Lorin A. Ferris

ROS1 Fusions Rarely Overlap with Other Oncogenic Drivers in Non–Small Cell Lung Cancer

Introduction: Chromosomal rearrangements involving the gene ROS1 define a distinct molecular subset of NSCLCs with sensitivity to ROS1 inhibitors. Recent reports have suggested a significant overlap between ROS1 fusions and other oncogenic driver alterations, including mutations in EGFR and KRAS. Methods: We identified patients at our institution with ROS1‐rearranged NSCLC who had undergone testing for genetic alterations in additional oncogenes, including EGFR, KRAS, and anaplastic lymphoma receptor tyrosine kinase gene (ALK). Clinicopathologic features and genetic testing results were reviewed. We also examined a separate database of ROS1‐rearranged NSCLCs identified through the commercial FoundationOne assay (Foundation Medicine, Cambridge, MA). Results: Among 62 patients with ROS1‐rearranged NSCLC evaluated at our institution, none harbored concurrent ALK fusions (0%) or EGFR activating mutations (0%). KRAS mutations were detected in two cases (3.2%), one of which harbored a concurrent noncanonical KRAS I24N mutation of unknown biological significance. In a separate ROS1 fluorescence in situ hybridization–positive case, targeted sequencing failed to confirm a ROS1 fusion but instead identified a KRAS G13D mutation. No concurrent mutations in B‐Raf proto‐oncogene, serine/threonine kinase gene (BRAF), erb‐b2 receptor tyrosine kinase 2 gene (ERBB2), phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha gene (PIK3CA), AKT/serine threonine kinase 1 gene (AKT1), or mitogen‐activated protein kinase kinase 1 gene (MAP2K1) were detected. Analysis of an independent data set of 166 ROS1‐rearranged NSCLCs identified by FoundationOne demonstrated rare cases with co‐occurring driver mutations in EGFR (one of 166) and KRAS (three of 166) and no cases with co‐occurring ROS1 and ALK rearrangements. Conclusions: ROS1 rearrangements rarely overlap with alterations in EGFR, KRAS, ALK, or other targetable oncogenes in NSCLC.

Patterns of Metastatic Spread and Mechanisms of Resistance to Crizotinib in ROS1-Positive Non–Small-Cell Lung Cancer

PURPOSE The ROS1 tyrosine kinase is activated through ROS1 gene rearrangements in 1-2% of non-small cell lung cancer (NSCLC), conferring sensitivity to treatment with the ALK/ROS1/MET inhibitor crizotinib. Currently, insights into patterns of metastatic spread and mechanisms of crizotinib resistance among ROS1-positive patients are limited. PATIENTS AND METHODS We reviewed clinical and radiographic imaging data of patients with ROS1- and ALK-positive NSCLC in order to compare patterns of metastatic spread at initial metastatic diagnosis. To determine molecular mechanisms of crizotinib resistance, we also analyzed repeat biopsies from a cohort of ROS1-positive patients progressing on crizotinib. RESULTS We identified 39 and 196 patients with advanced ROS1- and ALK-positive NSCLC, respectively. ROS1-positive patients had significantly lower rates of extrathoracic metastases (ROS1 59.0%, ALK 83.2%, P=0.002), including lower rates of brain metastases (ROS1 19.4%, ALK 39.1%; P = 0.033), at initial metastatic diagnosis. Despite similar overall survival between ALK- and ROS1-positive patients treated with crizotinib (median 3.0 versus 2.5 years, respectively; P=0.786), ROS1-positive patients also had a significantly lower cumulative incidence of brain metastases (34% vs. 73% at 5 years; P<0.0001). Additionally, we identified 16 patients who underwent a total of 17 repeat biopsies following progression on crizotinib. ROS1 resistance mutations were identified in 53% of specimens, including 9/14 (64%) non-brain metastasis specimens. ROS1 mutations included: G2032R (41%), D2033N (6%), and S1986F (6%). CONCLUSIONS Compared to ALK rearrangements, ROS1 rearrangements are associated with lower rates of extrathoracic metastases, including fewer brain metastases, at initial metastatic diagnosis. ROS1 resistance mutations, particularly G2032R, appear to be the predominant mechanism of resistance to crizotinib, underscoring the need to develop novel ROS1 inhibitors with activity against these resistant mutants.

Sequential ALK Inhibitors Can Select for Lorlatinib-Resistant Compound ALK Mutations in ALK-Positive Lung Cancer.

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of ALK mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4-ALK. Under comparable conditions, N-ethyl-N-nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single ALK mutations. In similar screens with EML4-ALK containing single ALK resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound ALK mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound ALK mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of ALK mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound ALK mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies.Significance: Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound ALK mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance. Cancer Discov; 8(6); 714-29. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.

