A novel mass spectrometry-based assay for diagnosis of EML4-ALK-positive non-small cell lung cancer

Kazuko Sakai, Isamu Okamoto, Ken Takezawa, Tomonori Hirashima, Hiroyasu Kaneda, Masayuki Takeda, Kazuko Matsumoto, Hideharu Kimura, Yoshihiko Fujita, Kazuhiko Nakagawa, Tokuzo Arao, Kazuto Nishio

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)

Abstract

Introduction: The presence of the transforming fusion gene echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) in non-small-cell lung cancer (NSCLC) is a predictive marker for the efficacy of anaplastic lymphoma kinase inhibitors. However, the currently available assays for the detection of the different variants of EML4-ALK have limitations. Methods: We developed an assay system for the detection of EML4-ALK variants 1, 2, 3a, 3b, 4, 5a, 5b, 6, or 7 transcripts in total RNA obtained from formalin-fixed, paraffin-embedded (FFPE) specimens of NSCLC tissue. The assay is based on region-specific polymerase chain reaction amplification of EML4-ALK complementary DNA followed by specific single-base primer extension and analysis of the extension products by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The assay was validated by fluorescence in situ hybridization and the results confirmed by subcloning and sequencing of polymerase chain reaction products. Results: Evaluation of the analytic sensitivity of the assay with serial dilutions of plasmids containing EML4-ALK complementary DNA sequences revealed it to be capable of the reliable detection of one copy of each plasmid per reaction. The assay also detected EML4-ALK variants 1 or 3 in three FFPE samples of surgically resected NSCLC shown to be positive for anaplastic lymphoma kinase rearrangement by fluorescence in situ hybridization. Furthermore, the assay identified variant 1 of EML4-ALK in 3 of 20 FFPE biopsy samples from patients with advanced NSCLC. All positive samples were confirmed by subcloning and sequencing. Conclusions: Our novel assay is highly sensitive and effective for the detection of EML4-ALK in FFPE specimens.

Original languageEnglish
Pages (from-to)913-918
Number of pages6
JournalJournal of Thoracic Oncology
Volume7
Issue number5
DOIs
Publication statusPublished - 2012
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Oncology
  • Pulmonary and Respiratory Medicine

Fingerprint

Dive into the research topics of 'A novel mass spectrometry-based assay for diagnosis of EML4-ALK-positive non-small cell lung cancer'. Together they form a unique fingerprint.

Cite this