Detection of BRAF mutations in melanoma: Rate of mutation detection at codon 600 using Sanger sequencing as compared to the cobas 4800 method.

Melanoma/Skin Cancers
Session Type and Session Title: 
General Poster Session, Melanoma/Skin Cancers
Abstract Number: 



J Clin Oncol 30, 2012 (suppl; abstr 8596)


Kevin Z. Qu, Qiulu Pan, Xi Zhang, Luis Rodriguez, Jennifer Uyeji, Hairong Li, Albert Ho, Heather Sanders, Anthony Sferruzza, Daniel Jones, Frederic Waldman, Quest Diagnostics Nichols Institute; Nichols Institute, San Juan Capistrano, CA; Nichols Institute, Chantilly, VA; Nichols Instutute, Chantilly, VA; Quest Diagnostics Nichols Institute, Chantilly, VA; Quest Diagnostics, San Juan Capistrano, CA

Abstracts that were granted an exception in accordance with ASCO's Conflict of Interest Policy are designated with a caret symbol (^).

Abstract Disclosures


Background: Detection of BRAF V600 mutations is currently a prerequisite for approved use of vemurafenib in patients with metastatic melanoma. The cobas 4800 BRAF V600 Mutation Test (Roche Molecular Diagnostics), a PCR-based assay approved to aid in selecting patients for vemurafenib therapy, primarily detects V600E. It is also reported to detect V600K, which has been associated with vemurafenib response as well. We compared the mutation detection rate of the cobas assay with that of Sanger sequencing. Methods: 125 de-identified FFPE tissues submitted for BRAF mutation analysis that all showed histologically-confirmed melanoma were tested. BRAF mutations were detected using both the cobas kit and bidirectional Sanger sequencing using BigDye kits (Applied Biosystems). DNA was extracted from 5-um sections without macrodissection using the cobas DNA extraction kit (for the cobas test) or from 5-10-um sections using Agencourt extraction kits (Beckman Coulter) following macrodissection. Results: The two methods showed agreement in 104/125 (83.2%) of cases (Table). Sanger sequencing detected V600 dinucleotide mutations in 9 samples that were negative by the cobas assay. Sanger sequencing produced no results in 10 cases owing to suboptimal PCR, including 2 that were positive by the cobas assay. The cobas assay produced 2 invalid results, including 1 that was positive for V600E by Sanger.The cobas assay detected 7/11 V600K mutations. Conclusions: Overall agreement between cobas and Sanger sequencing was 83.2%. The Sanger method had higher analytic sensitivity, resulting in nine additional V600 mutations not called by cobas compared to the two seen by cobas but not Sanger sequencing. Thus, 16% (9/57) more patients would be identified as candidates for vemurafenib therapy using the Sanger method.
Not detected Sanger
V600E V600K V600R Non-600 No result Total
Not detected 56 3a 4b 2c 2d 7 74
cobas detected 40 7 2 49
Invalid 1 1 2
Total 56 44 11 2 2 10 125

a 2/3 had dinucleotide mutations (GTG>GAA) and 1 had V600_K601del. b All had dinucleotide mutations (GTG>AAG). c Both had GTG>AGG dinucleotide mutations. d 1) G469R; 2) E501G.