Rearrangements of ALK are established targets in the current therapy of advanced NSCLC and are predominantly detected by IHC and/or FISH. However, both methods occasionally produce discordant results. This may occur especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10–20% of tumour nuclei around the cutoff (15%) for ALK FISH-positivity. This leads to a diagnostic, and thus therapeutic, dilemma.
We selected 18 unequivocal samples (12 ALK IHC- and FISH-negative; 6 ALK IHC- and FISH-positive) and 15 equivocal samples with discordance between FISH (Vysis LSI ALK Dual Color) and IHC (D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analysed using a targeted multiplex-PCR panel followed by S5 sequencing and direct transcript counting using a digital probe-based assay (Nanostring). Sensitivity of both methods was defined using RNA obtained from an ALK-positive NSCLC cell-line dilution series.
Cases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods. Three IHC-negative/FISH-positive samples were negative with both RNA-based methods. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples, showed negative results by sequencing and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA level; whereas a tumour with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript.
The comparison of methods for the confirmation of ALK rearrangements revealed that the detection of ALK protein by IHC and ALK fusion transcripts on transcriptional level by sequencing and probe-based assay leads to concordant results. Only a small proportion of clearly ALK FISH-positive cases are unable to express the ALK protein and ALK fusion transcript, which might explain non-response to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should be based on IHC- or RNA-based methods, especially for ALK FISH BL cases.
Clinical trial identification
Legal entity responsible for the study
Roche Pharma AG, Germany.
Part of the study was sponsored by Roche Pharma AG, Germany (ALK-IHC-FISH-NGS Head to Head Study ML39478
Support for third-party editorial assistance for this abstract, furnished by John Carron, PhD, of Health Interactions, was provided by Roche Pharma AG, Germany.
M. Hummel: Expert testimony, travel, accommodation, expenses: ThermoFisher. M. Moebs: Consulting or advisory roles, expert testimony, travel, accommodation, expenses: Thermofisher Scientific. All other authors have declared no conflicts of interest.