title: Application of BRAF, NRAS, KRAS mutations as markers for the detection of papillary thyroid cancer from FNAB specimens by pyrosequencing analysis.
abstract: BACKGROUND
  BRAFV600E, the most common BRAF gene mutation, is detected in approximately 50% of sporadic papillary thyroid carcinoma (PTC) and may be associated with triggering tumorigenesis of PTC. The aim of our study was to discover additional mutations to increase the diagnostic performance of molecular tests in screening for thyroid cancer from fine needle aspiration biopsy (FNAB) specimens.
  
  
  METHODS
  DNA was extracted from 120 freshly obtained FNAB specimens selected according to cytopathology grades of the Bethesda system. A conventional BRAF V600E test was carried out with real-time PCR, and further mutation screening for BRAF mutations in codons 464, 466, 469, NRAS and KRAS codons 12/13 and 61 was done by pyrosequencing. Histopathology reports were reviewed for those who underwent thyroidectomy (n=83).
  
  
  RESULTS
  The real-time PCR method detected 45 BRAF V600E- positive cases whereas pyrosequencing detected 30 cases. Additional BRAF (n=4), NRAS (n=11) and KRAS (n=3) mutations were detected in 17 cases (one overlapping BRAF and NRAS mutation). Among 11 NRAS-mutated cases, eight were confirmed as PTC and one as FVPTC on histopathology reports. Five PTC-confirmed cases with BRAF V600E mutation showed additional mutations, all of which were NRAS mutations.
  
  
  CONCLUSIONS
  Despite the higher sensitivity of real-time PCR for detecting BRAFV600E mutations, pyrosequencing easily detected additional point mutations. NRAS mutations were the most prevalently identified additional mutations and were highly associated with malignancy. In conclusion, our findings demonstrate that additional mutations identified by pyrosequencing may help in the pre-operative process in determining the possibility of malignancy and further studies on the occurrence of simultaneous mutations of BRAF, KRAS and NRAS may be warranted.
authors.affiliation: Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea.
authors.forename: Seo-Jin
authors.initials: SJ
authors.lastname: Park
authors.forename: Je Young Hannah
authors.initials: JY
authors.lastname: Sun
authors.forename: Kyungran
authors.initials: K
authors.lastname: Hong
authors.forename: Jin Young
authors.initials: JY
authors.lastname: Kwak
authors.forename: Eun-Kyung
authors.initials: EK
authors.lastname: Kim
authors.forename: Woung Youn
authors.initials: WY
authors.lastname: Chung
authors.forename: Jong Rak
authors.initials: JR
authors.lastname: Choi
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D044963:Biopsy, Fine-Needle; D002277:Carcinoma / Q000175:diagnosis* / Q000235:genetics; D002291:Carcinoma, Papillary; D020558:GTP Phosphohydrolases / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D000077273:Thyroid Cancer, Papillary; D013964:Thyroid Neoplasms / Q000175:diagnosis* / Q000235:genetics; D018631:ras Proteins / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; C117307:KRAS protein, human; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins