Mohit Butaney

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.

Activation of the PD-1 Pathway Contributes to Immune Escape in EGFR-Driven Lung Tumors

UNLABELLED The success in lung cancer therapy with programmed death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between EGF receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, CTL antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased CTLs and increased markers of T-cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T-cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape and mechanistically link treatment response to PD-1 inhibition. SIGNIFICANCE We show that autochthonous EGFR-driven lung tumors inhibit antitumor immunity by activating the PD-1/PD-L1 pathway to suppress T-cell function and increase levels of proinflammatory cytokines. These findings indicate that EGFR functions as an oncogene through non-cell-autonomous mechanisms and raise the possibility that other oncogenes may drive immune escape.

A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI), including crizotinib, are effective treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms of ALK TKI resistance identified from a crizotinib-treated non-small cell lung cancer (NSCLC) patient and in a cell line generated from the resistant tumor (DFCI076) as well as from studying a resistant version of the ALK TKI (TAE684)-sensitive H3122 cell line. The crizotinib-resistant DFCI076 cell line harbored a unique L1152R ALK secondary mutation and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076 cell line was still partially dependent on ALK for survival, it also contained concurrent coactivation of epidermal growth factor receptor (EGFR) signaling. In contrast, the TAE684-resistant (TR3) H3122 cell line did not contain an ALK secondary mutation but instead harbored coactivation of EGFR signaling. Dual inhibition of both ALK and EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We further identified a subset (3/50; 6%) of treatment naive NSCLC patients with ALK rearrangements that also had concurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated by both ALK and by a bypass signaling pathway mediated by EGFR. These mechanisms can occur independently, or in the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effective therapy for these subsets of NSCLC patients.

Noninvasive Detection of Response and Resistance in EGFR-Mutant Lung Cancer Using Quantitative Next-Generation Genotyping of Cell-Free Plasma DNA

Purpose: Tumor genotyping using cell-free plasma DNA (cfDNA) has the potential to allow noninvasive assessment of tumor biology, yet many existing assays are cumbersome and vulnerable to false-positive results. We sought to determine whether droplet digital PCR (ddPCR) of cfDNA would allow highly specific and quantitative assessment of tumor genotype. Experimental Design: ddPCR assays for EGFR, KRAS, and BRAF mutations were developed using plasma collected from patients with advanced lung cancer or melanoma of a known tumor genotype. Sensitivity and specificity were determined using cancers with nonoverlapping genotypes as positive and negative controls. Serial assessment of response and resistance was studied in patients with EGFR-mutant lung cancer on a prospective trial of erlotinib. Results: We identified a reference range for EGFR L858R and exon 19 deletions in specimens from KRAS-mutant lung cancer, allowing identification of candidate thresholds with high sensitivity and 100% specificity. Received operative characteristic curve analysis of four assays demonstrated an area under the curve in the range of 0.80 to 0.94. Sensitivity improved in specimens with optimal cfDNA concentrations. Serial plasma genotyping of EGFR-mutant lung cancer on erlotinib demonstrated pretreatment detection of EGFR mutations, complete plasma response in most cases, and increasing levels of EGFR T790M emerging before objective progression. Conclusions: Noninvasive genotyping of cfDNA using ddPCR demonstrates assay qualities that could allow effective translation into a clinical diagnostic. Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks before radiographic progression. Clin Cancer Res; 20(6); 1698–705. ©2014 AACR.

A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response

Targeted therapies have demonstrated efficacy against specific subsets of molecularly defined cancers. Although most patients with lung cancer are stratified according to a single oncogenic driver, cancers harbouring identical activating genetic mutations show large variations in their responses to the same targeted therapy. The biology underlying this heterogeneity is not well understood, and the impact of co-existing genetic mutations, especially the loss of tumour suppressors, has not been fully explored. Here we use genetically engineered mouse models to conduct a ‘co-clinical’ trial that mirrors an ongoing human clinical trial in patients with KRAS-mutant lung cancers. This trial aims to determine if the MEK inhibitor selumetinib (AZD6244) increases the efficacy of docetaxel, a standard of care chemotherapy. Our studies demonstrate that concomitant loss of either p53 (also known as Tp53) or Lkb1 (also known as Stk11), two clinically relevant tumour suppressors, markedly impaired the response of Kras-mutant cancers to docetaxel monotherapy. We observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by Kras and Kras and p53 mutations, but mice with Kras and Lkb1 mutations had primary resistance to this combination therapy. Pharmacodynamic studies, including positron-emission tomography (PET) and computed tomography (CT), identified biological markers in mice and patients that provide a rationale for the differential efficacy of these therapies in the different genotypes. These co-clinical results identify predictive genetic biomarkers that should be validated by interrogating samples from patients enrolled on the concurrent clinical trial. These studies also highlight the rationale for synchronous co-clinical trials, not only to anticipate the results of ongoing human clinical trials, but also to generate clinically relevant hypotheses that can inform the analysis and design of human studies.

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.

Reactivation of ERK Signaling Causes Resistance to EGFR Kinase Inhibitors

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors is limited by the development of drug resistance. The irreversible EGFR kinase inhibitor WZ4002 is effective against the most common mechanism of drug resistance mediated by the EGFR T790M mutation. Here, we show, in multiple complementary models, that resistance to WZ4002 develops through aberrant activation of extracellular signal-regulated kinase (ERK) signaling caused by either an amplification of mitogen-activated protein kinase 1 (MAPK1) or by downregulation of negative regulators of ERK signaling. Inhibition of MAP-ERK kinase (MEK) or ERK restores sensitivity to WZ4002 and prevents the emergence of drug resistance. We further identify MAPK1 amplification in an erlotinib-resistant EGFR-mutant non-small cell lung carcinoma patient. In addition, the WZ4002-resistant MAPK1-amplified cells also show an increase both in EGFR internalization and a decrease in sensitivity to cytotoxic chemotherapy. Our findings provide insights into mechanisms of drug resistance to EGFR kinase inhibitors and highlight rational combination therapies that should be evaluated in clinical trials.

