High-sensitivity BRAF mutation analysis: BRAF V600E is acquired early during tumor development but is heterogeneously distributed in a subset of papillary thyroid carcinomas.
BACKGROUND
The homogeneous distribution of BRAF V600E in papillary thyroid carcinoma (PTC) has been called into question by recent reports. These studies claim that BRAF V600E is heterogeneous and is limited to tumor cell subsets in the majority of PTCs.


OBJECTIVE
The objective of the study was to understand the allele distribution of BRAF V600E by evaluating the percentage of mutated neoplastic cells in a group of PTCs using two different highly sensitive analytical approaches: allele-specific locked nucleic acid PCR and 454 next-generation sequencing targeted to BRAF exon 15.


METHODS
BRAF V600E was investigated using allele-specific locked nucleic acid PCR on 155 consecutive samples of PTC. Mutated cases were reanalyzed by 454 next-generation sequencing and immunohistochemistry. Because the evaluation of genetic heterogeneity in tumor samples can be profoundly biased by contamination with normal cells, all mutation frequency data were normalized to the real amount of neoplastic cells within each tumor.


RESULTS
Eighty-five of 155 PTCs (54.8%) were BRAF V600E mutated. The distribution of mutated neoplastic cells within the tumor was as follows: greater than 80% in 37 of 85 (43.5%), 30-80% in 39 of 85 (45.9%), and less than 30% in 9 of 85 (10.6%). In most of the PTCs with less than 80% BRAF V600E-positive neoplastic cells, the mutation was present in large neoplastic cell subpopulations. Tumors with less than 30% mutated neoplastic cells were smaller than tumors with a percentage of mutated cells greater than 80% or between 30% and 80% (P < .05).


CONCLUSIONS
BRAF V600E is heterogeneously distributed in some PTCs. The large BRAF V600E neoplastic cell subpopulations found in mutated cases is consistent with the view that the BRAF V600E is acquired early during PTC development.
Department of Medicine (D.d.B., V.C., G.T.), Anatomic Pathology Unit, Ospedale Bellaria, University of Bologna, 40139 Bologna, Italy; Department of Pharmacology and Biotechnology (V.C., M.V., A.P.), University of Bologna, 40100 Bologna, Italy; Anatomic Pathology Unit (G.P.C.) and Endocrinology Unit (N.C.), Ospedale Maggiore, 40133 Bologna, Italy; and Molecular Biology Laboratory (G.G., V.S., A.C.) and Anatomic Pathology Unit (M.R.), Istituto di Ricovero e Cura a Carattere Scientifico-Arcispedale Santa Maria Nuova, 42123 Reggio Emilia, Italy.
Dario
D
de Biase
Valentina
V
Cesari
Michela
M
Visani
Gian Piero
GP
Casadei
Nadia
N
Cremonini
Greta
G
Gandolfi
Valentina
V
Sancisi
Moira
M
Ragazzi
Annalisa
A
Pession
Alessia
A
Ciarrocchi
Giovanni
G
Tallini
D000328:Adult; D000368:Aged; D019943:Amino Acid Substitution; D002277:Carcinoma / Q000453:epidemiology / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002291:Carcinoma, Papillary; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D005787:Gene Frequency; D018740:Genetic Heterogeneity; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D000077273:Thyroid Cancer, Papillary; D013964:Thyroid Neoplasms / Q000453:epidemiology / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D014633:Valine / Q000235:genetics; D055815:Young Adult
D018698:Glutamic Acid; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D014633:Valine