Detection of KRAS G12D in colorectal cancer stool by droplet digital PCR

AIM To assess KRAS G12 D mutation detection by droplet digital PCR(dd PCR) in stool-derived DNA from colorectal cancer(CRC) patients.METHODS In this study, tumor tissue and stool samples were collected from 70 patients with stage Ⅰ-Ⅳ CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffinembedded(FFPE) tumor tissues. The KRAS G12 D mutation was then analyzed by dd PCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type(WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by dd PCR was performed for these patients as well.RESULTS Among the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32(45.71%), whereas 38(54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors(11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12 D(14.29%, n = 10), which was more frequent in early-stage tumors(I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12 D mutation was detected by dd PCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12 D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation. CONCLUSION dd PCR is a reliable and sensitive method to analyze KRAS G12 D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.

AIMTo assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients.METHODSIn this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type (WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well.RESULTSAmong the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32 (45.71%), whereas 38 (54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors (11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12D (14.29%, n = 10), which was more frequent in early-stage tumors (I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation.CONCLUSIONddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.