ALK FISH patterns and the detection of ALK fusions by next generation sequencing in lung adenocarcinoma

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.

A comparison of ALK gene rearrangement and ALK protein expression in primary lung carcinoma and matched metastasis

“…Alternatively, this case could be an example of the very rare occurrence of concurrent EGFR mutation and ALK translocation . Our group reported recently that some ALK FISH patterns, such as a single orange (5′ deletion) or ‘mixed patterns’ (a combination of single orange and split signal), are associated with higher discordance with ALK IHC and next‐generation sequencing for gene fusions, and that these cases are associated with a less favourable response to crizotinib …”

“…As mentioned earlier, this case probably represents a false positive ALK FISH result. It has been shown recently that NGS studies might be of help in resolving discordant ALK status cases …”

KRAS mutation is predictive of outcome in patients with pulmonary sarcomatoid carcinoma

“…DNA sequencing was performed using the Ion AmpliSeq Cancer Panel (Ion Torrent; Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), as reported previously . Briefly, 10 ng of DNA was amplified by polymerase chain reaction (PCR) using the AmpliSeq Cancer Panel Primers pool and Ion AmpliSeq Master Mix version 2.0.…”

ALK and ROS1 testing on lung cancer cytologic samples: Perspectives

“…In a study by Yamamoto and colleagues, RNA was extracted from cytologic specimens derived from fluid aspiration, namely, cerebrospinal fluid, pleural effusion, abdominal fluid, and pericardial effusion; and ALK fusion genes were detected in 2.4% of 365 patients . Various groups have used NGS to evaluate histologic samples of lung adenocarcinoma, mainly in cases of discordance between FISH and IHC results . For example, Jang et al analyzed 5 discordant cases using the HiSeq 2000 NGS platform (Illumina); 1 case was false‐positive on D5F3 IHC, and 3 of the 4 remaining cases were false‐positive on ALK FISH .…”

“…Pekar‐Zlotin and coworkers analyzed 6 discordant cases using the Illumina platform (HiSeq 2000) and more often confirmed the IHC results (5 cases) than the FISH results (1 case) . By using the Ion Torrent NGS technology (Thermo Fisher Scientific), Dacic et al demonstrated that the ALK 5′ deletion (single red) signal was more frequently false‐positive than were the various FISH patterns . Notably, NGS can be used when crush and thermal artifacts impair the performance of FISH or IHC.…”