Status quo of ALK testing in lung cancer: results of an EQA scheme based on in-situ hybridization, immunohistochemistry, and RNA/DNA sequencing

Alterations of the anaplastic lymphoma kinase (ALK), most commonly in form of a paracentric inversion resulting in an EML4-ALK fusion transcript, occur in about 4–6% of non-small cell lung cancer (NSCLC) [1, 2]. Patients harboring this alteration can benefit from therapy with various tyrosine kinase inhibitors (TKI). Fluorescence in situ hybridization (FISH) has been used in the clinical trials that led to the approval of the first ALK TKI Crizotinib and is still considered the gold standard [3, 4]. However, numerous studies also demonstrated the reliability of immunohistochemistry (IHC) to identify patients with ALK alterations [5‐11]. Furthermore, IHC is widely deployed and requires less technical expertise compared with FISH, making it a highly promising screening tool [12]. Beyond IHC and in situ hybridization (ISH), next generation-sequencing (NGS) panels are able to identify the distinct ALK-fusion transcripts, which seems of interest, as recent data showed that specific ALK-subtypes (e.g., EML4-ALK variant 3) may be clinically more aggressive and tend to show an earlier resistance to therapy [13]. Furthermore, nowadays, diagnostic cancer approaches need to cover far more than one alteration. This fact is of emerging importance as the number of genes of interest for targeted therapy keeps growing, while the amount of tissue available for investigation is often limited [14, 15]. Therefore, methods that can reliably identify multiple, predictive, or prognostic alterations within a single analysis are becoming increasingly important. Additionally, DNA/RNA sequencing methods have been shown to be very useful in cases where IHC or ISH give contradictory or inconclusive results [16, 17].

While several ALK ring trials have demonstrated a high interrater concordance between different institutions with regard to IHC and ISH, there is virtually no experience for RNA/DNA sequencing-based methods [6, 18, 19]. To address this, the Qualitätssicherungs-Initiative Pathologie GmbH (QuIP, Quality Assurance Initiative Pathology) initiated a ring trial, investigating the reliability of the three methods to correctly assess the ALK status of pretested NSCLC samples in a multicentric setting.

FISH is still regarded as the gold standard and diagnostic method of choice to detect ALK-positive NSCLC [3, 4]. However, in the last years, further methods such as IHC and NGS showed promising and even comparable results and have been integrated in the daily routine testing [16, 17]. EQA schemes may serve to show the status quo of the diagnostic standard (quality) in a multi-center setting. To this end, based on an initial so-called internal ring trial to choose and validate eligible tumor samples, we enrolled an external nationwide ring trial encompassing 57 participants. Thus, we were for the first time able to evaluate the interrater concordance of IHC, ISH, and RNA/DNA sequencing to reliably identify ALK alterations in NSCLC between different laboratories of pathology.

In line with previous reports, we observed a high sensitivity and interrater reliability for ISH [6, 18, 19]. Of note, only clearly positive cases were included in the ring trial. Therefore, the reported sensitivity for ISH does not account for so-called borderline cases with translocation signals near the cut-off or the rare but existing IHC negative but FISH positive cases [24, 25].

Implementation of diagnostic ALK IHC was initially complicated by the existence of a whole variety of different antibody clones as well as the lack of standardized protocols and scoring systems [19]. However, after several successful harmonization studies, IHC quickly became a reliable screening method [5, 6]. In our study, we observed an adequate sensitivity and interrater agreement demonstrating the reliability of IHC across different institutions. In comparison to ISH and NGS, these values (and also the number of institutions with successful participation) were relatively low. However, a central reevaluation of the IHC slides from the institutions that did not successfully participate in the ring trial revealed that false negative results were primarily due to misinterpretation and not only due to technical issues. In more than half of the re-evaluated false negative cases, we observed a weak or an aberrant (stipple staining) staining pattern. For these specimens, a second method should be used to determine if an ALK translocation is present or not [26, 27].

In line with current recommendations, the most commonly used antibody clone was D5F3 [28]. The D5F3 antibody by Ventana is also approved by the FDA for selection of patients to be treated with Crizotinib. In comparison to other investigations, the sensitivity of this clone in our study was lower, but still within the range of the values reported in literature [6, 18, 19, 27]. The second most commonly used antibody was 1A4. This clone has not yet been validated in a multicenter setting before but showed promising results in previous reports [29]. In line with these studies, we observed a higher sensitivity compared with D5F3, further supporting the suitability of 1A4 for diagnostic use.

Regarding RNA/DNA sequencing, this is the first study to investigate this method in a multicenter setting. Interestingly, the observed sensitivity and interrater agreement was higher than for IHC and ISH. In fact, only one false negative result was observed, which was most likely caused by low RNA input. Compared with IHC and ISH, the number of samples that were not evaluable was considerably larger, although this was mainly caused by one participant who was unable to extract a sufficient amount of RNA/DNA in six of ten cases. It is well known that formalin fixation causes molecular modification, fragmentation and degradation of nucleic acids [30]. However, as no other institution observed compromised RNA/DNA quality, this outlier could be caused by individual technical difficulties during the extraction process.

The use of NGS panels (DNA and/or RNA fusion panels) comes with multiple benefits. Most obviously, these panels usually cover other predictive and prognostic molecular alterations in multiple genes, such as EGFR, ROS1, RET or MET. Furthermore, in contrast to ISH and IHC, RNA/DNA sequencing can be used to determine the ALK fusion variant, which, in future, could be a relevant information for refined treatment decisions [13]. Despite these advantages, the use of RNA/DNA sequencing is also limited, mainly by the required technical expertise, relatively high cost and longer turn-around time as well as potentially by RNA/DNA quality. Therefore, ISH and IHC will probably still be required in the future.

In addition to the main results from the ring trial, we also gained detailed insight on the distribution and application of the different techniques for ALK testing. ALK IHC is a relatively cheap and reliable method, which is established in almost all institutions and is also most commonly used for ALK testing. In fact, more than half of the participating institutions exclusively used IHC in routine diagnostics. Our investigation also showed that over 50% of the participating institutions have already established RNA/DNA sequencing for the detection of ALK fusions. However, only half of them actually used this method in the routine diagnostic setting. The excellent results for RNA/DNA sequencing that were observed in the multicentric validation presented in this study further encourages the broad implementation and application of this technique in the routine diagnostic of ALK translocation in NSCLC, although this approach can be limited by the small size of most biopsy specimens.

A limitation of our study is the relatively small number of samples that have been used for this EQA scheme. Although the number is comparable with previous studies [6, 10], a larger set of cases might be helpful to improve the robustness of the obtained results.

In summary, we provide further proof that the ISH- and IHC-based identification of ALK translocations in NSCLC is highly reliable and reproducible between different pathology laboratories. Furthermore, we show that RNA/DNA sequencing might even be superior to IHC and ISH in terms of specificity and interrater reliability. However, the application of this method in routine diagnostics might be limited by relatively high cost, required technical expertise, and tissue quality.