Abstract
BackgroundRearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). However, both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10–20% of the tumor nuclei around the cutoff (15%). This leads to a diagnostic and thus to a therapeutic dilemma.MethodsWe selected 18 unequivocal (12 ALK IHC/FISH-negative; 6 ALK IHC/FISH-positive) and 15 equivocal samples with discordant results between FISH (Abbott, Vysis LSI ALK Dual Color) and IHC (Ventana, D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analyzed using a targeted multiplex-PCR panel followed by IonTorrent sequencing and by direct transcript counting using a digital probe-based assay (NanoString). Sensitivity of both methods was defined using RNA obtained from an ALK-positive cell line dilution series.ResultsCases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods, whereas the three IHC-negative/FISH-positive samples were negative. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples showed negative results by massive parallel sequencing (MPS) and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA-level, whereas a tumor with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript.ConclusionsThe 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 MPS and the 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 a non-responding to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should initially be based on IHC and/or RNA-based methods.
Highlights
Rearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH)
Besides clear cut ALK negative and positive samples by IHC and Fluorescence in-situ hybridization (FISH), a special focus was set on ALK FISH-borderline (BL) samples with split signal (SS)/single red signal (SRS) between 10 and 20%, which were further sub-classified in FISH-BL-negative
The massive parallel sequencing (MPS) assay showed 100% specificity for ALK fusion detection and a higher sensitivity for breakpoint than for 3′/5′ analysis (67% vs. 33%) with uncertain data stated as negative
Summary
Rearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). Both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10–20% of the tumor nuclei around the cutoff (15%). Two different diagnostic approaches (DNA and protein-based) might be used, if carefully validated [3], for patient care This is in line with the European Medicines Agency (EMA) authorization text that “only” requires the proof of an “advanced ALK positive NSCLC” [12]. As RNA-based assays were reported as promising tools for ALK testing in some case series [24,25,26,27], we performed massive parallel sequencing (MPS) using IonTorrent chemistry and a probe-based technology (NanoString) allowing a direct counting of RNA molecules in an IHC and FISH pretested NSCLC cohort [13, 21]
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