Identification and characterization of RET fusions in advanced colorectal cancer

There is an unmet clinical need for molecularly directed therapies available for metastatic colorectal cancer. Comprehensive genomic profiling has the potential to identify actionable genomic alterations in colorectal cancer. Through comprehensive genomic profiling we prospectively identified 6 RET fusion kinases, including two novel fusions of CCDC6-RET and NCOA4-RET, in metastatic colorectal cancer (CRC) patients. RET fusion kinases represent a novel class of oncogenic driver in CRC and occurred at a 0.2% frequency without concurrent driver mutations, including KRAS, NRAS, BRAF, PIK3CA or other fusion tyrosine kinases. Multiple RET kinase inhibitors were cytotoxic to RET fusion kinase positive cancer cells and not RET fusion kinase negative CRC cells. The presence of a RET fusion kinase may identify a subset of metastatic CRC patients with a high response rate to RET kinase inhibition. This is the first characterization of RET fusions in CRC patients and highlights the therapeutic significance of prospective comprehensive genomic profiling in advanced CRC.     

RET Fusion Lung Carcinoma: Response to Therapy and Clinical Features in a Case Series of 14 Patients

Background

RET (rearranged during transfection) fusions have been reported in 1% to 2% of lung adenocarcinoma (LADC) cases. In contrast, KIF5B-RET and CCDC6-RET fusion genes have been identified in 70% to 90% and 10% to 25% of tumors, respectively. The natural history and management of RET-rearranged LADC are still being delineated.

KIF5B‐RET fusions in Chinese patients with non–small cell lung cancer

BACKGROUND:

It has been established that “ret proto‐oncogene” (RET) fusions are oncogenic drivers in non–small cell lung cancer (NSCLC). The prevalence and clinicopathologic characteristics of RET fusions in Chinese patients with NSCLC remain unclear. The objective of the current study was to determine the prevalence and clinicopathologic characteristics of KIF5B‐RET fusions (fusions of the RET and kinesin family member 5B [KIF5B] genes) in Chinese patients with NSCLC.

METHODS:

The authors screened for KIF5B‐RET fusions in 392 patients with NSCLC using multiplex real‐time polymerase chain reaction assay and validated all positive samples using direct sequencing. The relations between KIF5B‐RET fusions and clinicopathologic characteristics were analyzed.

RESULTS:

In total, 6 patients (1.5%) were identified who harbored KIF5B‐RET fusions. Of these, 4 had adenocarcinoma, 1 had a malignant neuroendocrine tumor, and 1 had squamous cell carcinoma. All patients who were positive for a KIF5B‐RET fusion were never‐smokers. There was no statistically significant difference in age, sex, smoking status, pathologic stage, or histologic type between patients with and without KIF5B‐RET fusions. Patients without KIF5B‐RET fusions had a better prognosis than those with KIF5B‐RET fusions (median survival, 52.6 months vs 21.0 months; P = .06), with a hazard ratio of 2.398 (95% confidence interval, 0.982‐5.856; P = .055) on multivariate analysis. Disease stage (hazard ratio, 2.879) and younger age (<65 years; hazard ratio, 1.485) were identified as independent prognostic factors for better survival.

CONCLUSIONS:

KIF5B‐RET fusions were quite rare, with a prevalence of approximately 1.5% in Chinese patients with NSCLC, and they were a little more common in patients with adenocarcinoma than in those with squamous carcinoma (1.73% vs 0.84%). In addition, KIF5B‐RET fusions also existed in patients with low‐grade malignant neuroendocrine tumors. Cancer 2013. © 2013 American Cancer Society.     

