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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 < 0·001), without any change in the diagnostic specificity. When 'suspicious for malignancy' was considered a positive cytological outcome, pyrosequencing increased the diagnostic sensitivity of cytology from 95·8% to 96·9%; however, this improvement did not show statistical significance (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. [ABSTRACT FROM AUTHOR]

Context Dideoxy sequencing is the most commonly used method for detecting the BRAFV600E mutation in thyroid cancer and melanoma. However, this gold standard method often makes less definite results in detecting the BRAF V600E mutation when there are relatively low amounts of the mutant template in biopsy specimens, which are invariably contaminated with normal tissues. Pyrosequencing, which measures the incorporation of each of the four nucleotides at each template position and indicates the amounts of mutant template present, may be more useful in such situations. Objective To investigate the diagnostic efficiency of pyrosequencing for the mutant BRAF allele in ultrasound (US)-guided fine needle aspiration biopsies (FNABs) of thyroid incidentalomas. Design, setting and subjects A total of 101 thyroid incidentaloma cases were included prospectively. Cytological diagnoses of the FNAB samples were made according to the American Thyroid Association (ATA) guidelines, 2006. The presence of the BRAF V600E mutation was investigated by pyrosequencing and dideoxy sequencing. Results On the basis of cytological analysis, the thyroid incidentalomas were classified into benign ( n = 43), malignant ( n = 30), indeterminate or suspicious neoplasm ( n = 24), and nondiagnostic ( n = 4) categories. Pyrosequencing detected the BRAF V600E mutation in 30 cases: 22 malignant cases, 7 indeterminate cases, and 1 nondiagnostic case. Dideoxy sequencing also detected the BRAF V600E mutation in 28 of the same cases but failed to clearly distinguish the mutant allele from the wild-type allele in one indeterminate case and one nondiagnostic case. Histopathological analysis ascertained that all BRAF V600E-positive cases were papillary thyroid carcinomas. Conclusions Pyrosequencing may be suitable for detecting the BRAF V600E mutation in thyroid incidentaloma and may be superior to dideoxy sequencing when low amounts of the mutant template are present in the biopsy. [ABSTRACT FROM AUTHOR]