Abstract
BackgroundIdentification of targetable EML4-ALK fusion proteins has revolutionized the treatment of a minor subgroup of non-small cell lung cancer (NSCLC) patients. Although fluorescence in situ hybridization (FISH) is regarded as the gold standard for detection of ALK rearrangements, ALK immunohistochemistry (IHC) is often used as screening tool in clinical practice. In order to unbiasedly analyze the diagnostic impact of such a screening strategy, we compared ALK IHC with ALK FISH in three large representative Swedish NSCLC cohorts incorporating clinical parameters and gene expression data.MethodsALK rearrangements were detected using FISH on tissue microarrays (TMAs), including tissue from 851 NSCLC patients. In parallel, ALK protein expression was detected using IHC, applying the antibody clone D5F3 with two different protocols (the FDA approved Ventana CDx assay and our in house Dako IHC protocol). Gene expression microarray data (Affymetrix) was available for 194 patients.ResultsALK rearrangements were detected in 1.7 % in the complete cohort and 2.0 % in the non-squamous cell carcinoma subgroup. ALK protein expression was observed in 1.8 and 1.4 % when applying the Ventana assay or the in house Dako protocol, respectively. The specificity and accuracy of IHC was high (> 98 %), while the sensitivity was between 69 % (Ventana) and 62 % (in house Dako protocol). Furthermore, only 67 % of the ALK IHC positive cases were positive with both IHC assays. Gene expression analysis revealed that 6/194 (3 %) tumors showed high ALK gene expression (≥ 6 AU) and of them only three were positive by either FISH or IHC.ConclusionThe overall frequency of ALK rearrangements based on FISH was lower than previously reported. The sensitivity of both IHC assays was low, and the concordance between the FISH and the IHC assays poor, questioning current strategies to screen with IHC prior to FISH or completely replace FISH by IHC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2646-x) contains supplementary material, which is available to authorized users.
Highlights
Identification of targetable echinoderm microtubule associated protein like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion proteins has revolutionized the treatment of a minor subgroup of non-small cell lung cancer (NSCLC) patients
ALK status evaluated by fluorescence in-situ hybridization ALK status using fluorescence in situ hybridization (FISH) was assessable for 754 (88.6 %) patients on the tissue microarray (TMA)
In non-assessable cases, either all tumor cores were missing on the TMA, the tissue present on the TMA did not contain any tumor tissue, or the hybridization was insufficient for reliable evaluation
Summary
Identification of targetable EML4-ALK fusion proteins has revolutionized the treatment of a minor subgroup of non-small cell lung cancer (NSCLC) patients. In order to unbiasedly analyze the diagnostic impact of such a screening strategy, we compared ALK IHC with ALK FISH in three large representative Swedish NSCLC cohorts incorporating clinical parameters and gene expression data. The fusion of ALK with EML4 leads to constitutive activation of ALK, directly affecting downstream signaling and increasing cell proliferation and survival [6]. Since the discovery of the EML4-ALK fusion in 2007, several other ALK fusion partners have been described, such as kinesin family member 5B (KIF5B) [16], kinesin light chain 1 (KLC1) [17] and TRK-fused gene (TFG) [7], all fusion products leading to comparable kinase activation and transforming capacities [9, 18]
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