A feasibility study of a fully automated molecular diagnostic system for mutation analysis of colorectal cancer.

Abstract

e21074 Background: KRAS and BRAF mutations are strong molecular predictors for efficacy of cetuximab and panitumumab in colorectal cancer (CRC). We have developed a novel, simple, sensitive and fully automated DNA mutation detection system (Toppan Genotyping Analyzer, TGA) based on Invader Plus® technology. Here we report the validation study of our system, comparing it to DS in the detection of KRAS and BRAF mutations. Methods: Assays were set up using plasmids containing major KRAS (G12A, G12C, G12D, G12R, G12S, G12V and G13D) and BRAF (V600E) mutations. Sensitivity and accuracy of the detection method were evaluated with plasmids, as well as cancer cell lines with KRAS or BRAF mutations. DNA samples were extracted from frozen (n=31) and formalin fixed, paraffin embedded (FFPE) samples (n=36). Results: Sensitivity and accuracy assays using genomic DNA derived from cancer cell lines and plasmid DNA showed that the TGA system can detect mutations at a 5% level. The chip-based assay system allows for simultaneous analysis of 23 mutations in one hour (including the PCR process). The system was compared with two sets of DS, which had different primer sites. KRAS mutations detected in both frozen (DS1:12/31, DS2:11/31) and FFPE (DS1:7/36, DS2:8/36) samples were also successfully detected by the TGA. In the samples shown to be wild-type by DS, TGA was able to detect additional mutants in the frozen (vs. DS1:1/31, vs. DS2:2/31) and FFPE (vs. DS1:4/36, vs. DS2:3/36) samples. In addition, the frequency of BRAFmutation in frozen (1/31) and FFPE (0/36) was concordant between DS and TGA. Conclusions: In terms of detection of KRAS and BRAF mutations, TGA is a highly sensitive and accurate system compared to DS. It also possesses several other advantages including its all-in-one chip reaction, simple procedure and excellent reproducibility. The versatility in detecting mutations in DNA samples with different fixative forms makes the TGA a useful system that can be widely applied in both diagnosis and research areas. We hypothesize that TGA is superior to DS for predicting efficacy of molecularly targeted agents, since it has greater sensitivity for detection of mutations associated with drug resistance.