Abstract 1878: Performance comparison of BRAF V600E detection assays in malignant melanoma and colorectal cancer specimens
Authors: Loes Inger Marie , Immervoll Heike , Sorbye Halfdan , Angelsen Jon-Helge , Horn Arild , Lonning Per Eystein , Knappskog Stian 
Published: May 2015

Abstract
Personalized cancer care requires reliable biomarkers. One already implemented in the clinic is the BRAF V600E mutation. We present a comprehensive comparison of three detection methods including immunohistochemistry (IHC) using a mutation specific monoclonal antibody (VE1), Sanger sequencing and a probe-based high resolution melting assay, in melanoma and colorectal cancer specimens. Genomic DNA (gDNA) from fresh frozen tissue from 389 liver metastases and 6 primary tumors from 141 metastatic colorectal cancer (mCRC) patients as well as gDNA from 125 tumors from 75 malignant melanoma (MM) patients was available. Tissue microarrays (TMAs) containing formalin fixed paraffin embedded (FFPE) tissue from 285 of the CRC metastases from 123 patients were made. BRAF, exon 15 was amplified using the DyNazyme EXT polymerase system and sequenced by Sanger sequencing. IHC staining with the BRAF-VE1 antibody was performed on a Ventana BenchMark XT immunostainer. The intensity of staining was graded as 0 (no visible staining), grade 1 (weak diffuse background staining), 2 (moderate diffuse and granular) and 3 (strong granular staining). Grade 2 or 3 was regarded as positive. For high resolution melting analysis of V600-status, we used the LigthMix Kit BRAF V600E/K on a LightCycler 480 instrument.To establish the detection limits for the DNA-based methods used in this comparison, we analyzed a dilution series containing a ratio of 1:1- 1:107 BRAF V600E mutated DNA in BRAF wild-type DNA. We found the detection limit for the LightMix assay to be 1:1000 mutated alleles while it was 1:10 for Sanger sequencing. Among 125 samples from 75 MM patients, we found 60 samples from 31 patients to be positive for BRAF mutations by Sanger sequencing. By the LigthMix assay, we found 75 mutated samples from 37 patients. Among 395 samples form 141 mCRC patients, we found 22 samples from 7 patients to be BRAF V600E mutated by Sanger sequencing. 127 tumors from 42 of these patients were analyzed by the LigthMix assay and the BRAF V600E mutation was detected in 24 samples from the same 7 patients. IHC was performed on TMAs including tissue from 285 metastases from 123 of the mCRC patients. By this method 25 samples from 15 patients were found to be BRAF V600E mutated. These 15 patients included 5 of the 7 patients identified by DNA-based mutation analyses. Thus 8 patients revealing no mutation by either of the DNA-based methods showed positive immunostaining. Two patients found to carry the mutation by sequencing and melting point analysis were not identified by IHC.
Our data show differences in sensitivity and specificity between the three methods. Compared to the LigthMix, assay Sanger sequencing showed inferior sensitivity. Further, we observed a large discrepancy between IHC and the two DNA-based methods, indicating IHC with the current antibody not to be recommendable for clinical tests alone.
Citation Format: Inger Marie Loes, Heike Immervoll, Halfdan Sorbye, Jon-Helge Angelsen, Arild Horn, Per Eystein Lonning, Stian Knappskog. Performance comparison of BRAF V600E detection assays in malignant melanoma and colorectal cancer specimens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1878. doi:10.1158/1538-7445.AM2014-1878