroid cancer, follicular variant. The diagnosis confirmed by two university pathologists, without any evidence of MTC. Genetic testing revealed a germline mutation in RET oncogene exons 13. Further labs revealed normal metanephrines of 12 pg/mL and normal normetanephrine of 31 pg/mL, normal serum calcitonin was less than 2 pg/mL (0-5), normal serum CEA was 2.5 ng/mL (0-5), elevated anti- thyroglobulin (anti-TG) antibodies of 1234 iu/mL (0-0.9), and elevated thyroglobulin of 50 ng/mL (1.5-38.5). The patient was referred to ENT for total thyroidectomy. Further, she was treated with radioactive iodine ablation by 155 mCi of iodine 131. Follow up whole body thyroid scan revealed no evidence of residual or recurrence. The patient was treated with 112 mcg levothyroxine with a target of TSH less than 0.1. On six months follow up, repeated neck ultrasonography revealed no remnant of thyroid tissue. Follow up Thyroglobulin (TG) was less than 0.1 ng/mL, TSH 0.05 μU/mL, FT4 of 1.98 ng/dL, and calcitonin was still less than 2 pg/ml. Genetic testing of her daughters (24, 31) revealed germline mutations in RET-oncogene exons 13 and was referred for prophylactic thyroidectomy. Discussion: FMTC is an inherited syndrome characterized by the presence of only MTC without hyperparathyroidism or pheochromocytoma and is considered a variant of MEN2A. Most cases of MEN2A have been attributed to mutations in the intracellular portion of RET, and somatic mutations in RET have been identified in 50% of cases of sporadic FMTC. We recommend in patients with germline RET mutations and a family history of MTC, the diagnosis of papillary thyroid cancer should be confirmed by two pathologists, and patients should be tested for serum calcitonin, serum calcium, and metanephrines/normetanephrine levels. Genetic testing of first-degree relatives of FMTC at an early age is essential in guiding major management decisions such as the timing of prophylactic thyroidectomy.

Hypercalcemia in Pheochromocytoma: From MEN to VHL

Background: Co-occurrence of phaeochromocytoma and primary hyperparathyroidism is usually seen in patients of Multiple Endocrine neoplasia 2A(MEN2A) and is rare in Von Hippel Lindau disease (VHL). Parathyroid adenoma with pheochromocytoma in a genetically confirmed VHL has been reported only once till date (1). Clinical Case: A 30-year male was admitted for evaluation of hypertension and incidentally diagnosed adrenal mass on ultrasound. 9-years back, he was diagnosed as a case of VHL (right renal clear cell carcinoma, pancreatic cystadenoma, spinal hemangioblastoma and epididymal cysts). Right nephrectomy and pancreatic cyst excision had been done and past work-up for pheochromocytoma was normal. He also had been operated twice for recurrent renal calculi. Family history revealed surgery in mother for pancreatic mass. Current imaging revealed right adrenal mass (4.7*4.6 cm) with left renal cysts and calculi, pancreatic cysts and spinal- medullary hemangioblastoma and epididymal cysts. Fundus examination was normal. 24-hours urinary fractionated normetanephrines were elevated (2062 mcg/24 hours) and I131MIBG scan showed 4.7*4.6cm concentrating lesion in right renal fossa suggestive of right adrenal pheochromocytoma. However his biochemical evaluation revealed hypercalcemia (12.1 mg/dl), low phosphorus (3.2 mg/dl), low 25(OH) D (24.84 nmol/l), and raised PTH (121pg/ml). Ultrasound neck and Tc99m-Sestamibi localized left inferior parathyroid adenoma. DEXA scan showed severe osteoporosis. Genetic analysis confirmed VHL mutation in exon-1. Calcitonin and RET mutation were normal (ruled out MEN2A). Therapeutic approach was surgical excision of adrenal pheochromocytoma followed by parathyroidectomy. We report a case of pheochromocytoma with primary hyperparathyroidism (cause: left inferior parathyroid adenoma) in a patient of VHL (Renal clear cell carcinoma, pancreatic cystadenoma, epidydymal cysts and medullary and spinal hemangioblastoma). Hypercalcemia seen in patients of VHL is either due to bone metastasis/PTHrP/IL-6 secretion from RCC or due to PTHrP/PTH/calcitonin secretion from pheochromocytoma and rarely due to associated parathyroid adenoma. Literature search revealed four case reports of parathyroid adenoma with VHL. In only one of these, VHL had pheochromocytoma associated with parathyroid adenoma (1). Conclusion: Ours is the 2nd such case reported in literature of primary hyperparathyroidism in a genetically confirmed case of VHL with pheochromocytoma. This case highlights the overlap of tumorigenesis in two rare genetically divergent syndromes and importance of long-term follow-up for sequential development of new tumors. Reference: Arao T, Okada Yet al. A case of VHL disease with bilateral pheochromocytoma, renal cell carcinoma, pelvic tumor, spinal hemangioblastoma and primary hyperparathyroidism. Endocr J. 2002 Apr;49(2):181–8.

