Comparison between two widely used laboratory methods in BRAF V600 mutation detection in a large cohort of clinical samples of cutaneous melanoma metastases to the lymph nodes.
OBJECTIVE
The study compares detection rates of oncogenic BRAF mutations in a homogenous group of 236 FFPE cutaneous melanoma lymph node metastases, collected in one cancer center. BRAF mutational status was verified by two independent in-house PCR/Sanger sequencing tests, and the Cobas® 4800 BRAF V600 Mutation Test.


RESULTS
The best of two sequencing approaches returned results for 230/236 samples. In 140 (60.9%), the mutation in codon 600 of BRAF was found. 91.4% of all mutated cases (128 samples) represented p.V600E. Both Sanger-based tests gave reproducible results although they differed significantly in the percentage of amplifiable samples: 230/236 to 109/143. Cobas generated results in all 236 cases, mutations changing codon V600 were detected in 144 of them (61.0%), including 5 not amplifiable and 5 negative in the standard sequencing. However, 6 cases positive in sequencing turned out to be negative in Cobas. Both tests provided us with the same BRAF V600 mutational status in 219 out of 230 cases with valid results (95.2%).


CONCLUSIONS
The total BRAF V600 mutation detection rate didn't differ significantly between the two methodological approaches (60.9% vs. 61.0%). Sequencing was a reproducible method of V600 mutation detection and more powerful to detect mutations other than p.V600E, while Cobas test proved to be less susceptible to the poor DNA quality or investigator's bias. The study underlined an important role of pathologists in quality assurance of molecular diagnostics.
Genomed Health Care Centre Ponczowa 12, Warsaw 02-971, Poland.
Monika
M
Jurkowska
Department of Molecular and Translational Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Roentgena 5, Warsaw 02-781, Poland.
Aleksandra
A
Gos
Department of Pathology, Center of Postgraduate Medical Education Marymoncka 99/103, Warsaw 01-809, Poland.
Konrad
K
Ptaszyński
Department of Pathology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Roentgena 5, Warsaw 02-781, Poland.
Wanda
W
Michej
Department of Molecular and Translational Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Roentgena 5, Warsaw 02-781, Poland.
Andrzej
A
Tysarowski
Department of Molecular and Translational Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Roentgena 5, Warsaw 02-781, Poland.
Renata
R
Zub
Department of Molecular and Translational Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Roentgena 5, Warsaw 02-781, Poland.
Janusz A
JA
Siedlecki
Department of Soft Tissue/Bone Sarcoma and Melanoma, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Roentgena 5, Warsaw 02-781, Poland.
Piotr
P
Rutkowski
D014408:Biomarkers, Tumor / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods* / Q000592:standards; D005240:Feasibility Studies; D020022:Genetic Predisposition to Disease; D006801:Humans; D008207:Lymphatic Metastasis; D008545:Melanoma / Q000201:enzymology / Q000235:genetics* / Q000556:secondary; D009154:Mutation; D015588:Observer Variation; D010641:Phenotype; D011237:Predictive Value of Tests; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D011786:Quality Control; D015203:Reproducibility of Results; D012878:Skin Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology
D014408:Biomarkers, Tumor; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
BRAF; Melanoma; analytical sensitivity; detection; mutation; sequencing