Clinical, Pathologic, and Biologic Features Associated with BRAF Mutations in Non–Small Cell Lung Cancer

Purpose: BRAF mutations are found in a subset of non–small cell lung cancers (NSCLC). We examined the clinical characteristics and treatment outcomes of patients with NSCLC harboring BRAF mutations. Experimental Design: Using DNA sequencing, we successfully screened 883 patients with NSCLC for BRAF mutations between July 1, 2009 and July 16, 2012. Baseline characteristics and treatment outcomes were compared between patients with and without BRAF mutations. Wild-type controls consisted of patients with NSCLC without a somatic alteration in BRAF, KRAS, EGFR, and ALK. In vitro studies assessed the biologic properties of selected non-V600E BRAF mutations identified from patients with NSCLC. Results: Of 883 tumors screened, 36 (4%) harbored BRAF mutations (V600E, 18; non-V600E, 18) and 257 were wild-type for BRAF, EGFR, KRAS, and ALK negative. Twenty-nine of 36 patients with BRAF mutations were smokers. There were no distinguishing clinical features between BRAF-mutant and wild-type patients. Patients with advanced NSCLC with BRAF mutations and wild-type tumors showed similar response rates and progression-free survival (PFS) to platinum-based combination chemotherapy and no difference in overall survival. Within the BRAF cohort, patients with V600E-mutated tumors had a shorter PFS to platinum-based chemotherapy compared with those with non-V600E mutations, although this did not reach statistical significance (4.1 vs. 8.9 months; P = 0.297). We identified five BRAF mutations not previously reported in NSCLC; two of five were associated with increased BRAF kinase activity. Conclusions: BRAF mutations occur in 4% of NSCLCs and half are non-V600E. Prospective trials are ongoing to validate BRAF as a therapeutic target in NSCLC. Clin Cancer Res; 19(16); 4532–40. ©2013 AACR.

The Impact of Initial Gefitinib or Erlotinib versus Chemotherapy on Central Nervous System Progression in Advanced Non–Small Cell Lung Cancer with EGFR Mutations

Purpose: This retrospective study was undertaken to investigate the impact of initial gefitinib or erlotinib (EGFR tyrosine kinase inhibitor, EGFR-TKI) versus chemotherapy on the risk of central nervous system (CNS) progression in advanced non–small cell lung cancer (NSCLC) with EGFR mutations. Experimental Design: Patients with stage IV or relapsed NSCLC with a sensitizing EGFR mutation initially treated with gefitinib, erlotinib, or chemotherapy were identified. The cumulative risk of CNS progression was calculated using death as a competing risk. Results: One hundred and fifty-five patients were eligible (EGFR-TKI: 101, chemotherapy: 54). Twenty-four patients (24%) in the EGFR-TKI group and 12 patients (22%) in the chemotherapy group had brain metastases at the time of diagnosis of advanced NSCLC (P = 1.000); 32 of the 36 received CNS therapy before initiating systemic treatment. Thirty-three patients (33%) in the EGFR-TKI group and 26 patients (48%) in the chemotherapy group developed CNS progression after a median follow-up of 25 months. The 6-, 12-, and 24-month cumulative risk of CNS progression was 1%, 6%, and 21% in the EGFR-TKI group compared with corresponding rates of 7%, 19%, and 32% in the chemotherapy group (P = 0.026). The HR of CNS progression for upfront EGFR-TKI versus chemotherapy was 0.56 [95% confidence interval (CI), 0.34–0.94]. Conclusions: Our data show lower rates of CNS progression in EGFR-mutant advanced NSCLC patients initially treated with an EGFR-TKI compared with upfront chemotherapy. If validated, our results suggest that gefitinib and erlotinib may have a role in the chemoprevention of CNS metastases from NSCLC. Clin Cancer Res; 18(16); 4406–14. ©2012 AACR.

Amplification of CRKL Induces Transformation and Epidermal Growth Factor Receptor Inhibitor Resistance in Human Non–Small Cell Lung Cancers

UNLABELLED We previously identified a region of recurrent amplification on chromosome 22q11.21 in a subset of primary lung adenocarcinomas. Here we show that CRKL, encoding for an adaptor protein, is amplified and overexpressed in non-small cell lung cancer (NSCLC) cells that harbor 22q11.21 amplifications. Overexpression of CRKL in immortalized human airway epithelial cells promoted anchorage-independent growth and tumorigenicity. Oncogenic CRKL activates the SOS1-RAS-RAF-ERK and SRC-C3G-RAP1 pathways. Suppression of CRKL in NSCLC cells that harbor CRKL amplifications induced cell death. Overexpression of CRKL in epidermal growth factor receptor (EGFR)-mutant cells induces resistance to gefitinib by activating extracellular signal-regulated kinase and AKT signaling. We identified CRKL amplification in an EGFR inhibitor-treated lung adenocarcinoma that was not present before treatment. These observations demonstrate that CRKL overexpression induces cell transformation, credential CRKL as a therapeutic target for a subset of NSCLC that harbor CRKL amplifications, and implicate CRKL as an additional mechanism of resistance to EGFR-directed therapy. SIGNIFICANCE These studies credential CRKL as an oncogene in a subset of NSCLC. Overexpression of CRKL induces cell transformation and resistance to epidermal growth factor receptor inhibitor treatment and suggest that therapeutic interventions targeting CRKL may confer a clinical benefit in a defined subset of NSCLCs.