Never Forget the Family History: MEN2A Syndrome Presenting as Metastatic Medullary Thyroid Cancer and Bilateral Pheochromocytoma

Abstract Introduction: MEN 2A is an autosomal dominant hereditary syndrome considered part of the medullary thyroid carcinoma (MTC) syndromes. This is characterized by MTC, pheochromocytoma, and parathyroid hyperplasia or adenomas causing primary hyperparathyroidism (PHPT). Clinical Case: A 34 year old female was referred to our clinic for multi-nodular goiter diagnosed during routine gynecologic evaluation. A thyroid ultrasound revealed a heterogeneous right thyroid lobe with a hypoechoic 2.5 cm nodule, associated macro calcifications and increased vascularity; and a left nodule measuring 2.3 cm with the same characteristics. Bilateral thyroid nodule biopsies were performed, resulting in MTC confirmed by positive calcitonin staining. Pre-operative studies revealed serum calcitonin and carcinoembryonic antigen (CEA), both of which are considered serologic markers of MTC activity, at 4,340 pg/ml (n &amp;lt;= 5) and 276.2 ng/mL (n &amp;lt;= 2.5 in non-smokers) respectively. The patient reported father with history of unspecified thyroid cancer, and paternal uncle with history of pheochromocytoma with a p.Cys634Trp mutation in RET proto-oncogene. Due to her family history, pre-operative screening for primary hyperparathyroidism (PHPT) resulted in a calcium 10 mg/dL (n 8.6-10.2), PTH 34 pg/mL (n 14-64). Additionally, screening for pheocromocytoma revealed an elevated 24 hour urine metanephrines of 2,276 (n &amp;lt;= 49-290) ug/24h, plasma metanephrines, including fractionated metanephrine (MN) at 163 (n &amp;lt;= 57) pg/ml, fractionated nor-metanephrine (NMN) at 182 (n &amp;lt;= 148) pg/ml, and total, Free (MN+NMN) metanephrines at 345 (n &amp;lt;= 205) pg/ml. CT abdomen revealed bilateral adrenal nodules, right measuring 1.4 x 3.3 cm and left 2.4 x 3.3 cm. The patient underwent posterior retroperitoneoscopic adrenalectomy with cortex sparing prior to thyroidectomy. Adrenal pathology resulted in bilateral pheochromocytoma with peri-adrenal adipose tissue microscopic involvement, and positive synaptophysin and S-100 stain. Subsequently, she underwent total thyroidectomy with extensive cervical lymph node resection, with pathology resulting in MTC with lymph node metastasis, involving 5/18 cervical lymph nodes. Post-operative labs revealed serum calcitonin &amp;lt;= 2 pg/ml, CEA 26.8 ng/mL, MN &amp;lt; 25 pg/ml, NMN 102 pg/ml, and MN+NMN of 102 pg/ml, which suggested initial surgical success. Post-operative genetic test evaluation revealed abnormal RET oncogene testing compatible with MEN 2A, variant 1: c.1902C&amp;gt;G (p.Cys634Trp). Conclusion: This case illustrates that patients presenting with MTC and reporting family history of thyroid cancer should be screened for familial MTC syndrome. Patients with RET mutation should be screened for pheochromocytoma prior to surgery for MTC to prevent life-threatening hypertensive crisis.

