RNA-based analysis of ALK fusions in non-small cell lung cancer cases showing IHC/FISH discordance

Alterations (paracentric inversion/translocation) of the anaplastic lymphoma kinase (ALK) gene occur in about 3–4% of non-small cell lung cancers (NSCLC) and represent a drugable target [1, 2]. Since the approval of the first ALK tyrosine kinase inhibitor (TKI) in the US (2011) and in Europe (2012), as well as of further second and third line TKI, ALK testing in locally advanced or metastatic non-pure squamous NSCLC is currently the diagnostic standard [3‐5]. Fluorescence in-situ hybridization (FISH) was used as the diagnostic method of choice in the studies leading to the approval of Crizotinib [6, 7]. The Food and Drug Administration (FDA) reflected this aspect within the authorization text [8, 9] therefore FISH is still regarded as the gold standard for the detection of ALK rearrangements. However, meanwhile the FDA further approved the Ventana ALK (D5F3) CDx immunohistochemistry (IHC) assay as a companion diagnostics to identify patients eligible for ALK TKI treatment [10, 11]. Thus, 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]. However, even though validated and recommended [3, 13‐18], IHC and FISH might still produce discordant results [18‐23] leading to a diagnostic and therapeutic dilemma. As RNA-based assays were reported as promising tools for ALK testing in some case series [24‐27], 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].

Since the approval of Crizotinib, there had been much effort to improve, harmonize and even fasten the diagnostic process of ALK testing [13‐16, 31‐33]. In this context, several ring trials and multi-center studies have confirmed the diagnostic reliability and quality of IHC and FISH [13‐16, 31‐34]. Concerning in-situ hybridization it was further shown that CISH (chromogenic based) can be regarded as an alternative reliable method [35, 36]. However, due to technical reasons, there might be samples that will be misclassified if FISH or CISH was used as stand-alone test [21, 37]. This is especially true for samples that show SS/SRS around the 15% cutoff [21]. To this end, it was highly appreciated that several initiatives demonstrated ALK IHC as a dependable method, if carefully validated [10, 13, 15, 31‐33, 38‐40]. However, there are still several reports depicting discrepant results by IHC and FISH [18‐26], leading to diagnostic problem and a potential “wrong” therapeutically decision. First of all, this is due to the so-called BL tumors especially those with SS/SRS FISH signals between 15 and 20% of the tumor cells. The fact that “true” ALK negative tumors and normal lung tissue may harbor SS in up to 11% of the tumor cells and ~ 8.5% of the NSCLC show SS between 10 and 15% is further complicating this situation [41‐43]. To this end it was recommended, that in biopsy specimens (with a few tumor cells only), the unequivocal result of a validated IHC should be used for therapy planning in NSCLC cases even though if FISH was negative [22].

Our data underline that a validated ALK IHC produces dependable results in most cases. We only found one sample in our cohort leading to an unclear staining pattern due to some, focal positive cells (questionable tumor cells or macrophages). The Ventana Interpretation Guide for the D5F3-Optiview system defines IHC-positivity if even single tumor cells show a cytoplasmic staining [28]. However, in our experience ALK positive staining, if truly positive, will be found in all (almost all) tumor cells and not merely in a small subpopulation due to its role as driver mutation. This is further demonstrated by the homogenous staining for the ALK protein in all tumor cells of the IHC-positive/FISH-BL-positive cases and further underlines that the occurrence of discrepant ALK results for IHC and FISH is due to technical reasons of FISH. This assumption was confirmed by some studies, which were able to reproduce the IHC results on the RNA-level by means of RT-PCR or MPS [24‐27, 44, 45]. This is also true for our study that confirmed the IHC results by MPS and the probe-based NanoString assay and therefore strengthens the hypothesis that IHC and RNA-based methods reflect the true biological situation. Unfortunately, clinical data within our cohort were scarce for the most of the cases. However, at present four patients of our study with an ALK IHC-negative/FISH-BL status were treated with an ALK TKI at Charité University Hospital and all did not show any benefit based on this therapy.

Interestingly, we further characterized three other discrepant samples, which were ALK IHC-negative/FISH-positive and also negative at the transcriptional level by MPS and NanoString. Concerning the ALK FISH pattern, two of them displayed SRS in > 60% of the tumor cells. A comparable observation, with SRS-samples displaying no protein expression and no transcript was made by Gao et al. [45]. Therefore, it seems that IHC/FISH discrepancy might be caused in the context of a FISH BL-status, especially in the context of small biopsies [22], however, might be further seen in at first sight “true” FISH-positive cases with SRS-pattern. This situation is still insufficiently covered by clinical data but it is justified to conclude that no transcription or translation of the ALK fusion gene occurs [45, 46]. A possible explanation for this observation, the break might have occurred but the promoter might not have been translocated to the ALK gene or might even have been lost, thus preventing ALK expression despite the genomic ALK alteration (non-productive rearrangement). This might be one explanation for the documented non-responders in the FISH-based trials leading to the TKI approval (see waterfall blot in ref. [6, 7]) already discussed in the literature [47].

To conclude, as predictive testing in NSCLC becomes more and more complex and further treatable targets (besides EGFR, ALK, ROS1, MET, RET, BRAF, HER2, PD-L1) will arise in the nearer future [2], pragmatic approaches (reliable, not time and money consuming, multiplexable) are needed. Furthermore, current hybrid capture-based sequencing assays allow the additional detection of so far unknown fusion partners [48‐50]. As this might not always be applicable for routine analysis, this was beyond the scope of our investigations. However, these approaches will enable a further comprehensive fusion analysis, helping for a better understanding of the molecular mechanism in lung cancer [48‐51]. The results of this study show that ALK testing should be based on methods that reflect the functional level of ALK. As RNA-based methods confirmed the IHC-status, future diagnostic algorithms should be based on these approaches, whereas FISH, at least as a stand-alone test, seems not eligible anymore.

The comparison of different methods for the confirmation of ALK rearrangements revealed that the detection of the protein (IHC) and the fusion transcripts on transcriptional level (sequencing and probe-based assays) leads to concordant results. A small proportion of clearly ALK FISH-positive cases do not express the ALK protein and the fusion transcript, which might explain non-response to ALK inhibitors (non-productive rearrangement). Therefore, our findings led us to conclude that ALK testing should initially be based on IHC- and/or RNA-based methods. This might further avoid needless ALK-TKI therapies in ALK FISH-BL cases.