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 that do respond. This study aimed to define molecular mechanisms of resistance to crizotinib in ALK+ non-small cell lung cancer (NSCLC) patients.

Experimental Design

We analyzed tissue obtained from 14 ALK+ NSCLC patients demonstrating evidence of radiologic progression while on crizotinib in order 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 kinase 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 demonstrated outgrowth of 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 demonstrated the emergence of an ALK gene fusion negative tumor compared to 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.

The treatment of advanced non–small cell lung cancer (NSCLC) increasingly involves the use of molecularly targeted therapy with activity against either the tumor directly, or indirectly, through activity against host-derived mechanisms of tumor support such as angiogenesis. The most well studied signaling pathway associated with angiogenesis is the vascular endothelial growth factor (VEGF) pathway, and the only antiangiogenic agent currently approved for the treatment of NSCLC is bevacizumab, an antibody targeted against VEGF. More recently, preclinical data supporting the role of fibroblast growth factor receptor (FGFR) and platelet-derived growth factor receptor (PDGFR) signaling in angiogenesis have been reported. The platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) pathways may also stimulate tumor growth directly through activation of downstream mitogenic signaling cascades. In addition, 1 or both of these pathways have been associated with resistance to agents targeting the epidermal growth factor receptor (EGFR) and VEGF. A number of agents that target FGF and/or PDGF signaling are now in development for the treatment of NSCLC. This review will summarize the potential molecular roles of PDGFR and FGFR in tumor growth and angiogenesis, as well as discuss the current clinical status of PDGFR and FGFR inhibitors in clinical development.

Purpose

Erlotinib prolongs survival in patients with advanced non–small-cell lung cancer (NSCLC). We report the results of a randomized, phase II study of erlotinib alone or intercalated with chemotherapy (CT + erlotinib) in chemotherapy-naïve patients with advanced NSCLC who were positive for epidermal growth factor receptor (EGFR) protein expression and/or with high EGFR gene copy number.

Patients and Methods

A total of 143 patients were randomly assigned to either erlotinib 150 mg daily orally until disease progression (PD) occurred or to chemotherapy with paclitaxel 200 mg/m2 intravenously (IV) and carboplatin dosed by creatinine clearance (AUC 6) IV on day 1 intercalated with erlotinib 150 mg orally on days 2 through 15 every 3 weeks for four cycles followed by erlotinib 150 mg orally until PD occurred (CT + erlotinib). The primary end point was 6-month progression-free survival (PFS); secondary end points included response rate, PFS, and survival. EGFR, KRAS mutation, EGFR fluorescent in situ hybridization and immunohistochemistry, and E-cadherin and vimentin protein levels were also assessed.

Results

Six-month PFS rates were 26% and 31% for the two arms (CT + erlotinib and erlotinib alone, respectively). Both were less than the historical control of 45% (P = .001 and P = .011, respectively). Median PFS times were 4.57 and 2.69 months, respectively. Patients with tumors harboring EGFR activating mutations fared better on erlotinib alone (median PFS, 18.2 months v 4.9 months for CT + erlotinib).

Conclusion

The feasibility of a multicenter biomarker-driven study was demonstrated, but neither treatment arms exceeded historical controls. This study does not support combined chemotherapy and erlotinib in first-line treatment of EGFR-selected advanced NSCLC, and the patients with tumors harboring EGFR mutations had a better outcome on erlotinib alone.

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.

Purpose

PF299804 is a potent, orally available, irreversible inhibitor of tyrosine kinase human epidermal growth factor receptors (HER) 1 (EGFR), HER2, and HER4. This first-in-human study investigated the safety, tolerability, pharmacokinetics, and pharmacodynamics of PF299804 in patients with advanced solid malignancies.

Experimental Design

PF299804 was administered once daily continuously (schedule A), and intermittently (schedule B). Dose escalation proceeded until intolerable toxicities occurred. Skin biopsies were taken pre-dose and after 14 days of treatment, to establish a pharmacokinetic/pharmacodynamic relationship. Tumor response was measured once every 2 cycles. Efficacy was correlated with tumor genotypes in non-small cell lung cancer (NSCLC) patients.

Results

121 patients were included (111 in schedule A, 10 in schedule B). The maximum tolerated dose (MTD) was 45 mg/day. Dose-limiting toxicities included stomatitis and skin toxicities. Most adverse events were mild and comprised skin toxicities, fatigue, and gastrointestinal side-effects including diarrhea, nausea, and vomiting. Pharmacokinetic analyses revealed dose-dependent increases in PF299804 exposure associated with target inhibition in skin biopsy samples. Fifty-seven patients with non-small cell lung cancer (NSCLC) were treated in this study. Four patients, all previously treated with gefitinib or erlotinib (2 with exon 19 deletions, 1 with exon 20 insertion, 1 mutational status unknown), had a partial response to PF299804.

Conclusions

The MTD of PF299804 is 45 mg/day. Both continuous and intermittent treatment schedules were well tolerated, and encouraging signs of antitumor activity were observed in gefitinib/erlotinib treated NSCLC patients.

BACKGROUND

Oncogenic fusion genes consisting of EML4 and anaplastic lymphoma kinase (ALK) are present in a subgroup of non–small-cell lung cancers, representing 2 to 7% of such tumors. We explored the therapeutic efficacy of inhibiting ALK in such tumors in an early-phase clinical trial of crizotinib (PF-02341066), an orally available small-molecule inhibitor of the ALK tyrosine kinase.

METHODS

After screening tumor samples from approximately 1500 patients with non–small-cell lung cancer for the presence of ALK rearrangements, we identified 82 patients with advanced ALK-positive disease who were eligible for the clinical trial. Most of the patients had received previous treatment. These patients were enrolled in an expanded cohort study instituted after phase 1 dose escalation had established a recommended crizotinib dose of 250 mg twice daily in 28-day cycles. Patients were assessed for adverse events and response to therapy.

