ALK-FISH borderline cases in non-small cell lung cancer: Implications for diagnostics and clinical decision making

Background: Fluorescence in-situ hybridization (FISH) for the detection of ALK-rearrangements in non-small cell lung cancer (NSCLC) is based on at first sight clear cut-off criteria (>= 15% of tumor cells) for split signals (SS) and single red signals (SRS). However, NSCLC with SS-counts around the cut-off may cause interpretation problems. Material and methods: Tissue microarrays containing 753 surgically resected NSCLCs were independently tested for ALK-alterations by FISH and immunohistochemistry (IHC). Our analysis focused on samples with SS/SRS in the range between 10% and 20% (ALK-FISH borderline group). To better understand the role of these samples in routine diagnostics, we performed statistical analyses to systematically estimate the probability of ALK-FISH-misclassification (false negative or positive) for different numbers of evaluated tumor cell nuclei (30, 50, 100, and 200). Results: 94.3% (710/753) of the cases were classified as unequivocally (<10% or >= 20%) ALK-FISH-negative (93%; 700/753) or positive (1.3%; 10/753) and showed concordant IHC results. 5.7% (43/753) of the samples showed SS/SRS between 10% and 20% of the tumor cells. Out of these, 7% (3/43; ALK-FISH: 14%, 18% and 20%) were positive by ALK-IHC, while 93% (40/43) had no detectable expression of the ALK-protein. Statistical analysis showed that ALK-FISH misclassifications occur frequently for samples with rearrangements between 10% and 20% if ALK-characterization is based on a sharp cut-off point (15%). If results in this interval are defined as equivocal (borderline), statistical sampling-related ALK-FISH misclassifications will occur in less than 1% of the cases if 100 tumor cells are evaluated. Conclusion: While ALK status can be determined robustly for the majority of NSCLC by FISH our analysis showed that similar to 6% of the cases belong to a borderline group for which ALK-FISH evaluation has only limited reliability due to statistical sampling effects. These cases should be considered equivocal and therapy decisions should include additional tests and clinical considerations. (c) 2015 Elsevier Ireland Ltd. All rights reserved.