ALK Status Testing in Non–Small Cell Lung Carcinoma

Abstract

ALK gene rearrangements in advanced non-small cell lung carcinomas (NSCLC) are an indication for targeted therapy with crizotinib. Fluorescence in situ hybridization (FISH) using a recently approved companion in vitro diagnostic class FISH system commonly assesses ALK status. More accessible IHC is challenged by low expression of ALK-fusion transcripts in NSCLC. We compared ultrasensitive automated IHC with FISH for detecting ALK status on 318 FFPE and 40 matched ThinPrep specimens from 296 patients with advanced NSCLC. IHC was concordant with FFPE-FISH on 229 of 231 dual-informative samples (31 positive and 198 negative) and with ThinPrep-FISH on 34 of 34 samples (5 positive and 29 negative). Two cases with negative IHC and borderline-positive FFPE-FISH (15% and 18%, respectively) were reclassified as concordant based on negative matched ThinPrep-FISH and clinical data consistent with ALK-negative status. Overall, after including ThinPrep-FISH and amending the false-positive FFPE-FISH results, IHC demonstrated 100% sensitivity and specificity (95% CI, 0.86 to 1.00 and 0.97 to 1.00, respectively) for ALK detection on 249 dual-informative NSCLC samples. IHC was informative on significantly more samples than FFPE-FISH, revealing additional ALK-positive cases. The high concordance with FISH warrants IHC's routine use as the initial component of an algorithmic approach to clinical ALK testing in NSCLC, followed by reflex FISH confirmation of IHC-positive cases.

Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry

“…Finally, it must be emphasized that we (E.C, unpublished data) and others [14], [48], [49] have found positive ALK IHC particularly useful in limited samples or when FISH is not evaluable. However, a broadly held consensus on the number of positive cells required for an IHC positive score has yet to emerge.…”

“…Conversely, the specificity of these ultrasensitive IHC assays [14] obviates the need for FISH confirmation in positive IHC cases. In fact, there have been reports of dramatic responses to crizotinib in patients with IHC positive and FISH negative tumors [44].…”

“…This controversy is also entering the field of NSCLC ALK testing [7], with an increasing number of recent publications addressing discordances between in situ hybridization and IHC assays [8]–[14], further fuelled by the different regulatory approvals and the arrival of other ALK inhibitors [3], [15]. While some groups recommend initial IHC followed by FISH confirmation of some IHC-positive cases [14], [16], others believe the detection of ALK rearrangements is improved when using two methodologies [9], [17].…”

“…While some groups recommend initial IHC followed by FISH confirmation of some IHC-positive cases [14], [16], others believe the detection of ALK rearrangements is improved when using two methodologies [9], [17]. This situation prompted us to investigate two IHC antibodies, using a novel ultrasensitive detection-amplification kit, and an automated FISH scanning system in a series of tumor samples to obtain supporting data for an ALK testing algorithm [18].…”

Custom Gene Capture and Next-Generation Sequencing to Resolve Discordant ALK Status by FISH and IHC in Lung Adenocarcinoma

“…We repeated immunostaining on all discordant cases (except one case without available tissue) with D5F3 antibody, which has been shown to correlate better with ALK FISH. 27,31 In all four FISH+/IHC− cases, the negative IHC result by ALK1 antibody was confirmed by D5F3 antibody. On the other hand, the positive IHC result (all score 2) by ALK1 antibody reversed as negative by D5F3 antibody in 11 of 11 FISH−/IHC+ cases, where sufficient tissue was available for repeat staining.…”