Comprehensive Genomic Profiling Identifies a Subset of Crizotinib-Responsive ALK-Rearranged Non-Small Cell Lung Cancer Not Detected by Fluorescence In Situ Hybridization

Abstract

Introduction: For patients with non-small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care.

Materials and methods: Hybrid-capture-based CGP using next-generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements.

Results: A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4-ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK-ALK, PPM1B-ALK, and PRKAR1A-ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months.

Conclusion: Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH-positive cases.

Implications for practice: Comprehensive genomic profiling (CGP) that includes hybrid capture and specific baiting of intron 19 of ALK is a highly sensitive, alternative method for identification of drug-sensitive ALK fusions in patients with non-small cell lung cancer (NSCLC) who had previously tested negative using standard ALK fluorescence in situ hybridization (FISH) diagnostic assays. Given the proven benefit of treatment with crizotinib and second-generation ALK inhibitors in patients with ALK fusions, CGP should be considered in patients with NSCLC, including those who have tested negative for other alterations, including negative results using ALK FISH testing.

Keywords: ALK; Crizotinib; Fluorescence in situ hybridization; Fusion; Genomic profiling.

Figure 1.

Analytical validation of fusion gene detection by hybrid-capture-based comprehensive genomic profiling (CGP). (A): Five cell lines harboring characterized fusions were mixed into 22 variably sized pools such that each fusion was represented at 20%, 25%, 33%, 50%, and 100% cellular fraction at least once, for a total of 32 gene-fusion test cases. (B): Repeated testing of clinical formalin-fixed, paraffin-embedded (FFPE) specimens identified to harbor fusions demonstrated detection reproducibility within and across assay batches. (C): Summary of concordance data between fluorescence in situ hybridization and nonhybrid-capture-based CGP for 45 clinical FFPE specimens validated for the presence or absence of ALK rearrangements. (D): Breakpoints in ALK rearrangements detected by CGP.

Figure 2.

EML4-ALK variant fusions and non-EML4-ALK fusions detected in this series. (A): Of 41 cases harboring EML4-ALK fusions, there was a breakpoint in 17 in intron 13 (variant 1), 16 cases in intron 6 (variant 3), 4 cases in intron 20 (variant 2), 2 cases in intron 2 (rare variant 5a/b), and 2 cases in intron 18 (a heretofore unnamed variant). (B): The 6 cases harboring non-EML4-ALK fusions all had breakpoints in intron 19 of ALK, but breakpoints in the partners were as follows: KIF5-ALK (intron 24), CLTC-ALK (intron 31), TFG-ALK (intron 6), PRKAR1A-ALK (intron 5), PPM1B-ALK (intron 1), and EIF2AK3-ALK (intron 2).

Figure 3.

CONSORT diagram for this study. Abbreviations: CGP, comprehensive genomic profiling; FISH, fluorescence in situ hybridization.

Figure 4.

A patient harboring the novel FISH-negative EIF2AK4-ALK fusion responded to crizotinib (case 1). (A): EIF2AK4-ALK fusion results from a complex rearrangement leading to insertion of a 92-kb region in 2p23 encoding part of ALK and CLP4 into intron 2 of EIF2AK3 located in 2p11.2. Probe regions are not shown to scale. (B): Coronal image from a CT scan of the chest and upper abdomen before treatment with crizotinib. (C): Coronal image from a CT scan of the chest and upper abdomen 6 weeks after starting treatment with crizotinib, with significant interval decrease in pleural-based disease. Abbreviation: FISH, fluorescence in situ hybridization.

Figure 5.

Response to crizotinib in a patient harboring fluorescence in situ hybridization-negative EML4-ALK (case 2). Axial and coronal images from CT scans (A) 2 weeks before crizotinib was initiated and (B) after 8 weeks of crizotinib therapy.

Figure 6.

Oncogenic PRKAR1A-ALK fusion is sensitive to crizotinib in vitro and in vivo (case 3). (A): NIH3T3 cells stably expressing either empty vector or vector encoding V5-PRKAR1A-ALK fusion were plated for colony formation and stained to show colony growth. (B): NIH3T3 cells stably expressing either empty vector of V5 tagged-PRKAR1A-ALK were treated with crizotinib at doses shown and then processed for Western blotting using antibodies to V5, phosphor-ERK (pERK), or total ERK. (C, D): NIH3T3 cells transformed with V5-PRKAR1A-ALK fusion were treated with increasing concentrations of crizotinib; growth was assayed using colony formation. Representative plates stained for colonies are shown in (C). Quantization of colonies is plotted in (D). (E): Axial image from a CT scan of the chest and upper abdomen before treatment with crizotinib. (F): Axial image from a CT scan of the chest and upper abdomen 3 months after starting crizotinib, with resolution of mediastinal adenopathy.