title: Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine-needle aspirate samples.
abstract: BACKGROUND
  BRAF mutation V600E (substitution Val600Glu) is a molecular signature for papillary thyroid carcinoma (PTC). Testing for BRAF mutation is clinically useful in providing prognostic prediction and facilitating accurate diagnosis of PTC in thyroid fine-needle aspirate (FNA) samples.
  
  
  METHODS
  This study assessed the correlation of cellularity with DNA yield and compared 2 technical platforms with different sensitivities in detection of BRAF mutation in cytologic specimens. Cellularity was evaluated based on groups of 10+ cells on a ThinPrep slide: 1+ (1-5 groups), 2+ (6-10 groups), 3+ (11-20 groups), and 4+ (> 20 groups). Genomic DNA was extracted from residual materials of thyroid FNAs after cytologic diagnosis.
  
  
  RESULTS
  Approximately 49% of thyroid FNA samples had low cellularity (1-2+). DNA yield is proportionate with increased cellularity and increased nearly 4-fold from 1+ to 4+ cellularity in cytologic samples. When applied to BRAF mutational assay, using a cutoff of 6 groups of follicular cells with 10+ cells per group, 96.7% of cases yielded enough DNA for at least one testing for BRAF mutation. Five specimens (11.6%) with lower cellularity did not yield sufficient DNA for duplicate testing. Comparison of Sanger sequencing to allele-specific polymerase chain reaction methods shows the latter confers better sensitivity in detection of BRAF mutation, especially in limited cytologic specimens with a lower percentage of malignant cells.
  
  
  CONCLUSIONS
  This study demonstrates that by using 6 groups of 10+ follicular cells as a cutoff, nearly 97% of thyroid FNA samples contain enough DNA for BRAF mutational assay. Careful selection of a molecular testing system with high sensitivity facilitates the successful conduction of molecular testing in limited cytologic specimens. Cancer (Cancer Cytopathol) 2014;122:114-22 © 2013 American Cancer Society.
authors.affiliation: Department of Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Dyhdalo
authors.forename: Stephen
authors.initials: S
authors.lastname: Macnamara
authors.forename: Jennifer
authors.initials: J
authors.lastname: Brainard
authors.forename: Dawn
authors.initials: D
authors.lastname: Underwood
authors.forename: Raymond
authors.initials: R
authors.lastname: Tubbs
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D044963:Biopsy, Fine-Needle / Q000379:methods*; D002277:Carcinoma / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D002291:Carcinoma, Papillary; D004247:DNA / Q000032:analysis*; D004252:DNA Mutational Analysis / Q000379:methods; D005260:Female; D015894:Genome, Human; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D000077273:Thyroid Cancer, Papillary; D013961:Thyroid Gland / Q000473:pathology*; D013964:Thyroid Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology
chemical_list: D004247:DNA; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
keywords: 1790T>A; BRAF; DNA yield; L597Q; cellularity; fine-needle aspirate; molecular cytology; papillary thyroid carcinoma; polymerase chain reaction; thyroid cytology