Rearranged During Transfection Mutations Research Articles

Roles of the RET Proto-oncogene in Cancer and Development.

RET (REarranged during Transfection)is activated by DNA rearrangement of the 3′ fragment of the receptor tyrosine kinase gene, namely, RET proto-oncogene, with the 5′ fragment of various genes with putative dimerization domains, such as a coiled coil domain, that are necessary for constitutive activation. RET rearrangements have been detected in a variety of human cancers, including thyroid, lung, colorectal, breast, and salivary gland cancers. Moreover, point mutations in RET are responsible for multiple endocrine neoplasia types 2A and 2B, which can develop into medullary thyroid cancer and pheochromocytoma. Substantial effort is currently being exerted in developing RET kinase inhibitors. RET is also responsible for Hirschsprung’s disease, a developmental abnormality in the enteric nervous system. Gene knockout studies have demonstrated that RET plays essential roles in the development of the enteric nervous system and kidney as well as in spermatogenesis. Studies regarding RET continue to provide fascinating challenges in the fields of cancer research, neuroscience, and developmental biology.

Responsiveness to immune checkpoint inhibitors versus other systemic therapies in RET-aberrant malignancies

PurposeThe receptor tyrosine kinase rearranged during transfection (RET) can be oncogenically activated by gene fusions or point mutations. Multikinase inhibitors such as cabozantinib, lenvatinib and vandetanib have demonstrated activity in RET-dependent malignancies, and selective RET inhibitors (Selpercatinib and Pralsetinib) are in clinical trials. However, the responsiveness of RET-dependent malignancies to immune checkpoint inhibitors (ICIs) is unknown. We compared the time to treatment discontinuation (TTD) for ICI versus non-ICI therapy in patients with malignancies harbouring activating RET mutations or fusions (RET+).MethodsA retrospective review of all RET+ patients who were referred to the phase I clinical trials programme at the University of Texas MD Anderson Cancer Center was conducted. TTD was estimated using Kaplan-Meier analysis. Multivariate analysis using the Cox proportional hazard model was performed to identify independent risk factors of treatment discontinuation.ResultsOf 70 patients who received systemic therapy for RET+ malignancies, 20 (28.6%) received ICI and 50 (71.4%) received non-ICI therapy. Non-ICI therapy was associated with decreased risk for treatment discontinuation compared with ICI in the overall population (HR=0.31; 95% CI 0.16–0.62; p=0.000834) and in patients with RET point mutations (HR=0.13; 95% CI 0.04–0.45; p=0.00134). In patients with RET fusions, non-ICI therapy was associated with a non-statistically significant decreased risk of treatment discontinuation (HR=0.59; 95% CI 0.25–1.4; p=0.24). ICI therapy and a diagnosis other than medullary thyroid cancer (MTC) were independent risk factors for treatment discontinuation.ConclusionOur study supports the prioritisation of non-ICI over ICI therapy in patients with RET+ tumours.

363O - Treatment with pralsetinib (formerly BLU-667), a potent and selective RET inhibitor, provides rapid clearance of ctDNA in patients with RET-altered non-small cell lung cancer (NSCLC) and medullary thyroid cancer (MTC)

363O - Treatment with pralsetinib (formerly BLU-667), a potent and selective RET inhibitor, provides rapid clearance of ctDNA in patients with RET-altered non-small cell lung cancer (NSCLC) and medullary thyroid cancer (MTC)

1966P - Treatment with BLU-667, a potent and selective RET inhibitor, provides rapid clearance of ctDNA in patients with RET-altered non-small cell lung cancer (NSCLC) and thyroid cancer

