FIRST STEPS FOR THE IMPLEMENTATION OF A NON-INVASIVE SCREENING PLATFORM FOR COLORECTAL CANCER USING DIGITAL PCR

BOGADO, JOSEFINA; JULIANA I. SESMA; J. GIRARDINI; HECKEL, SOFIA

Rosario

Congreso; XXIV Congreso y XLII Reunion Anual de la SBR; 2022

Colorectal cancer (CRC) is the second cause of cancer-related death in Argentina and the world. Early diagnosis is critical as it increases up to 90% the chances of cure. Currently, colonoscopy is the gold standard method for CRC diagnosis. However, due to its invasive nature and technical complexity, it has low adhesion rate, reducing the chances of survival. These difficulties, added to the cost of the intervention, limit its use in mass monitoring programs. Consequently, it is a priority to develop non-invasive methodologies for CRC diagnosis.In Argentina, the non-invasive monitoring method used is the fecal occult blood test, which presents insufficient sensitivity levels to provide an accurate diagnosis. The presence of DNA derived from tumor cells in feces or fluids has revolutionized the concept of diagnosis. The identification of genetic and epigenetic alterations in DNA derived from the primary tumor present in fluids and feces constitutes a target of great clinical potential. Mutations in KRAS and TP53 genes are among the most common in CRC. On the other hand, alterations in the methylation patterns of the promoters of specific genes are related to the development of the pathology. So far, there are no non-invasive methods with sufficient sensitivity to give an accurate diagnosis.For this reason, we propose to develop a non-invasive quantitative diagnostic method for CRC using an ultrasensitive and quantitative technology: droplet digital PCR (ddPCR). We started with biomarkers that have demonstrated their diagnostic potential in other non-invasive methods, with the aim of looking for the best combination of them. We have achieved a first set-up for the selective detection of one of the most frequent KRAS mutations, G13D, working with the HCT-116 cell line that presents an allele with the G13D mutation and a wild-type (WT) allele for KRAS. DNA extraction was performed by extraction columns, and two methods of DNA disruption (sonication and use of restriction enzymes) we used to improve detection by Real Time-PCR. Once the purification of the DNA was set up, we carried out the assays by ddPCR. We used different concentrations of DNA from the HCT-116 cell line and in all cases, both alleles could be detected in 1:1 ratios. The next tests to be carried out are to reduce the G13D/WT ratio, forcing the method to know its detection limit and validate it. In this pilot test, we fine-tuned the first determinations to detect a KRAS mutant allele in the presence of KRAS WT by ddPCR.