Therapeutic Potential of Afatinib in NRG1 Fusion‐Driven Solid Tumors: A Case Series

BackgroundNeuregulin 1 (NRG1) fusions, which activate ErbB signaling, are rare oncogenic drivers in multiple tumor types. Afatinib is a pan‐ErbB family inhibitor that may be an effective treatment for NRG1 fusion‐driven tumors.Patients and MethodsThis report summarizes pertinent details, including best tumor response to treatment, for six patients with metastatic NRG1 fusion‐positive tumors treated with afatinib.ResultsThe six cases include four female and two male patients who ranged in age from 34 to 69 years. Five of the cases are patients with lung cancer, including two patients with invasive mucinous adenocarcinoma and three patients with nonmucinous adenocarcinoma. The sixth case is a patient with colorectal cancer. NRG1 fusion partners for the patients with lung cancer were either CD74 or SDC4. The patient with colorectal cancer harbored a novel POMK‐NRG1 fusion and a KRAS mutation. Two patients received afatinib as first‐ or second‐line therapy, three patients received the drug as third‐ to fifth‐line therapy, and one patient received afatinib as fifteenth‐line therapy. Best response with afatinib was stable disease in two patients (duration up to 16 months when combined with local therapies) and partial response (PR) of >18 months in three patients, including one with ongoing PR after 27 months. The remaining patient had a PR of 5 months with afatinib 40 mg/day, then another 6 months after an increase to 50 mg/day.ConclusionThis report reviews previously published metastatic NRG1 fusion‐positive tumors treated with afatinib and summarizes six previously unpublished cases. The latter include several with a prolonged response to treatment (>18 months), as well as the first report of efficacy in NRG1 fusion‐positive colorectal cancer. This adds to the growing body of evidence suggesting that afatinib can be effective in patients with NRG1 fusion‐positive tumors.Key Points

• NRG1 fusions activate ErbB signaling and have been identified as oncogenic drivers in multiple solid tumor types. Afatinib is a pan‐ErbB family inhibitor authorized for the treatment of advanced non‐small cell lung cancer that may be effective in NRG1 fusion‐driven tumors.
• This report summarizes six previously unpublished cases of NRG1 fusion‐driven cancers treated with afatinib, including five with metastatic lung cancer and one with metastatic colorectal cancer.
• Several patients showed a prolonged response of >18 months with afatinib treatment. This case series adds to the evidence suggesting a potential role for afatinib in this area of unmet medical need.

     

RET fusions in solid tumors

The RET proto-oncogene has been well-studied. RET is involved in many different physiological and developmental functions. When altered, RET mutations influence disease in a variety of organ systems from Hirschsprung’s disease and multiple endocrine neoplasia 2 (MEN2) to papillary thyroid carcinoma (PTC) and non-small cell lung cancer (NSCLC). Changes in RET expression have been discovered in 30–70% of invasive breast cancers and 50–60% of pancreatic ductal adenocarcinomas in addition to colorectal adenocarcinoma, melanoma, small cell lung cancer, neuroblastoma, and small intestine neuroendocrine tumors. RET mutations have been associated with tumor proliferation, invasion, and migration. RET fusions or rearrangements are somatic juxtapositions of 5′ sequences from other genes with 3′ RET sequences encoding tyrosine kinase. RET rearrangements occur in approximately 2.5–73% of sporadic PTC and 1–3% of NSCLC patients. The most common RET fusions are CDCC6-RET and NCOA4-RET in PTC and KIF5B-RET in NSCLC. Tyrosine kinase inhibitors are drugs that target kinases such as RET in RET-driven (RET-mutation or RET-fusion-positive) disease. Multikinase inhibitors (MKI) target various kinases and other receptors. Several MKIs are FDA-approved for cancer therapy (sunitinib, sorafenib, vandetanib, cabozantinib, regorafenib, ponatinib, lenvatinib, alectinib) and non-oncologic disease (nintedanib). Selective RET inhibitor drugs LOXO-292 (selpercatinib) and BLU-667 (pralsetinib) are also undergoing phase I/II and I clinical trials, respectively, with preliminary results demonstrating partial response and low incidence of serious adverse events. RET fusions provide a viable therapeutic target for oncologic treatment, and further study is warranted into the prevalence and pathogenesis of RET fusions as well as development of current and new tyrosine kinase inhibitors.     