Abstract Background BLU667, an investigational agent, is a potent and selective inhibitor of oncogenic rearranged during transfection (RET) alterations and predicted resistance mutations. Up to 90% of advanced medullary thyroid cancer (MTC) is characterized by single nucleotide variants and short insertions/deletions in the RET gene. In NSCLC, 1-2% of patients (pts) harbor rearrangements resulting in RET fusions. In the first-in-human ARROW study (NCT03037385), BLU-667 has demonstrated significant clinical activity in RET-altered NSCLC and MTC and has been well tolerated. Previous data has shown that early declines in circulating tumor DNA (ctDNA) may predict for treatment outcome. We investigated the change in ctDNA levels from baseline following treatment with BLU-667 and whether early changes in ctDNA during treatment were associated with clinical responses and outcomes. Methods Blood was collected at baseline and prespecified time points during treatment. Plasma from 111 pts with locally documented RET-altered MTC and NSCLC were profiled with the Personal Genome Diagnostics PlasmaSELECT™ R64 sequencing panel. Results RET fusions were detected at baseline in 45/63 (71%) pts with NSCLC and RET mutations in 35/48 (73%) pts with MTC. Baseline ctDNA mutant allele fraction (MAF; MTC) or unique fusion reads (NSCLC) correlated with the sum of target lesions (p Conclusions Treatment with BLU-667 led to a robust and rapid decline in ctDNA in almost all patients regardless of treatment dose or tumor diagnosis and in NSCLC irrespective of fusion partner studied. Clinical trial identification NCT03037385. Legal entity responsible for the study Blueprint Medicines Corporation. Funding Blueprint Medicines Corporation. Disclosure G. Curigliano: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Seattle Genetics; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Roche; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Novartis; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Lilly; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: BMS; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Pfizer. V. Subbiah: Advisory / Consultancy: MedImmune; Research grant / Funding (institution): Novartis (Inst), GlaxoSmithKline (Inst), NanoCarrier (Inst), Northwest Biotherapeutics (Inst), Genentech (Inst), Roche (Inst), Berg Pharma (Inst), Bayer AG (Inst), Incyte (Inst), Fujifilm (Inst), PharmaMar (Inst), D3 Oncology Solutions (Inst), Pfizer (Ins; Travel / Accommodation / Expenses: PharmaMar, Bayer AG. J.F. Gainor: Honoraria (self): Merck, Incyte, ARIAD Pharmaceuticals, Novartis, Pfizer; Advisory / Consultancy: Genentech, Bristol-Myers Squibb, Theravance, Loxo, Takeda, Array BioPharma, Amgen, Merck, Agios, Regeneron, Oncorus, Jounce Therapeutics; Research grant / Funding (institution): Merck (Inst), Novartis (Inst), Genentech (Inst), Bristol-Myers Squibb (Inst), Adaptimmune (Inst), AstraZeneca (Inst), ARIAD Pharmaceuticals (Inst), Jounce Therapeutics (Inst), Blueprint Medicines (Inst), Moderna Therapeutics (Inst), Tesaro (Inst), Alexo T. D.H. Lee: Honoraria (self): AstraZeneca, Boehringer-Ingelheim, Bristol-Myers Squibb, CJ Healthcare, Eli Lilly, Janssen, Merck, MSD, Mundipharma, Novartis, Ono, Pfizer, Roche, Samyang Biopharm and ST Cube; Advisory / Consultancy: Ministry of Food and Drug Safety, Korea, Health Insurance Review and Assessment Service, Korea, National Evidence-based Collaborating Agency, Korea, and National Cancer Control Planning Board, Korea. M.H. Taylor: Honoraria (self), Advisory / Consultancy: BMS, Eisai Inc, Array Biopharma, Bayer, LOXO, Blueprint, Arqule, Novartis; Speaker Bureau / Expert testimony: BMS, Eisai Inc; Research grant / Funding (institution): BioAtla; Travel / Accommodation / Expenses: BMS, Eisai Inc, Array Biopharma, Bayer, Loxo, Blueprint. V. Zhu: Honoraria (self), Speaker Bureau / Expert testimony: AstraZeneca, Roche-Foundation Medicine, Roche/Genentech, Takeda; Advisory / Consultancy, Shareholder / Stockholder / Stock options: TP Therapeutics. R.C. Doebele: Honoraria (self): Pfizer, AstraZeneca, ARIAD Pharmaceuticals, Guardant Health, Takeda Pharmaceuticals, Spectrum Pharmaceuticals, Trovagene; Advisory / Consultancy: Pfizer, OncoMed Pharmaceuticals, Trovagene, Ignyta, GreenPeptide, AstraZeneca; Research grant / Funding (institution): Ignyta (Inst); Travel / Accommodation / Expenses: Ignyta, ARIAD Pharmaceuticals, Guardant Health; Shareholder / Stockholder / Stock options: Rain Therapeutics; Licensing / Royalties: Other Intellectual Property: Licensing fees from Abbott Molecular for patent PCT/US2013/057495, licensing fees from Ignyta for biologic materials (Inst). G. Lopes: Advisory / Consultancy: Pfizer; Research grant / Funding (self): AstraZeneca; Research grant / Funding (institution): Merck Sharp & Dohme (Inst); EMD Serono (Inst), AstraZeneca (Inst), AstraZeneca, Blueprint Medicines (Inst), Tesaro (Inst), Bavarian Nordic (Inst), Novartis (Inst), G1 Therapeutics (Inst). E. Garralda: Research grant / Funding (self), Research grant / Funding (institution): Novartis; Travel / Accommodation / Expenses: BMS, Menarini, Glycotope; Licensing / Royalties: MSD. S.M. Gadgeel: Advisory / Consultancy: Pfizer, Genentech, ARIAD Pharmaceuticals, AstraZeneca, Bristol-Myers Squibb, AbbVie; Speaker Bureau / Expert testimony: AstraZeneca; Research grant / Funding (self), Research grant / Funding (institution): Merck; Research grant / Funding (institution): Pfizer (Inst), Merck, Genentech (Inst), Blueprint Medicines (Inst), ARIAD Pharmaceuticals (Inst), Takeda (Inst); Travel / Accommodation / Expenses: ARIAD Pharmaceuticals, Takeda. C.D. Turner: Full / Part-time employment: Blueprint Medicines Corporation. M. Palmer: Full / Part-time employment: Blueprint Medicines Corporation. S. Miller: Full / Part-time employment: Blueprint Medicines Corporation. All other authors have declared no conflicts of interest.

