Chromogenic in situ hybridization is a reliable assay for detection of ALK rearrangements in adenocarcinomas of the lung

Abstract

Reliable detection of anaplastic lymphoma kinase (ALK) rearrangements is a prerequisite for personalized treatment of lung cancer patients, as ALK rearrangements represent a predictive biomarker for the therapy with specific tyrosine kinase inhibitors. Currently, fluorescent in situ hybridization (FISH) is considered to be the standard method for assessing formalin-fixed and paraffin-embedded tissue for ALK inversions and translocations. However, FISH requires a specialized equipment, the signals fade rapidly and it is difficult to detect overall morphology and tumor heterogeneity. Chromogenic in situ hybridization (CISH) has been successfully introduced as an alternative test for the detection of several genetic aberrations. This study validates a newly developed ALK CISH assay by comparing FISH and CISH signal patterns in lung cancer samples with and without ALK rearrangements. One hundred adenocarcinomas of the lung were included in this study, among them 17 with known ALK rearrangement. FISH and CISH were carried out and evaluated according to the manufacturers' recommendations. For both assays, tumors were considered positive if ≥15% of tumor cells showed either isolated 3' signals or break-apart patterns or a combination of both. A subset of tumors was exemplarily examined by using a novel EML4 (echinoderm microtubule-associated protein-like 4) CISH probe. Red, green and fusion CISH signals were clearcut and different signal patterns were easily recognized. The percentage of aberrant tumor cells was statistically highly correlated (P<0.001) between FISH and CISH. On the basis of 86 samples that were evaluable by ALK CISH, we found a 100% sensitivity and 100% specificity of this assay. Furthermore, EML4 rearrangements could be recognized by CISH. CISH is a highly reliable, sensitive and specific method for the detection of ALK gene rearrangements in pulmonary adenocarcinomas. Our results suggest that CISH might serve as a suitable alternative to FISH, which is the current gold standard.

Chromogenic in situ hybridisation (CISH) is a powerful method to detect ALK-positive non-small cell lung carcinomas

“…Probably, the clearer two signals are separated, the more counterintuitive it is to judge them as a fusion signal. We therefore advocate the ‘one signal diameter’ criterion in CISH, in agreement with other studies 19 23. As all seven cases found positive by CISH were confirmed by qPCR and Sanger sequencing, the ‘one signal diameter’ criterion seems to be appropriate for the ALK break apart probe we used in our study.…”

Multicenter ALK Testing in Non–Small-Cell Lung Cancer: Results of a Round Robin Test

“…Furthermore even though, only two of the 32 certified institutes performed ALK testing by CISH, correct ALK classification does not seem to be restricted to a fluorescencebased detection system. 16,23 A further striking result was the broad range of tumor cells classified as ALK-break positive within one and the same case among the participants (Tables 2 and 3). This is especially interesting since only cases with high numbers of SS and/or SRS (41-73%) were included in our study.…”

“…Therefore our round robin test was based on the detection of genomic ALK breaks by means of FISH or CISH, as the latter was recently described as a reliable and suitable alternative option in ALK diagnostics. 16,23 Nevertheless, our ALK-round robin test revealed a quite high failure rate of 39.7% (21 of 53) to identify ALK-positive cases, despite the usage of unequivocal and intensively pretested cases. Due to the clearness of the cases, a successful participation could only be certified if 19 or 20 points (maximum 20 points) had been reached.…”

“…at least as a prescreening approach followed by ALK FISH (or CISH?) 16,23 in case of protein expression. [31][32][33][34][35][36] In essence, our ALK ISH round robin test clearly demonstrates that there is a need to improve the ALK testing quality.…”

MET Amplification Status in Therapy-Naïve Adeno- and Squamous Cell Carcinomas of the Lung

“…All tumor tissues were fixed in buffered formalin and embedded in paraffin blocks. Mutational analyses for EGFR and KRAS were carried out as previously described (28).…”