Selective RET kinase inhibition for patients with RET-altered cancers

BackgroundAlterations involving the RET kinase are implicated in the pathogenesis of lung, thyroid and other cancers. However, the clinical activity of multikinase inhibitors (MKIs) with anti-RET activity in RET-altered patients appears limited, calling into question the therapeutic potential of targeting RET. LOXO-292 is a selective RET inhibitor designed to inhibit diverse RET fusions, activating mutations and acquired resistance mutations.Patients and methodsPotent anti-RET activity, high selectivity, and central nervous system coverage were confirmed preclinically using a variety of in vitro and in vivo RET-dependent tumor models. Due to clinical urgency, two patients with RET-altered, MKI-resistant cancers were treated with LOXO-292, utilizing rapid dose-titration guided by real-time pharmacokinetic assessments to achieve meaningful clinical exposures safely and rapidly.ResultsLOXO-292 demonstrated potent and selective anti-RET activity preclinically against human cancer cell lines harboring endogenous RET gene alterations; cells engineered to express a KIF5B-RET fusion protein −/+ the RET V804M gatekeeper resistance mutation or the common RET activating mutation M918T; and RET-altered human cancer cell line and patient-derived xenografts, including a patient-derived RET fusion-positive xenograft injected orthotopically into the brain. A patient with RET M918T-mutant medullary thyroid cancer metastatic to the liver and an acquired RET V804M gatekeeper resistance mutation, previously treated with six MKI regimens, experienced rapid reductions in tumor calcitonin, CEA and cell-free DNA, resolution of painful hepatomegaly and tumor-related diarrhea and a confirmed tumor response. A second patient with KIF5B-RET fusion-positive lung cancer, acquired resistance to alectinib and symptomatic brain metastases experienced a dramatic response in the brain, and her symptoms resolved.ConclusionsThese results provide proof-of-concept of the clinical actionability of RET alterations, and identify selective RET inhibition by LOXO-292 as a promising treatment in heavily pretreated, multikinase inhibitor-experienced patients with diverse RET-altered tumors.     

Antitumor activities of the targeted multi-tyrosine kinase inhibitor lenvatinib (E7080) against RET gene fusion-driven tumor models

RET gene fusions are recurrent oncogenes identified in thyroid and lung carcinomas. Lenvatinib is a multi-tyrosine kinase inhibitor currently under evaluation in several clinical trials. Here we evaluated lenvatinib in RET gene fusion-driven preclinical models. In cellular assays, lenvatinib inhibited auto-phosphorylation of KIF5B-RET, CCDC6-RET, and NcoA4-RET. Lenvatinib suppressed the growth of CCDC6-RET human thyroid and lung cancer cell lines, and as well, suppressed anchorage-independent growth and tumorigenicity of RET gene fusion-transformed NIH3T3 cells. These results demonstrate that lenvatinib can exert antitumor activity against RET gene fusion-driven tumor models by inhibiting oncogenic RET gene fusion signaling.     

RET Solvent Front Mutations Mediate Acquired Resistance to Selective RET Inhibition in RET-Driven Malignancies