Thyroid Paraganglioma- An Unusual Head and Neck Tumour

Paragangliomas are neuroendocrine tumour originating from the neural crest-derived paraganglia with majority arising from the head and neck. (1)Thyroid paraganglioma are exceedingly rare neuroendocrine tumours accounting for <0.1% of thyroid malignancy (1)We present a 57 years old gentleman who was referred to ENT surgeon following discovery of a two month history of lump on his left neckIt has not changed in size and not caused any symptoms such as anxiety, sweats, palpitations, dizziness or unexplained headaches. He has a Past medical history of epilepsy following a Road traffic accident 28 years ago leaving him seizure prone. He has no family history of neuroendocrine tumours His ultrasound scan of his thyroid gland showed a 25 x 23 x 15mm lesion lying anteriorly within the left thyroid lobe. There are two highly reflective foci which could represent microcalcification. It was classified as U5 lesion He proceeded with fine needle aspiration which confirmed carcinoma of the left thyroid gland with no clear differentiation between follicular or papillary carcinoma. He undergone a total thyroidectomy and left central level VI lymph node dissection His histology confirmed a thyroid paraganglioma staining strongly positive for neuroendocrine markers (Synaptophysin and chromogranin) while S-100 shows positivity in the sustentacular cells. He was referred for genetic testing which demonstrate no evidence of mutation in FH, SDHAF2, SDHB/C/D, RET, MAX, TMEM127 and VHL gene. He was commenced on levothyroxine replacement at a dose of 150micrograms OD. His urine metanephrines is 178.1pmol/L (0-510), urine normetanephrines 192.9pmol/L (0-1180) and 3-methoxytyramine <75pmol/ L (0-180) (all normal). His MRI neck revealed no synchronous tumour. He continues to be followed up under our endocrine clinic.Conclusion: Due to the rarity of these tumours, their natural history is mostly unknown. Nevertheless, postoperative surveillance should include plasma or urinary metanephrines and ultrasonography.