Medullary thyroid carcinoma beyond surgery: advances, challenges, and perspectives.

Medullary thyroid carcinoma (MTC) is a rare type of tumor that originates from thyroid C cells and accounts for 2-4% of all malignant thyroid neoplasms. MTC may occur sporadically or be inherited, as part of the MEN 2 syndrome. Germline mutations of the RET (REarranged during Transfection) proto-oncogene cause hereditary cancer, whereas somatic RET mutations and, less frequently, RAS mutations have been described in sporadic MTC samples. Since early surgery with complete resection of tumor mostly determines the likelihood of attaining cure for MTC, the broader use of RET genetic screening has dramatically changed the prognostic of gene carriers in hereditary MTC. Nevertheless, despite recent advances, the management of advanced, progressive MTC remains challenging. The multikinase inhibitors (MKI), vandetanib and cabozantinib, were approved for the treatment of progressive or symptomatic MTC, and several other compounds have exhibited variable efficacy. Although these drugs have been shown to improve progression-free survival, no MKI has been shown to increase the overall survival. As these drugs are nonselective, significant off-target toxicities may occur, limiting achievement of the required TK-specific inhibition. Recently, next-generation small-molecule TKI has been developed. These TKI are specifically designed for highly potent and selective targeting of oncogenic RET alterations, making them promising drugs for the treatment of advanced MTC. Here, we summarize the current understanding of the intracellular signaling pathways involved in MTC pathogenesis as well as the therapeutic approaches and challenges for the management of advanced MTC, focusing on targeted molecular therapies.

An investigation on the expression of miRNAs including miR-144 and miR-34a in plasma samples of RET-positive and RET-negative medullar thyroid carcinoma patients.