IntroductionNovel rearranged in transfection (RET)-specific tyrosine kinase inhibitors (TKIs) such as selpercatinib (LOXO-292) have shown unprecedented efficacy in tumors positive for RET fusions or mutations, notably RET fusion-positive NSCLC and RET-mutated medullary thyroid cancer (MTC). However, the mechanisms of resistance to these agents have not yet been described.MethodsAnalysis was performed of circulating tumor DNA and tissue in patients with RET fusion-positive NSCLC and RET-mutation positive MTC who developed disease progression after an initial response to selpercatinib. Acquired resistance was modeled preclinically using a CCDC6-RET fusion-positive NSCLC patient-derived xenograft. The inhibitory activity of anti-RET multikinase inhibitors and selective RET TKIs was evaluated in enzyme and cell-based assays.ResultsAfter a dramatic initial response to selpercatinib in a patient with KIF5B-RET NSCLC, analysis of circulating tumor DNA revealed emergence of RET G810R, G810S, and G810C mutations in the RET solvent front before the emergence of clinical resistance. Postmortem biopsy studies reported intratumor and intertumor heterogeneity with distinct disease subclones containing G810S, G810R, and G810C mutations in multiple disease sites indicative of convergent evolution on the G810 residue resulting in a common mechanism of resistance. Acquired mutations in RET G810 were identified in tumor tissue from a second patient with CCDC6-RET fusion-positive NSCLC and in plasma from patients with additional RET fusion-positive NSCLC and RET-mutant MTC progressing on an ongoing phase 1 and 2 trial of selpercatinib. Preclinical studies reported the presence of RET G810R mutations in a CCDC6-RET patient-derived xenograft (from a patient with NSCLC) model of acquired resistance to selpercatinib. Structural modeling predicted that these mutations sterically hinder the binding of selpercatinib, and in vitro assays confirmed loss of activity for both anti-RET multikinase inhibitors and selective RET TKIs.ConclusionsRET G810 solvent front mutations represent the first described recurrent mechanism of resistance to selective RET inhibition with selpercatinib. Development of potent inhibitor of these mutations and maintaining activity against RET gatekeeper mutations could be an effective strategy to target resistance to selective RET inhibitors.     

Gefitinib Combined with Cetuximab for the Treatment of Lung Adenocarcinoma Harboring the EGFR –Intergenic Region ( SEC61G ) Fusion and EGFR Amplification

EGFR fusions are rare genomic events in non‐small cell lung cancer (NSCLC), and a total of nine types have been previously reported in lung adenocarcinoma: EGFR‐RAD51, EGFR‐PURB, EGFR‐ANXA2, EGFR‐ZNF713, EGFR‐YAP1, USP42‐EGFR, EGFR‐SEPTIN14, EGFR‐TNS3, and EGFR‐ZCCHC6. EGFR fusion mutations combined with EGFR amplification are even rarer in NSCLC. The EGFR–intergenic region (IGR) fusion mutation is unreported, and thus, there are no studies targeting this fusion together with EGFR amplification in lung adenocarcinoma. Our brief study provides clinical evidence that combined targeted therapy with gefitinib and cetuximab could result in a significant antitumor response in patients with the EGFR‐IGR fusion and EGFR amplification.Key Points

• EGFR fusion mutations are rare, and EGFR fusion mutations combined with EGFR amplification are even rarer in non‐small cell lung cancer (NSCLC). To the authors’ knowledge, there is no previous report on the coexistence of the EGFR–intergenic region (IGR) fusion and EGFR amplification.
• This is the first report of a patient with NSCLC with the EGFR‐IGR fusion and EGFR amplification who achieved a significant antitumor response from treatment with gefitinib combined with cetuximab.

     

Clinical Characteristics and Molecular Patterns of <i>RET</i>-Rearranged Lung Cancer in Chinese Patients

RET rearrangement has been proven as an oncogenic driver in patients with lung cancer. However, the prevalence, clinical characteristics, molecular features, and therapeutic options in RET-rearranged patients remain unclear, especially in Chinese lung cancer patients. We retrospectively collected 6,125 Chinese lung cancer patients who have been profiled using next-generation sequencing (NGS). The clinical demographics and molecular features of RET rearrangement-positive patients were analyzed. RET rearrangements were identified in 84 patients with a proportion of 1.4% in our cohort. The median age at diagnosis was 58 years, and it mainly occurred in females with adenocarcinoma histology. KIF5B-RET was the most frequent fusion type and accounted for 53.8% (57/106) of all RET fusions identified, with K15-R12 as the most frequent variant (71.9%). Among 47 RET+ patients profiled with larger panels, 72.3% (34/47) harbored concurrent alterations. TP53 ranked as the most common concurrent alteration, and concomitant EGFR oncogenic alterations were identified in seven patients. Moreover, an adenocarcinoma patient harboring concurrent RET fusion and EGFR L858R responded to combinatorial treatment of cabozantinib and osimertinib, with a progression-free survival of 5 months. Our study improved knowledge of clinical characteristics and molecular features of RET-rearranged lung cancers in China. It might be helpful to guide clinicians for more effective personalized diagnostic and therapeutic approaches.     

