An ALK rearrangement was found
An ALK rearrangement was found in all the analyzable ‘truly positive’ IHC+/FISH+ samples, and the proportion of the various transcripts was in accordance with the literature [, , , ]. In these samples, the RNA-seq technique was therefore 100% sensitive and specific. Additional non-targeted analyses are required in order to identify the fusion transcripts in the cases for which an ALK 3′5′ imbalance was detected. However, this additional information comes at a cost, as these techniques often require at least 5 times more RNA (50–200 ng) than the targeted panel used (10 ng) in the present study, and tend to be more expensive. RNA-seq proved to be an effective rescue technique for the non-clear-cut (equivocal and/or borderline-positive) IHC/FISH cases as it VPS34-IN1 receptor allowed us to eventually qualify each of these samples as either being ALK-positive (4/10) or ALK-negative (6/10) on the basis of the presence or absence of an ALK fusion transcript. Moreover, in one case (negative IHC and borderline FISH), a KIF5B-RET fusion was found, pointing towards a false-positivity of the ALK FISH, and highlighting the importance of multiplex testing. On the other hand, the RNA-seq assay used in the present study was not as helpful in the analysis of the IHC/FISH discordant cases, as no results could be obtained for the IHC-negative/FISH-positive samples tested, because of a too low RNA concentration. This could be because these samples were repetitively tested, or, more probably, because of preanalytical issues, as highlighted in another study of such discordant cases , our samples coming from various pathology laboratories. For the two IHC-positive/FISH-negative samples analyzed, an ALK fusion transcript was detected in one case (IHC 90% 2/3+, FISH 1.5%), the other case (IHC 50% 1+, FISH 0%) was negative, leading us to conclude that the 50% 1+ IHC staining with the 5A4 antibody was probably not specific in this case, even though ALK fusion transcripts were found in 3 other cases with an IHC intensity of 30% 1+ and 50% 1+. The concordance between the FISH technique and the RNA-seq results obtained with our cohort was of 96%, as 21/22 FISH-negative cases were RNA-seq negative. The FISH-negative case which was found to harbor an ALK fusion transcript was IHC positive, thus probably harboring a complex rearrangement not detected by FISH, or an alternative mechanism leading to an overexpression of the ALK protein [, ]. For the ‘truly’ FISH-positive cases (>20%), an ALK rearrangement was found by RNA-seq in 27/29 (93%) cases, with the 2 RNA-seq negative cases showing a doubtful IHC staining. For the FISH borderline-positive samples (FISH 15–20%), an ALK fusion was found only in 2/6 (33%) samples; the only two samples for which the IHC was positive. These results, along with those obtained by other teams, highlight the challenges posed by borderline-positive FISH results for the molecular diagnosis of ALK, but also of ROS1 and RET rearrangements [, , , , , , ]. In these cases, the use of techniques which allow the simultaneous detection of all targetable gene fusions, such as RNA-seq, can be very helpful. Concerning the concordance between ALK IHC and RNA-seq results, an ALK fusion transcript was found in 3/8 (38%) cases with a low (≤50% 1+) staining intensity, 29/30 (97%) cases with an intensity >50% 1+, and no ALK fusions were found in the IHC-negative cases. These results confirm the importance of testing ALK IHC-positive cases for the presence of an ALK-rearrangement, especially for 1+ and 2+ scores, as recently highlighted by Marchetti et al. . However, and in accordance with our previous results with this antibody [, ], staining with the 5A4 antibody gave good results in our hands as the staining intensity (3+, 2+ or 0/1+) was correlated with crizotinib efficacy in terms of response, PFS and DOT. Finally, the real-time PCR results obtained on this cohort (all ALK-positive sample were analyzed, data not shown) gave limited information as only the 4 more frequent EML4-ALK transcript variants were tested (variants v1, v2, v3a/b and v5). However, for the samples for which an ALK fusion transcript was detected by real-time PCR and by RNA-seq, there were no discordances between the fusion transcripts found by both techniques.