Medullary thyroid carcinoma (MTC) is a scarce cancerous disease, originating from parafollicular C cells of the thyroid gland. MTC can be manifested as an aggressive carcinoma with metastasis, especially in sporadic forms. Mutations of the rearranged during transfection (RET) proto-oncogene occurs in all hereditary and a few somatic MTCs, so detection of RET mutations is needed for prompt and appropriate treatment. MicroRNAs (miRNAs) are noncoding regulatory RNAs. Extensive studies have done in progress or suppression of several types of cancers such as MTCs with the remarkable application as prognostic markers. Of the effective miRNAs in cancers, miR-144 and miR-34 were evaluated in our study. Blood samples of 25 RET-positive and 25 RET-negative blood samples of patients with MTC were evaluated for these miRNAs, using quantitative real-time polymerase chain reaction (RT-qPCR). Analysis of the results was performed by the 2 -ΔΔCt method, showing that miR-144 and miR-34a expression had a relative increase in patients with MTC compared with normal control samples and also in RET positives versus RET negatives. We recruited 50 out of 350 MTC plasma samples (27 female and 23 male) which were selected based on RET mutation in exon 11 (25 RET-positive and 25 RET-negative), with a mean ± SD age of 37.04 ± 1.74 years. Receiver operating characteristic (ROC) curve analysis was done to investigate the prognostic value of these miRNAs; although, they showed no significant prognostic value as MTC biomarkers in plasma samples. In conclusion, miRNAs can be used as biomarkers of cancers such as MTC; however, more studies are needed to find the best candidate miRNAs for the diagnosis of cancers.

Abstract 2209: Novel therapeutic agents for RET-driven cancers, which is highly potent and selective inhibitor to RET kinase

Abstract The rearranged during transfection (RET) tyrosine kinase is mostly altered by fusion and it makes RET as a attractive therapeutic target for medullary thyroid cancer and non-small cell lung cancer (NSCLC). Multi-kinase inhibitors (mKIs) such as cabozantinib and vandetanib expressed poor tolerability and limited efficacy due to off-target kinase inhibitory activity in academia and clinical cases. As for selective RET inhibitor, compounds such as BLU-667 and LOXO-292 are under development, currently in clinical trials. We are currently developing potent and selective RET inhibitors to address unmet medical needs for RET-driven cancers. Our team has validated its high potency and selectivity via kinase profiling and in-vitro assay results. We also anticipate our compound to overcome certain mutation, such as V804M. Our compounds strongly suppress phosphorylation of RET in tumor cells with various RET fusions and mutations by inhibiting proliferation at below 10 nM. This potency result is higher than those of other mKIs with RET inhibitory activity. The inhibitory concentration of our compounds to VEGFR2 is more than dozens of fold compared with RET. In addition, our compounds reveal low cytotoxicity in Ba/F3 naïve cells in the 1 µM concentration. These findings lead us to conclude that our compounds can be potent and highly selective inhibitors of RET kinase, a potential novel therapeutic agents for RET-driven cancers. Citation Format: Hua Li, SeoHyun Jo, Woomi Do, Hyunkyung Kim, Hwan Kim, Kyung-ah Seo, Ji-hoon Oh, Jieun Choi, Jaeyoung Ahn, Jung Beom Son, Nam Doo Kim. Novel therapeutic agents for RET-driven cancers, which is highly potent and selective inhibitor to RET kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2209.

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.

Investigating the expression and promoter methylation of RET gene in patients with medullary thyroid cancer with unmutated RET.

Thyroid cancer is one of the most common endocrine malignancies. Mutations in the rearranged during transfection (RET) gene, especially in exon 10, 11, and 16, as well as epigenetic modifications, constitute the major underlying molecular events leading to medullary thyroid cancer (MTC). There are few studies on the mutations and epigenetic changes of RET gene in Iranian patients with MTC. In the present study, we aimed to address this question and explore the clinical relevance of such genetic alternations in an Iranian population. Thirty-three patients with confirmed MTC who underwent thyroidectomy surgery in Imam Khomeini Hospital (Tehran, Iran) were enrolled. DNA extracted from cancerous tissues was amplified by polymerase chain reaction (PCR) and then was sequenced for identification of RET mutations. In patients with no identified mutations, the methylation status of RET promoter and its expression were further investigated using methylation-specific PCR and real-time PCR methods, respectively. In MTC patients with no RET mutations, the promoter of the proto-oncogene was hypomethylated. Furthermore, RET gene expression was elevated in patients who revealed no mutations in neither of exon 10, 11, or 16 of the RET gene. Hypomethylation of RET promoter may contribute to MTC pathogenesis. The methylation status of RET promoter could be a new potential prognostic, diagnostic and therapeutic marker in MTC.

Long-term follow-up of RET Y791F carriers in Denmark 1994-2017: A National Cohort Study.