Promoter CpG island methylation of RET predicts poor prognosis in stage II colorectal cancer patients

Improved prognostic stratification of patients with TNM stage II colorectal cancer (CRC) is desired, since 20–30% of high‐risk stage II patients may die within five years of diagnosis. This study was conducted to investigate REarranged during Transfection (RET) gene promoter CpG island methylation as a possible prognostic marker for TNM stage II CRC patients.The utility of RET promoter CpG island methylation in tumors of stage II CRC patients as a prognostic biomarker for CRC related death was studied in three independent series (including 233, 231, and 294 TNM stage II patients, respectively) by using MSP and pyrosequencing. The prognostic value of RET promoter CpG island methylation was analyzed by using Cox regression analysis.In the first series, analyzed by MSP, CRC stage II patients (n = 233) with RET methylated tumors had a significantly worse overall survival as compared to those with unmethylated tumors (HR_{multivariable} = 2.51, 95%‐CI: 1.42–4.43). Despite a significant prognostic effect of RET methylation in stage III patients of a second series, analyzed by MSP, the prognostic effect in stage II patients (n = 231) was not statistically significant (HR_{multivariable} = 1.16, 95%‐CI 0.71–1.92). The third series (n = 294), analyzed by pyrosequencing, confirmed a statistically significant association between RET methylation and poor overall survival in stage II patients (HR_{multivariable} = 1.91, 95%‐CI: 1.04–3.53). Our results show that RET promoter CpG island methylation, analyzed by two different techniques, is associated with a poor prognosis in stage II CRC in two independent series and a poor prognosis in stage III CRC in one series. RET methylation may serve as a useful and robust tool for clinical practice to identify high‐risk stage II CRC patients with a poor prognosis. This merits further investigation.     

RET-MAP: An International Multicenter Study on Clinicobiologic Features and Treatment Response in Patients With Lung Cancer Harboring a RET Fusion

IntroductionNearly 1% to 2% of NSCLCs harbor RET fusions. Characterization of this rare population is still incomplete.MethodsThis retrospective multicenter study included patients with any-stage RET positive (RET+) NSCLC from 31 cancer centers. Molecular profiling included DNA/RNA sequencing or fluorescence in situ hybridization analyses. Clinicobiological features and treatment outcomes (per investigator) with surgery, chemotherapy (CT), immune checkpoint blockers (ICBs), CT-ICB, multityrosine kinase inhibitors, and RET inhibitors (RETis) were evaluated.ResultsFor 218 patients included between February 2012 and April 2022, median age was 63 years, 56% were females, 93% had adenocarcinoma, and 41% were smokers. The most frequent fusion partner was KIF5B (72%). Median tumor mutational burden was 2.5 (range: 1–4) mutations per megabase, and median programmed death-ligand 1 expression was 10% (range: 0%–55%). The most common metastatic sites were the lung (50%), bone (43%), and pleura (40%). Central nervous system metastases were found at diagnosis of advanced NSCLC in 21% of the patients and at last follow-up or death in 31%. Overall response rate and median progression-free survival were 55% and 8.7 months with platinum doublet, 26% and 3.6 months with single-agent CT, 46% and 9.6 months with CT-ICB, 23% and 3.1 months with ICB, 37% and 3 months with multityrosine kinase inhibitor, and 76% and 16.2 months with RETi, respectively. Median overall survival was longer in patients treated with RETi versus no RETi (50.6 mo [37.7–72.1] versus 16.3 mo [12.7–28.8], p < 0.0001).ConclusionsPatients with RET+ NSCLC have mainly thoracic and bone disease and low tumor mutational burden and programmed death-ligand 1 expression. RETi markedly improved survival, whereas ICB may be active in selected patients.     

