Dara L. Aisner

Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer.

Purpose: Patients with anaplastic lymphoma kinase (ALK) gene rearrangements often manifest dramatic responses to crizotinib, a small-molecule ALK inhibitor. Unfortunately, not every patient responds and acquired drug resistance inevitably develops in those who do respond. This study aimed to define molecular mechanisms of resistance to crizotinib in patients with ALK+ non–small cell lung cancer (NSCLC). Experimental Design: We analyzed tissue obtained from 14 patients with ALK+ NSCLC showing evidence of radiologic progression while on crizotinib to define mechanisms of intrinsic and acquired resistance to crizotinib. Results: Eleven patients had material evaluable for molecular analysis. Four patients (36%) developed secondary mutations in the tyrosine kinase domain of ALK. A novel mutation in the ALK domain, encoding a G1269A amino acid substitution that confers resistance to crizotinib in vitro, was identified in two of these cases. Two patients, one with a resistance mutation, exhibited new onset ALK copy number gain (CNG). One patient showed outgrowth of epidermal growth factor receptor (EGFR) mutant NSCLC without evidence of a persistent ALK gene rearrangement. Two patients exhibited a KRAS mutation, one of which occurred without evidence of a persisting ALK gene rearrangement. One patient showed the emergence of an ALK gene fusion–negative tumor compared with the baseline sample but with no identifiable alternate driver. Two patients retained ALK positivity with no identifiable resistance mechanism. Conclusions: Crizotinib resistance in ALK+ NSCLC occurs through somatic kinase domain mutations, ALK gene fusion CNG, and emergence of separate oncogenic drivers. Clin Cancer Res; 18(5); 1472–82. ©2012 AACR.

Rociletinib in EGFR-mutated non-small-cell lung cancer.

BACKGROUND Non-small-cell lung cancer (NSCLC) with a mutation in the gene encoding epidermal growth factor receptor (EGFR) is sensitive to approved EGFR inhibitors, but resistance develops, mediated by the T790M EGFR mutation in most cases. Rociletinib (CO-1686) is an EGFR inhibitor active in preclinical models of EGFR-mutated NSCLC with or without T790M. METHODS In this phase 1-2 study, we administered rociletinib to patients with EGFR-mutated NSCLC who had disease progression during previous treatment with an existing EGFR inhibitor. In the expansion (phase 2) part of the study, patients with T790M-positive disease received rociletinib at a dose of 500 mg twice daily, 625 mg twice daily, or 750 mg twice daily. Key objectives were assessment of safety, side-effect profile, pharmacokinetics, and preliminary antitumor activity of rociletinib. Tumor biopsies to identify T790M were performed during screening. Treatment was administered in continuous 21-day cycles. RESULTS A total of 130 patients were enrolled. The first 57 patients to be enrolled received the free-base form of rociletinib (150 mg once daily to 900 mg twice daily). The remaining patients received the hydrogen bromide salt (HBr) form (500 mg twice daily to 1000 mg twice daily). A maximum tolerated dose (the highest dose associated with a rate of dose-limiting toxic effects of less than 33%) was not identified. The only common dose-limiting adverse event was hyperglycemia. In an efficacy analysis that included patients who received free-base rociletinib at a dose of 900 mg twice daily or the HBr form at any dose, the objective response rate among the 46 patients with T790M-positive disease who could be evaluated was 59% (95% confidence interval [CI], 45 to 73), and the rate among the 17 patients with T790M-negative disease who could be evaluated was 29% (95% CI, 8 to 51). CONCLUSIONS Rociletinib was active in patients with EGFR-mutated NSCLC associated with the T790M resistance mutation. (Funded by Clovis Oncology; ClinicalTrials.gov number, NCT01526928.).

Local Ablative Therapy of Oligoprogressive Disease Prolongs Disease Control by Tyrosine Kinase Inhibitors in Oncogene-Addicted Non-Small-Cell Lung Cancer

Introduction: Many patients with oncogene-driven non–small-cell lung cancer (NSCLC) treated with tyrosine kinase inhibitors experience limited sites of disease progression. This study investigated retrospectively the benefits of local ablative therapy (LAT) to central nervous system (CNS) and/or limited systemic disease progression and continuation of crizotinib or erlotinib in patients with metastatic ALK gene rearrangement (ALK+) or EGFR-mutant (EGFR-MT) NSCLC, respectively. Methods: Patients with metastatic ALK+ NSCLC treated with crizotinib (n = 38) and EGFR-MT NSCLC treated with erlotinib (n = 27) were identified at a single institution. Initial response to the respective kinase inhibitors, median progression-free survival (PFS1), and site of first progression were recorded. A subset of patients with either nonleptomeningeal CNS and/or four sites or fewer of extra-CNS progression (oligoprogressive disease) suitable for LAT received either radiation or surgery to these sites and continued on the same tyrosine kinase inhibitors. The subsequent median progression-free survival from the time of first progression (PFS2) and pattern of progression were recorded. Results: Median progression-free survival in ALK+ patients on crizotinib was 9.0 months, and 13.8 months for EGFR-MT patients on erlotinib. Twenty-five of 51 patients (49%) who progressed were deemed suitable for local therapy (15 ALK+, 10 EGFR-MT; 24 with radiotherapy, one with surgery) and continuation of the same targeted therapy. Post-LAT, 19 of 25 patients progressed again, with median PFS2 of 6.2 months. Discussion: Oncogene-addicted NSCLC with CNS and/or limited systemic disease progression (oligoprogressive disease) on relevant targeted therapies is often suitable for LAT and continuation of the targeted agent, and is associated with more than 6 months of additional disease control.

Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer

We identified new gene fusions in patients with lung cancer harboring the kinase domain of the NTRK1 gene that encodes the high-affinity nerve growth factor receptor (TRKA protein). Both the MPRIP-NTRK1 and CD74-NTRK1 fusions lead to constitutive TRKA kinase activity and are oncogenic. Treatment of cells expressing NTRK1 fusions with inhibitors of TRKA kinase activity inhibited autophosphorylation of TRKA and cell growth. Tumor samples from 3 of 91 patients with lung cancer (3.3%) without known oncogenic alterations assayed by next-generation sequencing or fluorescence in situ hybridization demonstrated evidence of NTRK1 gene fusions.

Identifying and targeting ROS1 gene fusions in non-small cell lung cancer.

Purpose: Oncogenic gene fusions involving the 3′ region of ROS1 kinase have been identified in various human cancers. In this study, we sought to characterize ROS1 fusion genes in non–small cell lung cancer (NSCLC) and establish the fusion proteins as drug targets. Experimental Design: An NSCLC tissue microarray (TMA) panel containing 447 samples was screened for ROS1 rearrangement by FISH. This assay was also used to screen patients with NSCLC. In positive samples, the identity of the fusion partner was determined through inverse PCR and reverse transcriptase PCR. In addition, the clinical efficacy of ROS1 inhibition was assessed by treating a ROS1-positive patient with crizotinib. The HCC78 cell line, which expresses the SLC34A2–ROS1 fusion, was treated with kinase inhibitors that have activity against ROS1. The effects of ROS1 inhibition on proliferation, cell-cycle progression, and cell signaling pathways were analyzed by MTS assay, flow cytometry, and Western blotting. Results: In the TMA panel, 5 of 428 (1.2%) evaluable samples were found to be positive for ROS1 rearrangement. In addition, 1 of 48 patients tested positive for rearrangement, and this patient showed tumor shrinkage upon treatment with crizotinib. The patient and one TMA sample displayed expression of the recently identified SDC4–ROS1 fusion, whereas two TMA samples expressed the CD74–ROS1 fusion and two others expressed the SLC34A2–ROS1 fusion. In HCC78 cells, treatment with ROS1 inhibitors was antiproliferative and downregulated signaling pathways that are critical for growth and survival. Conclusions: ROS1 inhibition may be an effective treatment strategy for the subset of patients with NSCLC whose tumors express ROS1 fusion genes. Clin Cancer Res; 18(17); 4570–9. ©2012 AACR.

Epithelioid GBMs show a high percentage of BRAF V600E mutation

BRAF V600E mutation has been identified in up to 2/3 of pleomorphic xanthoastrocytomas (PXAs), World Health Organization grade II, as well as in varying percentages of PXAs with anaplastic features (PXA-A), gangliogliomas, extracerebellar pilocytic astrocytomas, and, rarely, giant cell glioblastoma multiforme (GC-GBMs). GC-GBMs and epithelioid GBMs (E-GBMs) can be histologically challenging to distinguish from PXA-A. We undertook this study specifically to address whether these 2 tumor types also showed the mutation. We tested our originally reported cohort of 8 E-GBMs and 2 rhabdoid GBMs (R-GBM) as well as 5 new E-GBMs (1 pediatric, 4 adult) and 9 GC-GBMs (2 pediatric, 7 adult) (n=24) for BRAF V600E mutational status. Twenty-one of 24 had sufficient material for IDH-1 immunostaining, which is usually absent in PXAs, PXA-As, and primary GBMs but present in secondary GBMs. Patients ranged in age from 4 to 67 years. BRAF V600E mutation was identified in 7/13 of E-GBMs, including 3 of our original cases; patients with mutation were aged 10 to 50 years. None of the 9 GC-GBMs or 2 R-GBMs manifested this mutation, including pediatric patients. The sole secondary E-GBM was the single case manifesting positive IDH-1 immunoreactivity. A high percentage of E-GBMs manifest BRAF V600E mutation, paralleling PXAs. All R-GBMs and GC-GBMs were negative, although larger multi-institutional cohorts will have to be tested to extend this result. BRAF V600E mutational analyses should be performed on E-GBMs, particularly in all pediatric and young-aged adults, given the potential for BRAF inhibitor therapy in this subset of GBM patients.

Multi-institutional Oncogenic Driver Mutation Analysis in Lung Adenocarcinoma: The Lung Cancer Mutation Consortium Experience

Introduction: Molecular genetic analyses of lung adenocarcinoma have recently become standard of care for treatment selection. The Lung Cancer Mutation Consortium was formed to enable collaborative multi-institutional analyses of 10 potential oncogenic driver mutations. Technical aspects of testing and clinicopathologic correlations are presented. Methods: Mutation testing in at least one of the eight genes (epidermal growth factor receptor [EGFR], KRAS, ERBB2, AKT1, BRAF, MEK1, NRAS, and PIK3CA) using SNaPshot, mass spectrometry, Sanger sequencing+/− peptide nucleic acid and/or sizing assays, along with anaplastic lymphoma kinase (ALK) and/or MET fluorescence in situ hybridization, were performed in six labs on 1007 patients from 14 institutions. Results: In all, 1007 specimens had mutation analysis performed, and 733 specimens had all 10 genes analyzed. Mutation identification rates did not vary by analytic method. Biopsy and cytology specimens were inadequate for testing in 26% and 35% of cases compared with 5% of surgical specimens. Among the 1007 cases with mutation analysis performed, EGFR, KRAS, ALK, and ERBB2 alterations were detected in 22%, 25%, 8.5%, and 2.4% of cases, respectively. EGFR mutations were highly associated with female sex, Asian race, and never-smoking status; and less strongly associated with stage IV disease, presence of bone metastases, and absence of adrenal metastases. ALK rearrangements were strongly associated with never-smoking status and more weakly associated with presence of liver metastases. ERBB2 mutations were strongly associated with Asian race and never-smoking status. Two mutations were seen in 2.7% of samples, all but one of which involved one or more of PIK3CA, ALK, or MET. Conclusion: Multi-institutional molecular analysis across multiple platforms, sample types, and institutions can yield consistent results and novel clinicopathological observations.

