Pyrosequencing cut-off value identifying BRAFV600E mutation in fine needle aspiration samples of thyroid nodules

• Published in: Clinical endocrinology (Oxford) 2011-10, Vol.75 (4), p.555-560
• Main Authors: Yeo, Min-Kyung, Liang, Zhe Long, Oh, Taejeong, Moon, Youngho, An, Sungwhan, Kim, Min Kyeong, Kim, Koon Soon, Shong, Minho, Kim, Jin-Man, Jo, Young Suk
• Format: Article
• Language: English
• Subjects: Adult; Biological and medical sciences; Biopsy, Fine-Needle; DNA Mutational Analysis; Endocrinopathies; Female; Fundamental and applied biological sciences. Psychology; Humans; Male; Medical sciences; Middle Aged; Mutation; Non tumoral diseases. Target tissue resistance. Benign neoplasms; Proto-Oncogene Proteins B-raf - genetics; Thyroid Nodule - genetics; Thyroid. Thyroid axis (diseases); Vertebrates: endocrinology
• ISSN: 0300-0664; 1365-2265
• DOI: 10.1111/j.1365-2265.2011.04115.x

Summary Context  Recently, tremendous efforts have been made towards the development of sensitive techniques to detect the BRAFV600E mutation in fine needle aspiration biopsy (FNAB) samples. However, newly developed quantitative and semi‐quantitative methods, such as dual‐priming oligonucleotide (DPO)‐based multiplex polymerase chain reaction (PCR), have the potential to generate false‐positive (FP) results. Objectives  To eliminate the possibility of FP results, we generated a receiver operating characteristic (ROC) curve to investigate the diagnostic accuracy of pyrosequencing using quantitative data. Design  Cytological diagnoses of 983 thyroid nodules were made according to the Bethesda System 2007. The BRAFV600E mutation was analysed by pyrosequencing, and statistical analyses were performed. Results  Of the 983 nodules, 902 were adopted to evaluate the diagnostic value of pyrosequencing. The number of pathologically confirmed malignancies was 192, of which 182 were papillary thyroid cancer (PTC). By generating an ROC curve, we defined the optimal cut‐off value of the mutant allele peak as 5·95% (area under the curve, 0·849; sensitivity, 0·55; 1‐specificity, 0). When we applied this selective cut‐off value, the number of PTCs positive for BRAFV600E was 99 (54·4% of the total number of PTCs). With cytology alone, the diagnostic sensitivity and specificity of detecting malignancy were 71·2% and 100%, respectively. Pyrosequencing improved the diagnostic sensitivity from 71·2% to 78·5% (McNemar’s test, P  0·05). Conclusions  Pyrosequencing is an effective method for detecting the BRAFV600E mutation in FNAB samples. By allowing the optimal cut‐off value to be determined, pyrosequencing improves the diagnostic sensitivity while eliminating the possibility of FP results.