High Sensitivity EGFR Mutation Detection in Cytologic Preparations Enabled by Laser Capture Microdissection (LCM).
Sinchita Roy Chowdhuri, Liqiang Xi, Trinh Pham, Jeffrey Hanson, Giuseppe Giaccone, Michael Emmerert-Buck, Mark Raffeld, Armando C Filie. National Cancer Institute, Bethesda, MD
Background: The discovery of activating EGFR mutations in a subset of non-small cell lung carcinoma (NSCLC) was a major advance in our understanding of NSCLC biology, and has led to groundbreaking studies that have demonstrated the effectiveness of the tyrosine kinase inhibitors gefitinib and erlotinib in this disease. Fine needle aspirates (FNA) and other cytologic procedures have become increasingly popular for obtaining diagnostic material in NSCLC. However, frequently the small amount of material or sparseness of tumor cells obtained from cytologic preparations limit the number of specialized studies, such as EGFR mutation testing, that can be performed. In this study, we report a method, using laser capture microdissection (LCM), to assess EGFR mutations in cytologic preparations from small numbers of tumor cells.
Design: Cytology specimens including 3 FNAs and 2 pleural effusions from NSCLC patients with known EGFR mutations were reviewed. One case (lung FNA) containing an L858R mutation with sufficient material was selected to perform sensitivity assays. LCM was performed to obtain decreasing numbers of cells (250, 200, 150, 100, and 50) to determine the sensitivity of the method. 300-500 cells from the remaining 4 cases were obtained for study. DNA was extracted and divided directly into two reactions to detect the two most common EGFR mutations (exon 19 deletion and the L858R mutation), using capillary electrophoresis or pyrosequencing, respectively, after PCR.
Results: EGFR mutations were detected in all five cases, and were identical to the original mutations detected at the initial clinical evaluation. The sensitivity assay revealed that the L858R mutation could be from as few as 50 malignant cells. In one case, a pleural effusion, whole slide scraping (4 slides) of the cell block failed to detect the mutation; whereas LCM assisted analysis using a single slide (approximately 300 cells) was able to identify the appropriate mutation.
Conclusions: In this study we report a method that allows for detection of EGFR mutations in limited cytologic material using as few as 50 tumor cells. As more patients are diagnosed by minimally invasive procedures, such as FNAs and pleural taps, LCM can be a valuable tool to maximize the information that can be obtained from these small samples.
Tuesday, March 1, 2011 9:00 AM
Platform Session: Section F, Tuesday Morning