In:
Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 31, No. 15_suppl ( 2013-05-20), p. e22185-e22185
Abstract:
e22185 Background: The management of patients for NSCLC requires appropriate molecular pathology assessment to determine treatment. However current methods require the use of more than one technology including sequencing for EGFR and KRAS mutation and in situ hybridization for EML-ALK fusion, resulting in slow turnaround, and technical as well as logistical complexities. Here, we report the development of an automated microarray based quantitative nuclease protection assay that determines gene fusions, deletions and point mutations in target genes such as EML4/ALK, EGFR and KRAS from expressed mRNA directly from FFPE tissue without prior RNA extraction. Methods: We designed mutation and fusion specific probes for nuclease protection and measured the sensitivity, specificity, and reproducibility of gene mutations and fusions using in vitro transcribed RNA and cell lysates known to contain the target mutations. In addition to synthetic samples, we also screened 88 lung cancer specimen for EML-ALK fusions. Results: Mutations in KRAS (G12D, G12C, G12V); EGFR (D761Y, T790M, L858R); EML-ALK fusions (v1 v2, v3a, v3b-3, v4, v5a, v5b-3 and v6) were detected at concentrations of 3 copies/cell in 15,000 cells even when present as low as 5% of wild type IVT RNA. The sensitivity corresponds to an amount of FFPE sample ranging from 0.3 to 0.6 square cm of a 5mm FFPE section per replicate assay making it suitable for needle biopsy specimen. Six samples with probable EML4-ALK fusions were identified in the 88 adenomatous NSCLC samples and will be subjected to further confirmation. Additional clinical validation of all the assays will be presented in our poster. Conclusions: An automated, multiplexed, EGFR and KRAS mutation and EML-ALK gene fusion from expressed mRNA is feasible. The assay can be run directly on FFPE sections, does not require RNA extraction or amplification, and is completed within 24 hours.
Type of Medium:
Online Resource
ISSN:
0732-183X
,
1527-7755
DOI:
10.1200/jco.2013.31.15_suppl.e22185
Language:
English
Publisher:
American Society of Clinical Oncology (ASCO)
Publication Date:
2013
detail.hit.zdb_id:
2005181-5