Clinical Validation of a Novel Commercial Reverse Transcription–Quantitative Polymerase Chain Reaction Screening Assay for Detection of ALK Translocations and Amplifications in Non–Small Cell Lung Carcinomas

Abstract

Context.-EGFR mutations and anaplastic lymphoma kinase (ALK) translocations have significant biologic and therapeutic implications in lung cancers, particularly lung adenocarcinomas. ALK translocations are less frequent compared with EGFR mutations; interestingly, these two abnormalities are most commonly mutually exclusive. The 2013 College of American Pathologists/Association for Molecular Pathology/International Association for the Study of Lung Cancer molecular testing guideline for lung cancers recommend a testing algorithm in which detection of ALK translocations using fluorescence in situ hybridization (FISH) is to be performed following testing for EGFR mutations. Such an algorithm is cost-effective but potentially slows down turnaround time; and as a secondary test, ALK FISH assay may not be completed because it requires the use of additional tissue, and the small biopsies or cytology specimens may have been exhausted in the extraction of nucleic acid for EGFR mutation screening.Objective.-To provide efficient testing of both EGFR and ALK genetic alterations in small biopsies and cytology specimens.Design.-We validated a highly sensitive ALK reverse transcription-quantitative polymerase chain reaction (RTqPCR) assay as a screening tool for ALK translocations and amplifications.Results.-We performed a retrospective review of cases previously tested by FISH and found that all FISH ALK translocation-positive specimens were RT-qPCR positive, and all FISH ALK translocation-negative cases were RTqPCR negative (the sensitivity and specificity of the ALK RT-qPCR assay were 100%).Conclusion.-This assay allows rapid identification of ALK alterations, can be performed in conjunction with EGFR testing, and does not require use of valuable additional tumor tissue.

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer

“…RT-PCR is a sensitive and rapid detection assay when compared to FISH for the detection of ALK in NSCLC tumor samples. Earlier concerns of false-negative results due to allele-specific primer selection that permits detection of only a subset of the many ALK rearrangements have been obviated by the use of assays employing a primer set that detects the 3′ kinase domain conserved among all known ALK rearrangements regardless of the fusion gene partner [ 14 , 15 , 16 ]. Issues regarding fixation, storage and age of FFPE samples and their impact on RNA-based RT-PCR are also relevant for DNA and protein-based ALK detection methods.…”

“…RT-PCR-based assays have not been as widely used as FISH and IHC for the detection of ALK rearrangements in NSCLC. However, recent studies using FISH and IHC or FISH alone in comparison to RT-PCR have demonstrated that RT-PCR has a high sensitivity and specificity [ 14 , 15 , 16 ]. These tests have the advantages of a rapid turn-around time and ease of analysis, and can be used on biopsy or cytology specimens with lower tumor content than needed for accurate FISH and IHC testing [ 14 , 15 , 17 ].…”

“…However, recent studies using FISH and IHC or FISH alone in comparison to RT-PCR have demonstrated that RT-PCR has a high sensitivity and specificity [ 14 , 15 , 16 ]. These tests have the advantages of a rapid turn-around time and ease of analysis, and can be used on biopsy or cytology specimens with lower tumor content than needed for accurate FISH and IHC testing [ 14 , 15 , 17 ]. In the study described in this report, we compare a commercially available RT-PCR assay (ALK RGQ RT-PCR Kit, QIAGEN, Manchester, UK) designed to identify mRNA produced by all ALK rearrangements regardless of the fusion partner or variant ( Figure 1 ), with IHC that detects the ALK protein and FISH that directly identifies ALK genomic rearrangements in FFPE samples from patients with NSCLC.…”

ALK and ROS1 testing on lung cancer cytologic samples: Perspectives

“…For example, the assay used by Wang et al targets only 21 types of EML4 ‐ ALK fusion transcripts, not including the rarer types of rearrangements. In general, the current view is to use RT‐PCR in a multistep diagnostic algorithm in which FISH and IHC are integrated in the rare cases of discordant FISH and IHC results . Marchetti and colleagues proposed that young or nonsmoking patients with NSCLC, those who have tumors with a mucinous cribriform pattern or signet ring cells, or patients with TTF1‐positive/p63‐positive tumors should be tested for ALK rearrangements using a combined approach that includes RT‐PCR, FISH, and IHC.…”

“…In general, the current view is to use RT-PCR in a multistep diagnostic algorithm in which FISH and IHC are integrated in the rare cases of discordant FISH and IHC results. 19,31,81 Marchetti and colleagues 19 proposed that young or nonsmoking patients with NSCLC, those who have tumors with a mucinous cribriform pattern or signet ring cells, or patients with TTF1-positive/p63-positive tumors should be tested for ALK rearrangements using a combined approach that includes RT-PCR, FISH, and IHC. Like ALK rearrangements, ROS1 rearrangements have highly conserved breakpoints in the 5 0 gene region and a retained 3 0 kinase domain.…”