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Sensitive allele-specific real-time PCR test for mutations in BRAF codon V600 in skin melanoma

Pisareva, Ekaterinaa; Gutkina, Nadezhdaa; Kovalenko, Sergeia,b,d; Kuehnapfel, Sarahe; Hartmann, Arndte; Heinzerling, Lucief; Schneider-Stock, Reginee; Lyubchenko, Lyudmilac; Shamanin, Vladimir A.a,b

doi: 10.1097/CMR.0000000000000090

Mutations at BRAF codon V600 are used as predictive biomarkers for targeted therapy of skin melanoma. Here, a simple sensitive test to detect mutations of BRAF-V600 was developed using real-time PCR with allele-specific primers and TaqMan probes. Two versions of the test using sense and antisense allele-specific primers were designed and evaluated. The test detected 1% mutant allele V600E/K in 10 ng DNA standard made from wild-type human DNA spiked with BRAF-V600E or the V600K plasmid. The test was validated on clinical formalin-fixed paraffin-embedded samples of skin melanoma using pyrosequencing as a reference method. In the clinical samples, we detected the common mutation V600E, as well as the rare mutations V600K, V600E2 (codon GAA), V600E2 K601del, V600D-K601del, and V600R. In comparison with pyrosequencing, both versions of the test had 100% specificity with sensitivities of 97 and 86% for sense and antisense allele-specific primers, respectively. Using the PCR test with sense allele-specific primers, mutations in V600 were found in 33 of 51 Russian patients (64.7%) with cutaneous melanoma. This closed-tube real-time PCR test can be used as a simple and sensitive assay for mutations of BRAF-V600 in cutaneous melanoma.

aInstitute of Molecular Biology and Biophysics

bBioLink Ltd, Novosibirsk

cN.N.Blokhin Russian Cancer Research Center, Moscow, Russia

dBioron GmbH, Ludwigshafen

eInstitute of Pathology

fDepartment of Dermatology, University Hospital Erlangen, Erlangen, Germany

Correspondence to Vladimir A. Shamanin, PhD, BioLink Ltd, ul.Timakova 2, Novosibirsk 630117, Russia Tel/fax: +7 383 334 8614; e-mail:

Received July 26, 2013

Accepted April 28, 2014

© 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins