Robert C. Doebele

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.

Effect of crizotinib on overall survival in patients with advanced non-small-cell lung cancer harbouring ALK gene rearrangement: a retrospective analysis

BACKGROUND ALK gene rearrangement defines a new molecular subtype of non-small-cell lung cancer (NSCLC). In a recent phase 1 clinical trial, the ALK tyrosine-kinase inhibitor (TKI) crizotinib showed marked antitumour activity in patients with advanced, ALK-positive NSCLC. To assess whether crizotinib affects overall survival in these patients, we did a retrospective study comparing survival outcomes in crizotinib-treated patients in the trial and crizotinib-naive controls screened during the same time period. METHODS We examined overall survival in patients with advanced, ALK-positive NSCLC who enrolled in the phase 1 clinical trial of crizotinib, focusing on the cohort of 82 patients who had enrolled through Feb 10, 2010. For comparators, we identified 36 ALK-positive patients from trial sites who were not given crizotinib (ALK-positive controls), 67 patients without ALK rearrangement but positive for EGFR mutation, and 253 wild-type patients lacking either ALK rearrangement or EGFR mutation. To assess differences in overall survival, we assessed subsets of clinically comparable ALK-positive and ALK-negative patients. FINDINGS Among 82 ALK-positive patients who were given crizotinib, median overall survival from initiation of crizotinib has not been reached (95% CI 17 months to not reached); 1-year overall survival was 74% (95% CI 63-82), and 2-year overall survival was 54% (40-66). Overall survival did not differ based on age, sex, smoking history, or ethnic origin. Survival in 30 ALK-positive patients who were given crizotinib in the second-line or third-line setting was significantly longer than in 23 ALK-positive controls given any second-line therapy (median overall survival not reached [95% CI 14 months to not reached] vs 6 months [4-17], 1-year overall survival 70% [95% CI 50-83] vs 44% [23-64], and 2-year overall survival 55% [33-72] vs 12% [2-30]; hazard ratio 0·36, 95% CI 0·17-0·75; p=0·004). Survival in 56 crizotinib-treated, ALK-positive patients was similar to that in 63 ALK-negative, EGFR-positive patients given EGFR TKI therapy (median overall survival not reached [95% CI 17 months to not reached] vs 24 months [15-34], 1-year overall survival 71% [95% CI 58-81] vs 74% [61-83], and 2-year overall survival 57% [40-71] vs 52% [38-65]; p=0·786), whereas survival in 36 crizotinib-naive, ALK-positive controls was similar to that in 253 wild-type controls (median overall survival 20 months [95% CI 13-26] vs 15 months [13-17]; p=0·244). INTERPRETATION In patients with advanced, ALK-positive NSCLC, crizotinib therapy is associated with improved survival compared with that of crizotinib-naive controls. ALK rearrangement is not a favourable prognostic factor in advanced NSCLC. FUNDING Pfizer Inc, V Foundation for Cancer Research.

Crizotinib in ROS1-Rearranged Non–Small-Cell Lung Cancer

BACKGROUND Chromosomal rearrangements of the gene encoding ROS1 proto-oncogene receptor tyrosine kinase (ROS1) define a distinct molecular subgroup of non-small-cell lung cancers (NSCLCs) that may be susceptible to therapeutic ROS1 kinase inhibition. Crizotinib is a small-molecule tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK), ROS1, and another proto-oncogene receptor tyrosine kinase, MET. METHODS We enrolled 50 patients with advanced NSCLC who tested positive for ROS1 rearrangement in an expansion cohort of the phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg twice daily and assessed for safety, pharmacokinetics, and response to therapy. ROS1 fusion partners were identified with the use of next-generation sequencing or reverse-transcriptase-polymerase-chain-reaction assays. RESULTS The objective response rate was 72% (95% confidence interval [CI], 58 to 84), with 3 complete responses and 33 partial responses. The median duration of response was 17.6 months (95% CI, 14.5 to not reached). Median progression-free survival was 19.2 months (95% CI, 14.4 to not reached), with 25 patients (50%) still in follow-up for progression. Among 30 tumors that were tested, we identified 7 ROS1 fusion partners: 5 known and 2 novel partner genes. No correlation was observed between the type of ROS1 rearrangement and the clinical response to crizotinib. The safety profile of crizotinib was similar to that seen in patients with ALK-rearranged NSCLC. CONCLUSIONS In this study, crizotinib showed marked antitumor activity in patients with advanced ROS1-rearranged NSCLC. ROS1 rearrangement defines a second molecular subgroup of NSCLC for which crizotinib is highly active. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

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.