Clinical Activity of Selpercatinib in RET Mutant Pheochromocytoma-Case Reports

Activating RET gene alterations have been reported in solid tumors including the rare cancer, pheochromocytoma (PHEO) found sporadically and in familial multiple endocrine neoplasia type 2 (MEN2) syndromes. Selpercatinib is a highly selective and potent small molecule RET kinase inhibitor that has demonstrated marked and durable anti-tumor activity in diverse RET-altered solid tumors. Described are the initial 3 PHEO patients treated with selpercatinib (LIBRETTO-001/NCT03157128). Case 1: 70-year-old white male with MEN2A and a history of medullary thyroid cancer (MTC) and PHEO s/p thyroidectomy and adrenalectomy, received MIBG in 1991 and 2016 due to symptom reoccurrence. Progressive metastatic disease associated with severe hypertension was treated with Lutate in 2017 and germline RET mutation p.Cys634Phe was confirmed. After developing severe back pain due to a T6 vertebral metastasis, he began selpercatinib treatment. As of Mar 2020, he has a partial response (PR) as assessed by investigator; his back pain resolved, normetanephrine and metanephrine levels decreased, and has ceased alpha and beta blockers. He remains on treatment with only grade 1-2 adverse events, none requiring interruption or dose modification. Case 2: 51-year-old white female with MEN2A and history of MTC and PHEO s/p thyroidectomy and adrenalectomy in 2010. She developed metastatic PHEO in 2013, with multiple bone, omentum, lung, liver, and spleen metastases. Between 2013 and 2018 she was treated with multiple courses of radiation and, additional surgical resections; a PR with sunitinib lasted 13 months followed by temozolomide/capecitabine treatment. A bone lesion biopsy in 2018 confirmed RET C618S mutation and with her disease progression and uncontrolled bone pain, she began selpercatinib treatment, experiencing a PR. After 5.5 months in the study, she discontinued treatment due to disease progression. Case 3: 45-year-old African American female diagnosed with sporadic PHEO in 1996, s/p multiple surgical resections. She received 2 cycles of cyclophosphamide/vincristine/dacarbazine without clinical benefit. I-131-MIBG therapy with autologous stem cell rescue in 2017 improved blood pressure, palpitations, and flushing but without tumor shrinkage while abdominal pain persisted. Somatic M918T RET-mutation was confirmed, and she began selpercatinib treatment in 2018 with symptom resolution and improved plasma metanephrine levels. She required dose reduction for grade 3 palmar-plantar erthrodysesthesia and had stable disease for 22 months until a new bone metastasis was identified. Due to ongoing clinical benefit, she remains on treatment despite disease progression. Conclusion: These are the initial reports of RET-mutant PHEO patients treated with selpercatinib adding to the diversity of RET-altered tumor types that may benefit from a selective RET inhibitor.

Biochemical and Genomic Changes of a Devastating Metastatic Medullary Thyroid Cancer

Abstract Introduction: Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor of thyroid parafollicular C cells. About 25% of cases are hereditary, associated with MEN2 syndromes. RET M918 mutation (somatic or germline) is associated with the most aggressive form of MTC. If MTC is hereditary, further tumor genomic analysis is not routinely performed. Therefore, the somatic mutational landscape of hereditary MTCs is not frequently described in the literature. We present an unusual case of MTC with a germline M918 RET mutation, displaying an initial smoldering course followed by rapidly fatal widespread metastases. Whole exome sequencing (WES) of primary tumor and metastases revealed intertumoral heterogeneity with many somatic mutations including p53 mutation. Clinical Case: The patient with characteristic facies of MEN 2B presented with a neck mass at age 28. She had no family history of MEN. At diagnosis, calcitonin was 11,392 pg/mL and CEA was 729.7 ug/L. Pathology showed MTCs of 5 cm (right) and 2.1 cm (left) with lymph node metastases (pT3N1bMx). Genetic testing showed RET M918 mutation. Postoperative calcitonin and CEA were 3.3 pg/ml and 1.7 ug/L respectively. Tumor markers doubling time was initially about 1 year, but progressively shortened over time. Three years after surgery, calcitonin and CEA were 101 pg/ml and 387 ug/L respectively. CT chest and abdomen identified no distant metastasis. Over the next 4 months, calcitonin and CEA increased dramatically to 1821pg/ml and 3974 ug/L. The patient was admitted for acute liver failure and passed away. Autopsy revealed widely metastatic disease to liver, lungs, spleen, pituitary, right adrenal gland, bones, and brain. WES revealed 87, 283 and 591 somatic mutations in the primary tumor, lung and liver metastases respectively. The primary tumor had chromosome 1p loss whereas liver and lung metastases had somatic mutation of TP53 and single copy loss of chromosome 17 (biallelic inactivation of p53). Conclusion: This case illustrates intertumoral spatial and temporal genetic heterogeneity. Mutations detected by WES may or may not be cancer-related and their clinical relevance is often uncelar. We hypothesize TP53 biallelic loss might have contributed to the aggressive phenotype of this tumor. Also of note, even before the appearance of metastases, CEA was disproportionately higher than calcitonin, reflecting tumor dedifferentiation. Further studies are needed to address CEA threshold for additional imaging and imaging modality for early detection of metastases.