For the time being fluorescent in situ hybridization FISH re

For the time being, fluorescent in situ hybridization (FISH) remains the ‘gold standard’ for ALK rearrangements diagnosis but immunohistochemistry has become a widely used pre-screening tool and the FDA recently approved the Ventana ALK (D5F3) CDx Assay (Ventana Medical Systems, Tucson, AZ) as a companion diagnostic assay for crizotinib, alectinib and ceritinib. However the diagnosis of ALK rearrangements remains challenging [[18], [19], [20], [21], [22], [23]]. Moreover, neither IHC nor FISH allows the characterization of the EML4-ALK transcript variants produced by the gene fusion or the identification of other ALK fusion partners. Direct detection of ALK rearrangements using RT-PCR or next-generation sequencing (NGS) could thus not only allow the identification of the transcript variants, but also help elucidate potential differential responses to crizotinib in IHC/FISH discordant cases, and ultimately to better select the patients able to respond to ALK inhibitors. Indeed, recent clinical data [24] suggest that ubiquitin e3 ligase IHC-positive/FISH-negative tumors may respond to crizotinib in almost 100% of the cases, whereas responses to crizotinib could be observed in less than 50% of the IHC negative/FISH positive tumors. Moreover, following an in-vitro study by Heuckman et al. [25], four recent retrospective studies [[26], [27], [28], [29]] using RT-PCR for the detection of EML4-ALK variants have suggested that the variability in the fusion variants generated by ALK rearrangements could potentially influence the response to ALK inhibitors. However, conflicting results emerge from these studies and the clinical impact of non-EML4-ALK variants was not evaluated. In the past few years, NGS technologies have allowed vast improvements in genomic research, as well as in clinical molecular diagnosis. These technologies can be used for cancer genotyping, and in-house or commercial panels have also been developed for the detection of clinically targetable fusion transcripts [[30], [31], [32], [33], [34], [35], [36], [37], [38]]. The aim of the present study was to assess the yields of an amplicon-based parallel sequencing (RNA-seq) assay, more comprehensive than RT-PCR for fusion variants identification, for ALK fusion transcripts detection in comparison with IHC and FISH in formalin-fixed paraffin embedded (FFPE) specimens from a cohort of ALK-positive NSCLCs and to evaluate the impact of the ALK variant on crizotinib efficacy.
Material and methods
Results
Discussion In this study, we assessed the use in a clinical setting of an amplicon-based RNA parallel sequencing assay to detect ALK fusion transcripts in FFPE samples from a selected population of ALK-positive and ALK-negative cases. We found that RNA sequencing yielded a sensitivity of 80% and a specificity of 100% versus IHC and FISH combined and was a promising rescue technique in equivocal and/or borderline-positive IHC/FISH cases. Moreover, correlations between RNA-sequencing results and crizotinib efficacy suggest the potential of NGS as a diagnostic tool for the detection of ALK-rearranged NSCLCs. Fifty-seven out of seventy-six samples (75%) of our cohort were of sufficient quantity/quality to be analyzed by RNA-seq. Comparable results have been obtained in other retrospective studies [[32], [44]] in which 34% and 30% of samples could not be analyzed by NGS, and this can be explained by the exhaustion of the specimens by previous multiple assays testing, but also by the challenge posed by the necessity of obtaining RNA of sufficient integrity and usability from archival material [45]. In light of previous results obtained by our team and others [[20], [21], [22], [23], [33]], the RNA-seq results of the samples for which the IHC and FISH were clearly positive (‘truly positive’ cases) were interpreted separately from those with equivocal/borderline-positive or discordant IHC and FISH. This also gave us the opportunity to evaluate the use of RNA-seq as a rescue technique, with the aim of improving patient selection for ALK inhibitor therapy.