Recently, a comprehensive study presented evidence that a long-disputed REarranged during Transfection (RET) variant, RET Y791F, should be classified as nonpathogenic. In spite of this, several subsequently published papers, including the revised American Thyroid Association guidelines for medullary thyroid carcinoma, refer to the variant as pathogenic. This study presents data from a unique national Danish cohort of RET Y791F carriers who have been followed by watchful waiting instead of being subjected to early thyroidectomy, to determine if any carrier shows evidence of multiple endocrine neoplasia 2A (MEN2A) at long-term follow-up. A national cohort of all patients tested for RET mutations in Denmark from September 1994 to October 2017 was searched for carriers of RET Y791F. Medical records and laboratory reports of carriers were reviewed for signs of MEN2A at latest follow-up (medullary thyroid carcinoma, primary hyperparathyroidism, pheochromocytoma, cutaneous lichen amyloidosis, or Hirschsprung's disease). In total, twenty RET Y791F-carriers were identified, none of whom showed any evidence of MEN2A, despite an age range from 7 to 87 years. Our national cohort study of all Danish RET Y791F carriers substantiates the claim that the RET Y791F variant is nonpathogenic.

Genetic Profile of Indian Pheochromocytoma and Paraganglioma Patients - A Single Institutional Study.

Background and Aims:Pheochromocytomas (PCCs) and Paragangliomas (PGL) are rare catecholamine producing tumors that may present in sporadic or familial settings. Despite vast strides in understanding of PCC/PGL genetics in the last two decades, there is a dearth of information from India. The aim here is to study the prevalence of genetic mutations in Indian PCC/PGL patients.Settings and Design:Tertiary care academic hospital; prospective study.Methods:50 histopathologically diagnosed PCC/PGL patients formed the study group. Clinical, biochemical, pathological attributes and outcomes were documented and the phenotype was compared to the genotype. Succinyl dehydrogenase (SDH), Re-Arranged during Transfection (RET), Von-Hippel-Lindau (VHL) and NeuroFibromatosis-1 (NF1) mutations were studied. Additionally, immunohistochemisty for SDHB was also done, and the results compared to mutational analysis of SDH by MLPA (Multiplex Ligation-dependent Probe Activation).Statistical Analysis:Independent samples t-test and Fisher's exact test were used as appropriate. P values ≤0.05 were considered statistically significant.Results:The mean age was 34.3 years. Of the 50 patients, 27 were males and 23 females. 10 patients (20%) in all were detected to have a genetic mutation. 6 patients possessed a RET mutation, while two had VHL mutations. No patient presented with a NF1 mutation. 2 patients had a SDH mutation, and Immunohistochemistry for SDHB correlated with mutational analysis for these patients.Conclusions:The proportion of patients with a familial variant of PCC/PGL is more than what the historic “Rule of Ten” suggests. Our study found that one in five patients have a genetic mutation. PCC/PGL patients with genetic mutations not only require more stringent follow-up, but also screening of family members.

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.

Abstract 4784: TAS0286/HM05, a novel highly selective RET inhibitor, prominently inhibits various RET defective tumor growth

