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Journal of Thoracic Oncology:
doi: 10.1097/JTO.0000000000000030
Original Articles

Large-Scale Screening and Molecular Characterization of EML4-ALK Fusion Variants in Archival Non–Small-Cell Lung Cancer Tumor Specimens Using Quantitative Reverse Transcription Polymerase Chain Reaction Assays

Li, Tianhong MD, PhD*†; Maus, Martin K.H. MD‡§; Desai, Sonal J. PhD*†; Beckett, Laurel A. PhD*‖; Stephens, Craig PhD§; Huang, Eric§; Hsiang, Jack§; Zeger, Gary MD§¶; Danenberg, Kathleen D. MS#; Astrow, Stephanie H. PhD§; Gandara, David R. MD*†

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Abstract

Introduction:

The objective of this study was to identify and characterize echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase fusion (EML4-ALK+) cancers by variant-specific, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays in a large cohort of North American non–small-cell lung cancer (NSCLC) patients.

Methods:

We developed a panel of single and multiplex RT-PCR assays suitable for rapid and accurate detection of the eight most common EML4-ALK+ variants and ALK gene expression in archival formalin-fixed, paraffin-embedded NSCLC specimens. EGFR and KRAS genotyping and thymidylate synthase RNA level by RT-PCR assays were available in a subset of patients.

Results:

Between December 2009 and September 2012, 7344 NSCLC specimens were tested. An EML4-ALK+ transcript was detected in 200 cases (2.7%), including 109 V1 (54.5%), 20 V2 (10.0%), 68 V3 (34.0%), and three V5a (1.5%) variants. Median age was 54.5 years (range, 23–89), and 104 patients (52.0%) were women. The great majority (n=188, 94.0%) of EML4-ALK+ NSCLC tumors had adenocarcinoma histology. ALK expression level varied significantly among different EML4-ALK+ variants and individual tumors. Only one case each of concurrent EGFR or KRAS mutation was detected. The median thymidylate synthase RNA level from 85 EML4-ALK+ cancers was significantly lower compared with that of EML4-ALK-negative lung adenocarcinomas (2.02 versus 3.29, respectively, p<0.001).

Conclusions:

This panel of variant-specific, quantitative RT-PCR assays detects common EML4-ALK+ variants as well as ALK gene expression level in archival formalin-fixed paraffin-embedded NSCLC specimens. These RT-PCR assays may be useful as an adjunct to the standard fluorescence in situ hybridization assay to better understand biologic variability and response patterns to anaplastic lymphoma kinase inhibitors.

Copyright © 2013 by the International Association for the Study of Lung Cancer

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