Tracking the Evolution of Resistance to ALK Tyrosine Kinase Inhibitors Through Longitudinal Analysis of Circulating Tumor DNA

Purpose ALK rearrangements predict for sensitivity to ALK tyrosine kinase inhibitors (TKIs). However, responses to ALK TKIs are generally short-lived. Serial molecular analysis is an informative strategy for identifying genetic mediators of resistance. Although multiple studies support the clinical benefits of repeat tissue sampling, the clinical utility of longitudinal circulating tumor DNA analysis has not been established in ALK-positive lung cancer. Methods Using a 566-gene hybrid-capture next-generation sequencing (NGS) assay, we performed longitudinal analysis of plasma specimens from 22 ALK-positive patients with acquired resistance to ALK TKIs to track the evolution of resistance during treatment. To determine tissue-plasma concordance, we compared plasma findings to results of repeat biopsies. Results At progression, we detected an ALK fusion in plasma from 19 (86%) of 22 patients, and identified ALK resistance mutations in plasma specimens from 11 (50%) patients. There was 100% agreement between tissue- and plasma-detected ALK fusions. Among 16 cases where contemporaneous plasma and tissue specimens were available, we observed 100% concordance between ALK mutation calls. ALK mutations emerged and disappeared during treatment with sequential ALK TKIs, suggesting that plasma mutation profiles were dependent on the specific TKI administered. ALK G1202R, the most frequent plasma mutation detected after progression on a second-generation TKI, was consistently suppressed during treatment with lorlatinib. Conclusions Plasma genotyping by NGS is an effective method for detecting ALK fusions and ALK mutations in patients progressing on ALK TKIs. The correlation between plasma ALK mutations and response to distinct ALK TKIs highlights the potential for plasma analysis to guide selection of ALK-directed therapies.

Brigatinib in Patients With Alectinib-Refractory ALK-Positive NSCLC

Introduction: The second‐generation anaplastic lymphoma kinase (ALK) inhibitor alectinib recently showed superior efficacy compared to the first‐generation ALK inhibitor crizotinib in advanced ALK‐rearranged NSCLC, establishing alectinib as the new standard first‐line therapy. Brigatinib, another second‐generation ALK inhibitor, has shown substantial activity in patients with crizotinib‐refractory ALK‐positive NSCLC; however, its activity in the alectinib‐refractory setting is unknown. Methods: A multicenter, retrospective study was performed at three institutions. Patients were eligible if they had advanced, alectinib‐refractory ALK‐positive NSCLC and were treated with brigatinib. Medical records were reviewed to determine clinical outcomes. Results: Twenty‐two patients were eligible for this study. Confirmed objective responses to brigatinib were observed in 3 of 18 patients (17%) with measurable disease. Nine patients (50%) had stable disease on brigatinib. The median progression‐free survival was 4.4 months (95% confidence interval [CI]: 1.8–5.6 months) with a median duration of treatment of 5.7 months (95% CI: 1.8–6.2 months). Among 9 patients in this study who underwent post‐alectinib/pre‐brigatinib biopsies, 5 had an ALK I1171X or V1180L resistance mutation; of these, 1 had a confirmed partial response and 3 had stable disease on brigatinib. One patient had an ALK G1202R mutation in a post‐alectinib/pre‐brigatinib biopsy, and had progressive disease as the best overall response to brigatinib. Conclusions: Brigatinib has limited clinical activity in alectinib‐refractory ALK‐positive NSCLC. Additional studies are needed to establish biomarkers of response to brigatinib and to identify effective therapeutic options for alectinib‐resistant ALK‐positive NSCLC patients.

Brief Report: Increased Hepatotoxicity Associated with Sequential Immune Checkpoint Inhibitor and Crizotinib Therapy in Patients with Non-Small-Cell Lung Cancer

Introduction: Immune checkpoint inhibitors (ICIs) are standard therapies in advanced NSCLC. Although genotype‐directed tyrosine kinase inhibitors represent the standard of care for subsets of oncogene‐driven NSCLC, patients may receive ICIs during their disease course. The impact of sequential ICI and tyrosine kinase inhibitor therapy on the risk of hepatotoxicity has not been described. Methods: Patients with advanced ALK receptor tyrosine kinase (ALK)‐driven, ROS1‐driven, or MET proto‐oncogene, receptor tyrosine kinase (MET)‐driven NSCLC treated with crizotinib, with or without preceding ICI therapy, were identified. The cumulative incidences of crizotinib‐associated grade 3 or higher increases in transaminase level (per the Common Terminology Criteria for Adverse Events, version 4.0) were compared. Results: We identified 453 patients who had NSCLC with an oncogenic alteration in ALK receptor tyrosine kinase gene (ALK), ROS1, or MET proto‐oncogene, receptor tyrosine kinase gene (MET) and were treated with crizotinib (11 with and 442 without prior ICI therapy). Among the 11 patients treated with an ICI followed by crizotinib, five (cumulative incidence 45.5% [95% confidence interval (CI): 14.9–72.2]) experienced development of a grade 3 or 4 increase in alanine transaminase level and four (cumulative incidence 36.4% [95% CI: 10.0–64.2]) experienced development of a grade 3 or 4 increase in aspartate transaminase level. In comparison, among the 442 patients who received crizotinib only, a grade 3 or 4 increase in alanine transaminase level occurred in 34 patients (cumulative incidence 8.1% [95% CI: 5.7–11.0, p < 0.0001]) and a grade 3 or 4 increase in aspartate transaminase level occurred in 14 (cumulative incidence 3.4% [95% CI: 1.9–5.5, p < 0.0001]). There were no grade 5 transaminitis events. All cases of hepatotoxicity after sequential ICI and crizotinib use were reversible and nonfatal, and no case met the Hys law criteria. Conclusions: Sequential ICI and crizotinib treatment is associated with a significantly increased risk of hepatotoxicity. Careful consideration and monitoring for hepatotoxicity may be warranted in patients treated with crizotinib after ICI therapy.