Rapid KRAS, EGFR, BRAF and PIK3CA Mutation Analysis of Fine Needle Aspirates using allele-specific qPCR

Ronald van Eijk, Jappe Licht, Hans Morreau, Tom van Wezel
Leiden University Medical Center, Netherlands, The

Fine needle aspiration based novel techniques for the diagnosis and staging of diverse cancers have been incorporated into cancer staging guidelines. The concordance rate of the mutation analysis between these cytological aspirates and histological samples obtained by surgical staging is often unknown. Therefore, we studied the extent to which allele-specific quantitative real-time PCR with hydrolysis probes could be reliably performed on fine needle aspirates by comparing the results with histological material from the same patient. Assays for the detection of seven different KRAS, three PIK3CA and one BRAF variant together with two different EGFR assays have been used on a series of patients for whom cytological and histological material was available. Raw data from the LC480 or CFX384 software were imported into an in-house–created Microsoft Excel 2003 spreadsheet to define the mutation status. We demonstrated that these standard molecular techniques can be accurately applied on fine needle cytological aspirates.

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