An Oncogenic NTRK Fusion in a Patient with Soft-Tissue Sarcoma with Response to the Tropomyosin-Related Kinase Inhibitor LOXO-101

UNLABELLED Oncogenic TRK fusions induce cancer cell proliferation and engage critical cancer-related downstream signaling pathways. These TRK fusions occur rarely, but in a diverse spectrum of tumor histologies. LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors. Preclinical models of LOXO-101 using TRK-fusion-bearing human-derived cancer cell lines demonstrate inhibition of the fusion oncoprotein and cellular proliferation in vitro, and tumor growth in vivo. The tumor of a 41-year-old woman with soft-tissue sarcoma metastatic to the lung was found to harbor an LMNA-NTRK1 gene fusion encoding a functional LMNA-TRKA fusion oncoprotein as determined by an in situ proximity ligation assay. In a phase I study of LOXO-101 (ClinicalTrials.gov no. NCT02122913), this patients tumors underwent rapid and substantial tumor regression, with an accompanying improvement in pulmonary dyspnea, oxygen saturation, and plasma tumor markers. SIGNIFICANCE TRK fusions have been deemed putative oncogenic drivers, but their clinical significance remained unclear. A patient with a metastatic soft-tissue sarcoma with an LMNA-NTRK1 fusion had rapid and substantial tumor regression with a novel, highly selective TRK inhibitor, LOXO-101, providing the first clinical evidence of benefit from inhibiting TRK fusions.

RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK–positive lung cancer

One strategy for combating cancer-drug resistance is to deploy rational polytherapy up front that suppresses the survival and emergence of resistant tumor cells. Here we demonstrate in models of lung adenocarcinoma harboring the oncogenic fusion of ALK and EML4 that the GTPase RAS–mitogen-activated protein kinase (MAPK) pathway, but not other known ALK effectors, is required for tumor-cell survival. EML4-ALK activated RAS-MAPK signaling by engaging all three major RAS isoforms through the HELP domain of EML4. Reactivation of the MAPK pathway via either a gain in the number of copies of the gene encoding wild-type K-RAS (KRASWT) or decreased expression of the MAPK phosphatase DUSP6 promoted resistance to ALK inhibitors in vitro, and each was associated with resistance to ALK inhibitors in individuals with EML4-ALK–positive lung adenocarcinoma. Upfront inhibition of both ALK and the kinase MEK enhanced both the magnitude and duration of the initial response in preclinical models of EML4-ALK lung adenocarcinoma. Our findings identify RAS-MAPK dependence as a hallmark of EML4-ALK lung adenocarcinoma and provide a rationale for the upfront inhibition of both ALK and MEK to forestall resistance and improve patient outcomes.

ROS1 and ALK Fusions in Colorectal Cancer, with Evidence of Intratumoral Heterogeneity for Molecular Drivers

Activated anaplastic lymphoma kinase (ALK) and ROS1 tyrosine kinases, through gene fusions, have been found in lung adenocarcinomas and are highly sensitive to selective kinase inhibitors. This study aimed at identifying the presence of these rearrangements in human colorectal adenocarcinoma specimens using a 4-target, 4-color break-apart FISH assay to simultaneously determine the genomic status of ALK and ROS1. Among the clinical colorectal cancer specimens analyzed, rearrangement-positive cases for both ALK and ROS1 were observed. The fusion partner for ALK was identified as EML4 and the fusion partner for one of the ROS1-positive cases was SLC34A2, the partner for the other ROS1-positive case remains to be identified. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. In addition, rearrangements were detected in samples that also harbored KRAS and BRAF mutations in two of the three cases. Interestingly, the ALK-positive specimen displayed marked intratumoral heterogeneity and rearrangement was also identified in regions of high-grade dysplasia. Despite the additional oncogenic events and tumor heterogeneity observed, elucidation of the first cases of ROS1 rearrangements and confirmation of ALK rearrangements support further evaluation of these genomic fusions as potential therapeutic targets in colorectal cancer. Implications: ROS1 and ALK fusions occur in colorectal cancer and may have substantial impact in therapy selection. Mol Cancer Res; 12(1); 111–8. ©2013 AACR.