Abstract The rearranged during transfection (RET) gene is a well-known proto-oncogene and encodes a single-pass transmembrane receptor tyrosine kinase. RET fusions and point mutations are oncogenic drivers in NSCLC, medullary thyroid cancer and other solid tumors, and therefore potential targets for cancer therapy. Multikinase inhibitors targeting the RET mutations have been tested in clinical trials with moderate efficacy in terms of tumor shrinkage and PFS.Moreover, multikinase inhibitors are characterized by poor tolerability due to off-target kinase inhibitory activities. To widen the therapeutic index, a selective RET kinase inhibitor is highly desirable. TAS0286/HM05 is a novel highly selective and potent RET kinase inhibitor.RET kinase assay was performed by homogeneous time-resolved fluorescence (HTRF) method. In-vitro proliferation studies and pharmacodynamics analyses were conducted in cancer cell lines with the RET fusions and RET activating mutations. The antitumor efficacy of TAS0286/HM05 was evaluated using mice xenograft models implanted with cancer cells with various RET gene abnormality. TAS0286/HM05 was orally administered for 14 or 28 consecutive days after grouping. In-vitro and in-vivo RET phosphorylation was detected using western blot analysis. The IC50 value for RET kinase of TAS0286/HM05 was below 1 nM. TAS0286/HM05 showed highly selective RET inhibitory activity among 283 kinases. In cellular assay, TAS0286/HM05 strongly suppressed phosphorylation of RET expressed in cells with various RET fusions and activating mutations, and inhibited cell proliferation at around 10 nM. At this concentration, apoptosis was also observed. The potency was higher than other multikinase inhibitors with RET inhibitory activity. Furthermore, in in-vivo efficacy studies, TAS0286/HM05 significantly inhibited the growth of tumors harboring various RET fusions and activating mutations at a range of 20 to 100 mg/kg/day without any body weight loss. The antitumor efficacy of TAS0286/HM05 was more potent than pre-existing multikinase inhibitors at their maximum tolerated dose. In particular, TAS0286/HM05 dramatically induced tumor regression of 40% within 15 days in animals implanted with LC-2/ad cells, a human lung adenocarcinoma cell line with CCDC6-RET fusion gene. TAS0286/HM05 is a novel and highly selective RET inhibitor with prominent tolerability. The potency of TAS0286/HM05 against tumors with RET abnormalities was stronger than currently marketed multikinase inhibitors being tested in NSCLC patients with RET fusions. TAS0286/HM05 is a promising agent for future clinical development in patients with RET gene abnormalities. Citation Format: Hidenori Fujita, Isao Miyazaki, Masanori Kato, Yukari Yamada, Keiji Ishida, Tomonori Haruma, Haruka Nagasaki, Kenjiro Ito, Akihiro Hashimoto, Yasuo Kodama, Kaoru Funabashi, Emanuela Lovati, Kazutaka Miyadera, Kenichi Matsuo, Yoshikazu Iwasawa. TAS0286/HM05, a novel highly selective RET inhibitor, prominently inhibits various RET defective tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4784.

65 YEARS OF THE DOUBLE HELIX: Exploiting insights on the RET receptor for personalized cancer medicine.

The focus of precision cancer medicine is the use of patient genetic signatures to predict disease occurrence and course and tailor approaches to individualized treatment to improve patient outcomes. The rearranged during transfection (RET) receptor tyrosine kinase represents a paradigm for the power of personalized cancer management to change cancer impact and improve quality of life. Oncogenic activation of RET occurs through several mechanisms including activating mutations and increased or aberrant expression. Activating RET mutations found in the inherited cancer syndrome multiple endocrine neoplasia 2 permit early diagnosis, predict disease course and guide disease management to optimize patient survival. Rearrangements of RET found in thyroid and lung tumors provide insights on potential disease aggressiveness and offer opportunities for RET-targeted therapy. Aberrant RET expression in a subset of cases is associated with tumor dissemination, resistance to therapies and/or poorer prognosis in multiple cancers. The potential of RET targeting through repurposing of small-molecule multikinase inhibitors, selective RET inhibitors or other novel approaches provides exciting opportunities to individualize therapies across multiple pathologies where RET oncogenicity contributes to cancer outcomes.

Genotype-specific progression of hereditary medullary thyroid cancer.

Although already 25 years into the genomic era, age-related progression of hereditary medullary thyroid cancer (MTC), the prevalence of which is estimated at one in 80,000 inhabitants, remains to be delineated for most unique RET (REarranged during Transfection) mutations. Included in this study were 567 RET carriers. The age-related progression of MTC across histopathological groups (normal thyroid/C-cell hyperplasia; node-negative MTC; node-positive MTC) was statistically significant for 13 unique RET mutations (p.Cys611Phe/c.1832G>T; p.Cys611Tyr; p.Cys618Ser/c.1852T>A; p.Cys620Arg; p.Cys634Arg; p.Cys634Phe; p.Cys634Ser; p.Cys634Tyr; p.Glu768Asp; p.Leu790Phe/c.2370G>T; p.Val804Met; p.Ser891Ala; p.Met918Thr), whereas two unique RET mutations (p.Cys618Phe; p.Cys634Gly) trended toward statistical significance. When grouped by mutational risk (highest; high; moderate-high; low-moderate; polymorphism), the age-related progression of MTC was significant for all four categories of RET mutations, which differed significantly across and within the three histopathological groups. For high, for moderate-high, and for low-moderate risk RET mutations, the age-related progression of MTC by mutated codon was broadly comparable across and within the three histopathological groups, and essentially unaffected by the amino acid substitutions examined. These data argue in favor of splitting the American Thyroid Association's moderate-risk category into moderate-high and low-moderate risk categories, while emphasizing the need to contradistinguish the latter from rare nonpathogenic polymorphisms.

