Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Apr 10:3:10.
doi: 10.1186/1742-6413-3-10.

Utility of BRAF V600E mutation detection in cytologically indeterminate thyroid nodules

Affiliations

Utility of BRAF V600E mutation detection in cytologically indeterminate thyroid nodules

Leslie R Rowe et al. Cytojournal. .

Abstract

Background: Fine needle aspiration (FNA) is widely utilized for evaluation of patients with thyroid nodules. However, approximately 30% are indeterminate for malignancy. Recently, a mutation in the BRAF gene has been reported to be the most common genetic event in papillary thyroid carcinoma (PTC). In this retrospective study, we assessed the utility of BRAF V600E mutation detection for refining indeterminate preoperative cytologic diagnoses in patients with PTC.

Methods: Archival indeterminate thyroid FNAs and corresponding formalin-fixed, paraffin-embedded (FFPE) surgical samples with PTC were identified in our patient files. DNA extracted from slide scape lysates and 5 mum FFPE sections were evaluated for the BRAF V600E mutation using LightCycler PCR and fluorescent melting curve analysis (LCPCR). Amplification products that showed deviation from the wild-type genomic DNA melting peak, discordant FNA and FFPE matched pairs, and all benign control samples, underwent direct DNA sequencing.

Results: A total of 19 indeterminate thyroid FNAs demonstrating PTC on FFPE surgical samples were included in the study. Using BRAF mutation analysis, the preoperative diagnosis of PTC was confirmed in 3/19 (15.8%) FNA samples that could not be conclusively diagnosed on cytology alone. However, 9/19 (47.4%) FFPE tissue samples were positive for the V600E mutation. Of the discordant pairs, 5/6 FNAs contained less than 50% tumor cells.

Conclusion: When used with indeterminate FNA samples, BRAF mutation analysis may be a useful adjunct technique for confirming the diagnosis of malignancy in an otherwise equivocal case. However, overall tumor cell content of some archival FNA smear slides is a limiting factor for mutation detection.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Example of indeterminate thyroid fine needle aspiration (FNA). Fine needle aspirate sample from 28 year-old female with thyroid nodule. Case was interpreted as "fragments of atypical epithelial cells in a background of reactive lymphocytes. A follicular neoplasm or papillary carcinoma cannot be excluded." The thyroidectomy specimen showed a classical papillary thyroid carcinoma in the corresponding lobe of the thyroid.
Figure 2
Figure 2
Melting curve analysis of BRAF mutations in thyroid FNA samples. Overlapping fluorescein-labeled oligonucleotide probes were used to scan extracted DNA for mutations in exon 15 of BRAF. Multiple probes complementary to the wild-type (WT) sequences were placed within the same reaction, and the different sites were identified by their specific probe/target duplex melting temperatures. The position of each probe/target melting temperature and the relative ratio of the melting peak areas determined WT profiles. After amplification in a LightCycler, the instrument begins a melting program where the reactions are cooled to anneal the probes and then slowly heated (0.1°C/s) while fluorescence is continuously monitored. Somatic mutations are identified by changes from a characteristic WT melting curve profile. When melting curves from non-mutated and mutated samples are compared, additional melting peaks or changes in peak-area ratios indicate a sequence alteration (nucleotide mismatch) under the probe. Melting curve analysis revealed that for the WT BRAF sequence (GTG) Tm was 65.34°C ± 0.37°C, the GTG→GAG mutation at nucleotide 1799, resulted in a shift to 60.23°C ± 0.53°C.

Similar articles

Cited by

References

    1. Vander JB, Gaston EA, Dawber TR. The significance of nontoxic thyroid nodules. Final report of a 15-year study of the incidence of thyroid malignancy. Ann Intern Med. 1968;69:537–40. - PubMed
    1. Tyler DS, Shaha AR, Udelsman RA, Sherman SI, Thompson NW, Moley JF, et al. Thyroid cancer: 1999 update. Ann Surg Oncol. 2000;7:376–98. doi: 10.1007/s10434-000-0376-3. - DOI - PubMed
    1. Belfiore A, Giuffrida D, La Rosa GL, Ippolito O, Russo G, Fiumara A, et al. High frequency of cancer in cold thyroid nodules occurring at young age. Acta Endocrinol (Copenh) 1989;121:197–202. - PubMed
    1. Werk EE, Jr, Vernon BM, Gonzalez JJ, Ungaro PC, McCoy RC. Cancer in thyroid nodules. A community hospital survey. Arch Intern Med. 1984;144:474–6. doi: 10.1001/archinte.144.3.474. - DOI - PubMed
    1. Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, et al. Cancer statistics, 2004. CA Cancer J Clin. 2004;54:8–29. - PubMed