BRAF V600E Detection for Papillary Thyroid Carcinoma by LUNA ARMS PCR.
GDenice Smith, Luming Zhou, Leslie R Rowe, Barbara E Chadwick, Elke A Jarboe, Carl T Wittwer, Brian T Collins. ARUP Laboratories, Salt Lake City, UT; University of Utah, Salt Lake City
Background: Detection of the BRAF V600E mutation in a thyroid fine needle aspirate (FNA) can provide important guidance for the preoperative diagnosis of papillary thyroid carcinoma (PTC), particularly for indeterminate samples. Existing assays may have a low sensitivity for the detection of BRAF-mutant tumor cells in a background of wild type/normal cells. A modified PCR technique, LUNA ARMS (Zhou, et al., 2010), was used to selectively block amplification of wild type BRAF with an unlabeled probe and ARMS primer that favor amplification/detection of the 1799 T>A mutation in exon 15, which changes Val to Glu. Mutation detection was by probe melting analysis.
Design: Patient samples with confirmed PTC or benign findings were selected. Tumor cell containing regions of formalin-fixed, paraffin-embedded (FFPE) sections and FNA slides were used for slide scrape and proteinase K DNA extraction. Paired FNA/FFPE samples, previously evaluated by a FRET PCR assay, were tested to determine if the increase in sensitivity of LUNA ARMS would resolve discordant cases. Additional cases with FNA, FFPE, and needle wash samples were also tested.
Results: An analytical sensitivitiy of <0.01% was obtained using dilutions of wild-type control and BRAF V600E mutation-containing cell line DNA. Forty-two cases with paired FNA and FFPE specimens were evaluated for the BRAF V600E mutation by LUNA ARMS PCR. Of 37 cases diagnosed histologically as PTC, 23 (62.2%) carried the BRAF mutation. For the FRET PCR assay, concordance of mutation analysis between FNA and FFPE samples was 100% for cases that were judged to be negative or PTC by cytology, whereas agreement was 72% for cases that were indeterminate by cytology. Concordance between FNA and FFPE indeterminate samples using the LUNA ARMS PCR test increased to >90%. Although the mutation may be detected in some cases in FNA needle washes, detection is more reliable from slide scrape lysates of FNA smears.
Conclusions: Because the surgical approach to patients with thyroid nodules is significantly guided by FNA biopsy, tests for PTC-associated mutations, such as BRAF V600E, can be a useful diagnostic adjunct. The presence of contaminating normal cells in FNA smears, however, requires that sensitive PCR tests be employed to detect the mutation. This study has shown that using the LUNA ARMS test results in the increased detection of BRAF V600E in FNA specimens.
Wednesday, March 2, 2011 1:00 PM
Poster Session VI # 50, Wednesday Afternoon