EF24 (a Curcumin Analog) and ZSTK474 Emphasize the Effect of Cabozantinib in Medullary Thyroid Cancer.

XL184 is a small-molecule kinase inhibitor recently included in first-line systemic therapy for patients with advanced, progressive medullary thyroid cancer (MTC). EF24 is a curcumin analog with a high bioavailability, and ZSTK474 is an inhibitor of the phosphatidylinositol 3-kinase signaling pathway. We investigated the effect of these compounds, alone and in combination, in two rearranged during transfection (RET)-mutated TT and MZ-CRC-1 MTC cell lines and in six mostly RET wild-type human MTC primary cultures. Low IC50 values demonstrated the efficacy of the drugs, whereas the combination index revealed an important synergistic effect of combinations of XL184 + ZSTK474 and XL184 + EF24. Cell-cycle changes and the induction of apoptosis or necrosis were modulated by single compounds or combinations thereof. Both XL184 and EF24, alone or combined, were effective in reducing calcitonin secretion. Western blot and in-cell Western analysis showed that the compounds prompted a decrease in general reactivity to phosphorylated antibodies. Our data confirm XL184 alone as the reference drug for RET-mutated MTC, but we also demonstrated that EF24 alone is effective in inhibiting MTC cell viability. We tested the combinations XL184 + ZSTK474 and XL184 + EF24 too, finding that they act synergistically, irrespective of RET mutation status.

P.Val804Met, the Most Frequent Pathogenic Mutation in RET, Confers a Very Low Lifetime Risk of Medullary Thyroid Cancer

ContextTo date, penetrance figures for medullary thyroid cancer (MTC) for variants in rearranged during transfection (RET) have been estimated from families ascertained because of the presence of MTC.ObjectiveTo gain estimates of penetrance, unbiased by ascertainment, we analyzed 61 RET mutations assigned as disease causing by the American Thyroid Association (ATA) in population whole-exome sequencing data.DesignFor the 61 RET mutations, we used analyses of the observed allele frequencies in ∼51,000 individuals from the Exome Aggregation Consortium (ExAC) database that were not contributed via The Cancer Genome Atlas (TCGA; non-TCGA ExAC), assuming lifetime penetrance for MTC of 90%, 50%, and unbounded.SettingPopulation-based.ResultsTen of 61 ATA disease-causing RET mutations were present in the non-TCGA ExAC population with observed frequency consistent with penetrance for MTC of >90%. For p.Val804Met, the lifetime penetrance for MTC, estimated from the allele frequency observed, was 4% [95% confidence interval (CI), 0.9% to 8%].ConclusionsBased on penetrance analysis in carrier relatives of p.Val804Met-positive cases of MTC, p.Val804Met is currently understood to have high-lifetime penetrance for MTC (87% by age 70), albeit of later onset of MTC than other RET mutations. Given our unbiased estimate of penetrance for RET p.Val804Met of 4% (95% CI, 0.9% to 8%), the current recommendation by the ATA of prophylactic thyroidectomy as standard for all RET mutation carriers is likely inappropriate.

A large Chinese pedigree of multiple endocrine neoplasia type 2A with a novel C634Y/D707E germline mutation in RET exon 11.

