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

doi:10.1097/JTO.0000000000000030

Journal of Thoracic Oncology

Volume 9, Issue 1, January 2014, Pages 18-25

https://doi.org/10.1097/JTO.0000000000000030 Get rights and content

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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.

Key Words

Echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase fusion variants

Formalin-fixed

Paraffin-embedded

Non–small-cell lung cancer

Quantitative

Reverse transcription polymerase chain reaction

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In summary, we here report the detection of 200 EML4-ALK fusion variants in 7344 North American NSCLC patients (2.7%) using variant-specific, quantitative RT-PCR assays. ALK expression level varied significantly among different EML4-ALK-positive variants and individual NSCLC tumors. EML4-ALK-positive tumors had a significantly lower TS RNA level compared with that of EML4-ALK-negative lung adenocarcinomas, a potential molecular basis for clinical response of ALK-positive tumors to pemetrexed. Further evaluation of these variant-specific, quantitative RT-PCR assays as an adjuvant to the standard FISH assay is warranted to better understand biologic variability and response patterns to ALK inhibitors. It remains to be determined how to integrate these quantitative RT-PCR assays into the cost-effective diagnostic algorithm for ALK+ tumors and whether patient tumors detected by different methods are equally sensitive to ALK inhibitors.

Presented in part at the 2010–2012 Annual Meetings of the American Society of Clinical Oncology, Chicago, IL (Dannenberg et al., ASCO 2010: Abs #10535; Li et al., ASCO 2011: Abs #10520; and Li et al., ASCO 2012: Abs #7594; Gandara et al., ASCO 2012: Abs #7582).

Disclosure: TL is supported by UL1 RR024146 from the National Center for Research Resources, UC Davis Comprehensive Cancer Center Developmental Award (National Institutes of Health/National Cancer Institute P30CA093373), and the Hope Foundation (Southwest Oncology Group Young Investigator Award). MKH, CS, EH, JH, GZ, and SHA are employees of RGI. KDD is a former employee of RGI. DRG serves as a consultant for RGI. The remaining authors declare no conflict of interest.