RNA-based Analysis of Anaplastic Lymphoma Kinase (ALK) Fusions in Non-Small Cell Lung Cancer (NSCLC) Cases Showing Immunohistochemistry/fluorescence In-Situ Hybridisation (IHC/FISH) Discordance

Background: 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. Methods: 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. Results: 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. Conclusions: 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. Editorial acknowledgement: Support for third-party editorial assistance for this abstract, furnished by John Carron, PhD, of Health Interactions, was provided by Roche Pharma AG, Germany. Legal entity responsible for the study: Roche Pharma AG, Germany. Funding: Part of the study was sponsored by Roche Pharma AG, Germany (ALK-IHC-FISH-NGS Head to Head Study ML39478 Disclosure: 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.