Association Between RET Fusions and Efficacy of Pemetrexed-based Chemotherapy for Patients With Advanced NSCLC in China: A Multicenter Retrospective Study

BackgroundRearranged during transfection (RET) proto-oncogene gene fusions are rare in non–small-cell lung cancer (NSCLC). We compared the efficacy of pemetrexed-based chemotherapy with other chemotherapy regimens in patients with NSCLC with different RET fusion subtypes.Patients and MethodsA retrospective, multicenter study of patients with pathologically confirmed stage IIIB/IV lung adenocarcinomas was conducted. RET rearrangements were detected using next generation sequencing. We analyzed the clinical characteristics of patients with RET-rearranged NSCLC and the efficacy of chemotherapy regimens. We also evaluated the efficacy between groups of patients with and without KIF5B-RET–rearranged lung cancer.ResultsWe evaluated 62 patients with NSCLC and RET rearrangements, including 41 with KIF5B-RET, 15 with CCDC6-RET, and 6 with other rare fusion subtypes. Of these 62 patients, 50 had stage IIIB/IV. We also evaluated 40 patients with first-line chemotherapy information available. The median progression-free survival was significantly different between those receiving pemetrexed-based chemotherapy and those receiving other chemotherapy regimens (9.2 vs. 5.2 months; P = .007). The median progression-free survival for patients with KIF5B-RET fusion and non–KIF5B-RET fusion was not significantly different statistically (7.8 vs. 11.2 months; P = .847). For second-line chemotherapy, a statistically significant difference was found between the chemotherapy regimens (4.9 vs. 2.8 months; P = .049). Survival follow-up data were available for 38 patients with advanced NSCLC. The median overall survival was 26.4 months. The overall survival of the patients with RET-rearranged NSCLC who had received pemetrexed-based chemotherapy versus no pemetrexed-based chemotherapy was 35.2 versus 22.6 months (P = .052). No difference in survival was observed between the patients with KIF5B-RET and non–KIF5B-RET rearrangements.ConclusionsPemetrexed-based treatment should be considered first when selecting the chemotherapy regimen for patients with NSCLC and RET rearrangements.     

Utility of the Ba/F3 cell system for exploring on‐target mechanisms of resistance to targeted therapies for lung cancer

Molecular targeted therapies are the standard of care for front‐line treatment of metastatic non‐small‐cell lung cancers (NSCLCs) harboring driver gene mutations. However, despite the initial dramatic responses, the emergence of acquired resistance is inevitable. Acquisition of secondary mutations in the target gene (on‐target resistance) is one of the major mechanisms of resistance. The mouse pro‐B cell line Ba/F3 is dependent on interleukin‐3 for survival and proliferation. Upon transduction of a driver gene, Ba/F3 cells become independent of interleukin‐3 but dependent on the transduced driver gene. Therefore, the Ba/F3 cell line has been a popular system to generate models with oncogene dependence and vulnerability to specific targeted therapies. These models have been used to estimate oncogenicity of driver mutations or efficacies of molecularly targeted drugs. In addition, Ba/F3 models, together with N‐ethyl‐N‐nitrosourea mutagenesis, have been used to derive acquired resistant cells to investigate on‐target resistance mechanisms. Here, we reviewed studies that used Ba/F3 models with EGFR mutations, ALK/ROS1/NTRK/RET fusions, MET exon 14 skipping mutations, or KRAS G12C mutations to investigate secondary/tertiary drug resistant mutations. We determined that 68% of resistance mutations reproducibly detected in clinical cases were also found in Ba/F3 models. In addition, sensitivity data generated with Ba/F3 models correlated well with clinical responses to each drug. Ba/F3 models are useful to comprehensively identify potential mutations that induce resistance to molecularly targeted drugs and to explore drugs to overcome the resistance.