Abstract 2220: Insight ALK ScreenTM, a highly sensitive and specific RT-qPCR first-line screening assay for comprehensive detection of all oncogenic anaplastic lymphoma kinase (ALK) fusions: clinical validation
Authors: Hout David , Xue Liquan , Choppa Paul , Handshoe John , Bloom Kenneth , Ross Douglas , Nickols Josh , Morris Stephan W. 
Published: June 2012

More than 15 different ALK fusions (e.g., CLTC-ALK, multiple EML4-ALK variants, KIF5B-ALK, NPM-ALK, RanBP2-ALK, TFG-ALK, TPM4-ALK, others) cause malignancies including NSCLC, NHL, and inflammatory myofibroblastic tumors. Preliminary studies suggest several other cancer subsets may also express ALK fusions (e.g., breast, colorectal and esophageal cancers). Several ALK small-molecule inhibitors are in development, and patients treated with one clinical stage inhibitor have had marked antitumor responses. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for ALK have been used to select patients likely to benefit from ALK inhibitor therapy; however, these methods are problematic. Both methods have suboptimal sensitivity for certain ALK fusions (e.g., EML4-ALK), and FISH is technically demanding and has a prolonged turn-around time (5-7 days). To address the unmet need for a highly sensitive, practical assay for clinical ALK detection, we developed Insight ALK Screen™, a real-time PCR test for ALK fusions and wild-type ALK upregulation. The test operates by qPCR of two regions corresponding to the ALK extracellular and intracellular kinase domains; for example, EML4-ALK or other ALK fusion events result in a ΔCt (change in threshold cycle) increase in transcripts encoding the ALK kinase domain over wild-type ALK expression levels.This assay design enables Insight ALK Screen to detect any ALK fusion regardless of the ALK fusion partner. We are validating Insight ALK Screen in comparison to ALK IHC and FISH using NSCLC FFPE specimens from the Clarient tumor bank. Eighty-two NSCLCs were screened using Insight ALK Screen, and 17 of the 82 (20.7%) showed either an ALK fusion or wild-type ALK upregulation. All 17 ALK-expressing NSCLCs were next tested using an EML4-ALK variant-specific RT-PCR assay; 6 (35.3%) of the 17 cases (7.3% of the original 82 cases) were shown to be EML4-ALK-positive. Ongoing studies are assessing the genetic mechanisms (e.g., other EML4-ALK variants not tested, other ALK fusions, ALK gene amplification, chromosome 2 polysomy, etc.) underlying the aberrant ALK expression in the other 11 of 17 NSCLCs; these data will be presented at the meeting together with complete IHC, FISH and clinical-molecular correlative information. Together with its ease-of-use, small tissue requirements, multiplex compatibility, quantitation of ALK expression levels, lack of bias for specific fusions, and quick turn-around (24-48 hrs), our preliminary results support Insight ALK Screen as the first-line ALK diagnostic method of choice.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2220. doi:10.1158/1538-7445.AM2011-2220