Laser microdissection for the analysis of molecular heterogeneity in colorectal cancer

Abstract

Oncogenic driver mutations in RAS/RAF oncogenes are frequent in colorectal cancer (CRC). The presence of different subclones within asingle tumor can lead to treatment failure in anti-EGFR/epidermal growth factor receptor-directed antibody therapies. The identification of different subclones and their mutational profiles within asingle tumor and the identification of morphologically distinct tumor areas might help to unravel novel aspects of tumor biology and therapy resistance. The aim of this study was to identify intratumoral heterogeneity in CRC by using laser microdissection (LMD) in comparison to the routinely used method. We hereby applied LMD to identify and investigate tumor heterogeneity in CRC. We established LMD and purified DNA from several morphologically distinct tumor areas (n = 13) in CRCs from 2patients and compared the results from routine testing to our newly established LMD approach. LMD enabled the comparative analysis of small tumor areas by cutting histologically selected elements under microscopic control using alaser beam. In some cases, potential low-level mutations (PLLM) could not be detected using the routine method since they were masked by high-level mutations (HLM). The application of LMD enabled the identification of concomitant PLLM in NRAS and BRAF genes in the identical patient sample. LMD improved spatial resolution in the molecular analysis of CRC tumor tissue compared to routine methods. Our results confirmed the presence of molecular heterogeneity in CRC. This should be kept in mind when interpreting sequencing results, since low frequency mutations can have an impact on the effectiveness of targeted therapy.