The present study identified the clinical features of the largest multiple endocrine neoplasia type 2 (MEN2) A pedigree from China, with a novel double missense rearranged during transfection (RET) mutation (C634Y/D707E). To the best of our knowledge, the D707E mutation has not been identified to date. In the present study, a total of 101 family members who originated from a large pedigree (134 members in total) underwent RET mutation screening by next-generation sequencing and polymerase chain reaction (PCR) amplification, followed by direct bidirectional DNA sequencing. The clinical features of this pedigree were carefully reviewed retrospectively, and statistical analyses were conducted using SPSS software. A total of 33 (32.67%) carriers were identified to exhibit the C634Y/D707E RET germline mutation. The mean age of the patients with medullary thyroid carcinoma (MTC) identified by RET screening was 38.4±16.5 years (n=11; range, 14–65 years). Only 4 patients with pheochromocytoma with a median age of 37 years were identified. No hyperparathyroidism was diagnosed. Persistent or recurrent disease developed in the patients of the present study who underwent inappropriate initial thyroid surgeries that were performed in previous decades (III10, III23, III24, III27 and IV46, as they had undergone two surgeries prior to the present study). A total of 66.70% (6/9) of patients, following thyroidectomy, continued to develop persistent or recurrent disease during the present screening study. In total, 3 patients succumbed to MTC or distant metastasis in the present study. The increase in carcinoembryonic antigen (CEA) levels correlated with the increase in basal serum calcitonin (Ct) levels according to Pearson correlation analysis in patients with MTC without surgery. Ct and CEA levels were also significantly correlated with tumor volumes. To the best of our knowledge, the present study is the first to identify a novel double RET missense mutation in the largest MEN2A pedigree from China. Additional in-depth study is necessary to elucidate the molecular mechanisms of the D707E mutation and its potential joint effects with the other C634Y mutation in the pedigree of the present study.

Distribution of RET Mutations in Multiple Endocrine Neoplasia 2 in Denmark 1994-2014: A Nationwide Study.

Background: Germline mutations of the REarranged during Transfection (RET) proto-oncogene cause multiple endocrine neoplasia 2 (MEN2). It is unclear whether the distribution of RET mutations varies among populations. The first nationwide study of the distribution of RET mutations was conducted, and the results were compared to those of other populations.Methods: This retrospective cohort study included 1583 patients who underwent RET gene testing in one of three centers covering all of Denmark between September 1994 and December 2014. Primary testing method was Sanger sequencing, which included exons 8–11 and 13–16. Mutations were defined according to the ARUP database July 1, 2016.Results: RET mutations were identified in 163 patients from 36 apparently unrelated families. Among the 36 families 13 (36.1%) carried mutations in codon 611, four (11.1%) in codon 618, three (8.3%) in codon 620, one (2.8%) in codon 631, six (16.7%) in codon 634, one (2.8%) in codon 790, one (2.8%) in codon 804, one (2.8%) in codon 852, one (2.8%) in codon 883, and five (13.9%) in codon 918. Among the 13 families with codon 611 mutations, 12 had the p.C611Y mutation.Conclusions: The distribution of RET mutations in Denmark appears to differ from that of other populations. Mutations in codon 611 were the most prevalent, followed by more frequently reported mutations. This might be due to a possible founder effect for the p.C611Y mutation. However, further studies are needed to find possible explanations for the skewed mutational spectrum in Denmark.

Different RET gene mutation-induced multiple endocrine neoplasia type 2A in 3 Chinese families.

Backgroud:Multiple endocrine neoplasia type 2A (MEN2A) is a condition with inherited autosomal dominant mutations in RET (rearranged during transfection) gene that predisposes the carrier to extremely high risk of medullary thyroid cancer (MTC) and other MEN2A-associated tumors such as parathyroid cancer and/or pheochromocytoma. Little is reported about MEN2A syndrome in the Chinese population.Methods:All members of the 3 families along with specific probands of MEN2A were analyzed for their clinical, laboratory, and genetic characteristics. Exome sequencing was performed on the 3 probands, and specific mutation in RET was further screened on each of the family members.Results:Different mutations in the RET gene were identified: C634S in Family 1, C611Y in Family 2, and C634Y in Family 3. Proband 1 mainly showed pheochromocytoma with MTC, both medullary thyroid carcinoma and pheochromocytoma were seen in proband 2, and proband 3 showed medullary thyroid carcinoma.Conclusion:The genetic evaluation is strongly recommended for patients with a positive family history, early onset of age, or multiple sites of masses. If the results verified the mutations of RET gene, thyroidectomy should be undertaken as the guide for better prognosis.