The ORR obtained with crizotinib on

The ORR obtained with crizotinib on our cohort was low compared to other studies, such as the prospective studies of A. Shaw et al. (ORR = 65% in second line), B. Solomon et al. (ORR = 74% in first line) and the more comparable large retrospective study of M. Duruisseaux et al. on the French CLINALK cohort (ORR = 50.2% in first, second and further-line settings) [[3], [4], [17]]. However, compared to these studies, our cohort was enriched in IHC/FISH equivocal/borderline-positive cases, as one of our objectives was to evaluate the use of RNA-seq as a rescue technique for these difficult cases. Overall, the correlations we found between the RNA-seq results and the clinical outcomes in the limited number of patients of our cohort who received crizotinib highlight the potential of this technique. Indeed, if only the RNA-seq-positive patients are taken into account, the response rate obtained was comparable to that of the CLINALK study (56% and 50.2%, respectively) in which crizotinib was given in various lines of treatment in a “real-life” setting, as was the case in our study. We found that crizotinib was ineffective in the two cases with no ALK fusion according to RNA-seq. Interestingly, no responses were observed in the two patients with the KLC1(9)-ALK(20) rearrangements, suggesting the lack of efficacy of crizotinib in these non-EML4-ALK variants. Lastly, we found a trend for a better median PFS and DOT in v1/v2 EML4-ALK variants cases compared to v3a/b. These data should be carefully interpreted because of the small number of patients considered. However, these results are in line with the data recently published by Woo et al. [28], showing a longer PFS with crizotinib in patients with v1/v2/others EML4-ALK variants compared to v3a/b variants. An in-vitro study by Heuckman et al. [25] showed that the E-64-d of the different EML4-ALK rearrangement products conditioned the sensitivity to crizotinib, with EML4-ALK v2 being the most sensitive, v1 and v3b having intermediate sensitivity and v3a being the less sensitive. Our results, together with those of Woo et al. [28] support Turner syndrome assumption. Further studies on larger cohorts are warranted in order to address this question, and RNA-seq techniques offer a unique opportunity to decipher the role of EML4-ALK and non-EML4-ALK variants as predictive biomarkers of ALK inhibitors efficacy. As alectinib has recently proven its superiority to crizotinib in first-line treatment and could become the new standard of care [13], data are needed about ALK variants impact on alectinib and other next-generation ALK inhibitors sensitivity.
Conclusion The use of accurate and highly informative tools for the detection of ALK and other gene fusions has become a necessity for the management of advanced NSCLCs. By using an RNA parallel-sequencing assay, we were able to detect EML4-ALK and non-EML4-ALK fusion variants in our cohort, and along with others, our study suggests the potential of ALK fusion variants as predictive biomarkers of crizotinib efficacy [[26], [27], [28], [29]]. Further studies on larger cohorts are warranted in order to address this question and NGS-based techniques offer a unique opportunity to decipher the role of EML4-ALK and non-EML4-ALK variants as predictive biomarkers of ALK inhibitors efficacy. Overall, in light of the present results and those of other teams [[24], [26], [27], [28], [29]], we suggest that prescreening of advanced-stage lung tumor samples with a validated ALK IHC followed by a molecular testing technique, such as RNA-seq, to identify the nature of the fusion variant, would allow additional precision to the treatment of ALK-positive NSCLs, with the advantage of NGS-based techniques to test for other targetable molecular alterations in a single test.
Grants and funding This work received funding from the French Institut National du Cancer (INCa), the Grenoble-Alpes University Hospital (DRCI REALK project), the Grenoble Lions Club and the Groupement des Entreprises Françaises de Lutte Contre Cancer (GEFLUC) Grenoble Dauphiné-Savoie.