RESULTS

Patients with ALK rearrangements tended to be younger than those without the rearrangements, and most of the patients had little or no exposure to tobacco and had adenocarcinomas. At a mean treatment duration of 6.4 months, the overall response rate was 57% (47 of 82 patients, with 46 confirmed partial responses and 1 confirmed complete response); 27 patients (33%) had stable disease. A total of 63 of 82 patients (77%) were continuing to receive crizotinib at the time of data cutoff, and the estimated probability of 6-month progression-free survival was 72%, with no median for the study reached. The drug resulted in grade 1 or 2 (mild) gastrointestinal side effects.

CONCLUSIONS

The inhibition of ALK in lung tumors with the ALK rearrangement resulted in tumor shrinkage or stable disease in most patients.

Purpose

To evaluate the safety, maximum-tolerated dose (MTD), pharmacokinetics (PKs), pharmacodynamics, and preliminary anticancer activity of ramucirumab (IMC-1121B), a fully human immunoglobulin G1 monoclonal antibody targeting the vascular endothelial growth factor receptor (VEGFR)-2.

Patients and Methods

Patients with advanced solid malignancies were treated once weekly with escalating doses of ramucirumab. Blood was sampled for PK studies throughout treatment. The effects of ramucirumab on circulating vascular endothelial growth factor-A (VEGF-A), soluble VEGFR-1 and VEGFR-2, tumor perfusion, and vascularity using dynamic contrast-enhanced magnetic resonance imaging were assessed.

Results

Thirty-seven patients were treated with 2 to 16 mg/kg of ramucirumab. After one patient each developed dose-limiting hypertension and deep venous thrombosis at 16 mg/kg, the next lower dose (13 mg/kg) was considered the MTD. Nausea, vomiting, headache, fatigue, and proteinuria were also noted. Four (15%) of 27 patients with measurable disease had a partial response (PR), and 11 (30%) of 37 patients had either a PR or stable disease lasting at least 6 months. PKs were characterized by dose-dependent elimination and nonlinear exposure consistent with saturable clearance. Mean trough concentrations exceeded biologically relevant target levels throughout treatment at all dose levels. Serum VEGF-A increased 1.5 to 3.5 times above pretreatment values and remained in this range throughout treatment at all dose levels. Tumor perfusion and vascularity decreased in 69% of evaluable patients.

Conclusion

Objective antitumor activity and antiangiogenic effects were observed over a wide range of dose levels, suggesting that ramucirumab may have a favorable therapeutic index in treating malignancies amenable to VEGFR-2 inhibition.

Caspase-9 is involved in the intrinsic apoptotic pathway and suggested to play a role as a tumor suppressor. Little is known about the mechanisms governing caspase-9 expression, but post-transcriptional pre-mRNA processing generates 2 splice variants from the caspase-9 gene, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b. Here we demonstrate that the ratio of caspase-9 splice variants is dysregulated in non–small cell lung cancer (NSCLC) tumors. Mechanistic analysis revealed that an exonic splicing silencer (ESS) regulated caspase-9 pre-mRNA processing in NSCLC cells. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacted with this ESS, and downregulation of hnRNP L expression induced an increase in the caspase-9a/9b ratio. Although expression of hnRNP L lowered the caspase-9a/9b ratio in NSCLC cells, expression of hnRNP L produced the opposite effect in non-transformed cells, suggesting a post-translational modification specific for NSCLC cells. Indeed, Ser52 was identified as a critical modification regulating the caspase-9a/9b ratio. Importantly, in a mouse xenograft model, downregulation of hnRNP L in NSCLC cells induced a complete loss of tumorigenic capacity that was due to the changes in caspase-9 pre-mRNA processing. This study therefore identifies a cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase-9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.

Introduction

This phase I/II study evaluated the safety and anti-tumor effect of the combination of erlotinib with cixutumumab, a recombinant fully humanized anti-insulin-like growth factor-1 receptor IgG1 monoclonal antibody, in advanced non-small cell lung cancer (NSCLC).

Methods

Patients with advanced NSCLC were treated in an initial safety-lead and drop-down cohorts using erlotinib 150 mg/d with cixutumumab 6 or 5 mg/kg on days 1, 8, 15, and 22 in 28-day cycles (cohorts 1 and 2). Emerging pharmacokinetic data led to an additional cohort (3 + 3 design) with cixutumumab at 15 mg/kg on day 1 in 21-day cycles (cohort 3).

Results

Eighteen patients entered the study (6 at 6 mg/kg, 8 at 5 mg/kg, and 4 at 15 mg/kg), with median age of 65 years. Four of six patients at 6 mg/kg experienced dose-limiting toxicities (DLTs), whereas at 5 mg/kg, one of eight patients experienced DLT but three of eight patients still required a dose delay during cycle 1. At 15 mg/kg every 21 days, two of four patients experienced DLTs. In all cohorts, DLTs were either G3 rash or fatigue. Five patients had stable disease as best response and 14 patients had progressive disease. The median progression-free survival was 39 days (range 21–432+ days). Biomarkers analyses showed a trend toward better progression-free survival seen with higher free baseline insulin-like growth factor-1 levels as seen with other insulin-like growth factor-1R inhibitors.

Conclusions

The combinations of cixutumumab at 6 mg/kg every 7 days and 15 mg/kg every 21 days and full-dose erlotinib are not tolerable in unselected patients with NSCLC, as measured by DLT. Cixutumumab at 5 mg/kg every 7 days was tolerable per DLT, but dose delays were common. Efficacy in unselected patients with NSCLC seems to be low.

Summary

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.