Optimizing the Detection of Lung Cancer Patients Harboring Anaplastic Lymphoma Kinase (ALK) Gene Rearrangements Potentially Suitable for ALK Inhibitor Treatment

Purpose: Anaplastic lymphoma kinase (ALK) rearrangements, associated with sensitivity to an experimental ALK/MET inhibitor, occur in 3% to 5% of non–small cell lung cancers. Intratumoral fluorescence in situ hybridization (FISH) heterogeneity has been reported. We explored the heterogeneity basis, the requirements for accurately determining ALK FISH positivity, and the effect of enriching the tested population using clinical and molecular factors. Experimental Design: Lung cancer patients were screened by ALK and MET FISH and for EGFR and KRAS mutations. Results: Thirteen ALK-positive cases were identified from 73 screened patients. Gene copy number increases occurred together with classic rearrangements. All positive cases were adenocarcinomas, 12 were EGFR/KRAS wild-type, and 1 had a coexistent EGFR exon 20 mutation. No association with MET amplification occurred. ALK positivity was associated with <10–pack-year smoking status (P = 0.0004). Among adenocarcinomas, without KRAS or EGFR mutations, with <10–pack-year history, 44.8% of cases were ALK positive. ALK FISH positivity was heterogeneous, but mean values in tumor areas from ALK-positive patients (54% of cells; range, 22-87%) were significantly higher than in adjacent normal tissue or tumor/normal areas from ALK-negative patients (mean, 5-7%). Contiguous sliding field analyses showed diffuse heterogeneity without evidence of focal ALK rearrangements. One hundred percent sensitivity and specificity occurred when four or more fields (∼60 cells) were counted. Conclusions: Intratumoral ALK FISH heterogeneity reflects technique, not biology. The clinical activity of ALK/MET inhibitors in ALK-positive patients probably reflects ALK, but not MET, activity. Prescreening by histology, EGFR/KRAS mutations, and smoking status dramatically increases the ALK-positive hit rate compared with unselected series. Clin Cancer Res; 16(22); 5581–90. ©2010 AACR.

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.

Anaplastic Lymphoma Kinase Gene Rearrangements in Non-small Cell Lung Cancer are Associated with Prolonged Progression-Free Survival on Pemetrexed

Hypothesis: To explore whether the progression-free survival (PFS) with pemetrexed differs between anaplastic lymphoma kinase (ALK)-positive and other major molecular subtypes of non-small cell lung cancer. Methods: In an ALK-enriched population, patients with metastatic non-small cell lung cancer were screened by ALK fluorescence in situ hybridization and for epidermal growth factor receptor (EGFR) and KRAS mutations. Triple-tested, pemetrexed-treated patients (monotherapy or combination therapy) were identified and PFS with pemetrexed captured retrospectively. Results: Eighty-nine eligible cases were identified (19 ALK fluorescence in situ hybridization positive, 12 EGFR mutant, 21 KRAS mutant, and 37 triple negatives). Eighty-three cases (93%) were adenocarcinomas, two were adenosquamous, one squamous, and three had large cell histology. None of the ALK-positive patients had received crizotinib before pemetrexed. Pemetrexed was first-line therapy in 48% (72% as platinum-based combinations). Median PFS (95% confidence interval) data were EGFR mutant (5.5 months; 1–9), KRAS mutant (7 months; 1.5–10), ALK positive (9 months; 3–12), and triple negative (4 months; 3–5). In a multivariate analysis adjusting for line of therapy, mono- versus platinum and nonplatinum combination therapy, age, sex, histology, and smoking status, the only variable associated with prolonged PFS on pemetrexed was ALK+ (hazard ratio = 0.36 [95% confidence interval: 0.17–0.73], p = 0.0051). Conclusions: In this exploratory analysis, ALK-positive patients have a significantly longer PFS on pemetrexed compared with triple-negative patients, whereas EGFR or KRAS mutant patients do not. This information should be considered when sizing randomized studies in ALK-positive patients that involve pemetrexed. Pemetrexed should also be prioritized as a cytotoxic to explore further in patients with known ALK-positive disease.

Targeted Inhibition of the Molecular Chaperone Hsp90 Overcomes ALK Inhibitor Resistance in Non–Small Cell Lung Cancer

UNLABELLED EML4-ALK gene rearrangements define a unique subset of patients with non-small cell lung carcinoma (NSCLC), and the clinical success of the anaplastic lymphoma kinase (ALK) inhibitor crizotinib in this population has become a paradigm for molecularly targeted therapy. Here, we show that the Hsp90 inhibitor ganetespib induced loss of EML4-ALK expression and depletion of multiple oncogenic signaling proteins in ALK-driven NSCLC cells, leading to greater in vitro potency, superior antitumor efficacy, and prolonged animal survival compared with results obtained with crizotinib. In addition, combinatorial benefit was seen when ganetespib was used with other targeted ALK agents both in vitro and in vivo. Importantly, ganetespib overcame multiple forms of crizotinib resistance, including secondary ALK mutations, consistent with activity seen in a patient with crizotinib-resistant NSCLC. Cancer cells driven by ALK amplification and oncogenic rearrangements of ROS1 and RET kinase genes were also sensitive to ganetespib exposure. Taken together, these results highlight the therapeutic potential of ganetespib for ALK-driven NSCLC. SIGNIFICANCE In addition to direct kinase inhibition, pharmacologic blockade of the molecular chaperone Hsp90 is emerging as a promising approach for treating tumors driven by oncogenic rearrangements of ALK. The bioactivity profi le of ganetespib presented here underscores a new therapeutic opportunity to target ALK and overcome multiple mechanisms of resistance in patients with ALK-positive NSCLC.