Abstract
Break-apart ALK FISH probe is the FDA approved approach for detection of ALK rearrangements in lung carcinoma patients who may benefit from ALK kinase inhibitors. The FISH assay can be technically challenging and difficult to interpret. ALK immunohistochemistry and next generation sequencing have been proposed as alternative approaches. In this study, we compared various ALK –FISH patterns to next –generation sequencing (NGS) for gene fusion detection, ALK immunohistochemistry (IHC) and tumor responses to crizotinib. 72 (4%) of 2116 lung adenocarcinoma were positive by ALK- FISH. Of 28 ALK-FISH positive cases selected for the study, FISH patterns included 15 (54%) cases with split signal, 10 (36%) with single orange signal and 3 (10%) with “mixed pattern”. 12 (80%) cases with split signal and 4 (40%) cases with single orange signal were positive by NGS and IHC, while mixed cases were all negative. Mutation analysis of discordant cases revealed multiple mutations including oncogenic mutations in EGFR, KRAS, BRAF and ATM genes. All discordant cases in groups with split and mixed signal showed a lower number of cells with rearrangement (mean 28.5%; range 20.5-36.9%). No statistically significant association between response to crizotinib and FISH patterns was observed (p=0.73). In contrast, NGS fusion positive cases were associated with more responses to crizotinib than NGS negative cases (p= 0.016). Our study suggests that ALK FISH alone may not be the most reliable assay for detection of ALK gene rearrangements, and probably should be used in parallel with ALK IHC and NGS for detection of gene fusions and mutations.
Highlights
Rearrangements of the ALK gene occur in up to 5% of non-small cell lung carcinoma (NSCLC), and are associated with an objective response rate of about 65% in patients treated with the ALK inhibitor crizotinib [1,2,3]
The assay is considered to be positive for ALK rearrangement if at least 15% of tumor cells show rearrangement
We evaluated the detection of ALK gene fusions by a targeted next –generation sequencing (NGS) approach and compared the results with various ALK-FISH patterns, ALK IHC and response to crizotinib
Summary
Rearrangements of the ALK gene occur in up to 5% of non-small cell lung carcinoma (NSCLC), and are associated with an objective response rate of about 65% in patients treated with the ALK inhibitor crizotinib [1,2,3]. Many technical modifications including antigen retrieval and the development of new antibodies have been reported to increase the overall performance www.impactjournals.com/oncotarget of immunohistochemistry in the detection of ALK rearrangement. Strong staining seems to be specific for ALK rearrangement and ALK IHC was suggested as a cost effective screening method [10, 21]. Studies showed that positive ALK protein expression correlates with tumor response to ALK inhibitors [22]. Preliminary data showed that the patients with ALKATI may benefit from ALK inhibitors and authors suggested immunohistochemistry as a screening method [23]
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