Influence of mucinous and necrotic tissue in colorectal cancer samples on KRAS mutation analysis

Evaluation of the RAS mutation status is necessary for patients with advanced colorectal cancer to predict the response to anti-EGFR therapy. In routine diagnostics, FFPE tissue samples are tested by sequencing (amplicon-based NGS and Sanger) to obtain the RAS status of the patient. Samples that are collected after chemotherapy occasionally contain necrotic tissue. Furthermore, colorectal cancer tissue sometimes has mucinous components. This may pose a challenge to molecular analysis because mucinous tumor samples often contain only few tumor cells compared to solid tumor samples. Therefore, the aim of this study was to explore if mucin or necrosis affect mutation analysis and if mucinous tumor samples contain enough tumor cells for reliable mutation detection. To this end, we analyzed KRAS status in 10 samples showing mucin production and 10 samples with necrosis. In all 20 samples the tissue areas with the highest amount of mucin or necrosis were used for re-evaluation. These results show no differences with those obtained during routine diagnostics, where analysis of mucinous or necrotic areas was tried to avoid. Our study thus shows that mucin and necrosis have no influence on KRAS mutation analysis. Furthermore we were able to demonstrate that mucinous adenocarcinoma contains enough tumor cells for a valid mutation analysis. In addition, we also observed only minor differences in KRAS status results when comparing Sanger sequencing with NGS. Both methods detected the KRAS mutation in 19 of 20 tested samples, even for mutated samples with low allele-frequencies.