Liquid biopsy of colorectal cancer: a new approach to evaluation of aberrant methylation of the <i>SEPT9</i> gene

Abstract

Introduction. Hypermethylated CpG islands in the promoters of suppressor genes (in particular, SEPT9) are clinically significant markers of malignant growth that are widely used in liquid biopsy. Real-time polymerase chain reaction with methylation-specific primers is commonly used to quantify hypermethylated DNA. The method requires data normalization, depends on copy number variability of the calibrator genes, and is rather laborious.The study subject is to develop an alternative qDMA method (quantitative DNA Melting Analysis).Materials and methods. DNA samples isolated from blood plasma of healthy donors and colorectal cancer patients were analyzed by the method including: 1) asymmetric polymerase chain reaction with methylation-independent individually selected primers for the SEPT9 gene; 2) using the TaqMan probe hybridizing to two CpG dinucleotides in the amplicon; 3) post-amplification melting of probe/amplicon hybrids; 4) quantitative analysis of DNA melting.Results. The method was tested on the SEPT9 gene in liquid biopsy of colorectal cancer. Differences in SEPT9 methylation in healthy donors (n = 41) and cancer patients (n = 39) were statistically significant (p <0.0001). Analytical sensitivity and diagnostic efficiency of qDMA were determined: AUC (area under curve) ROC– 0.812 (according to the result of 10-fold cross-validation AUC ROC – 0.801), sensitivity – 90%, specificity – 66%.Conclusion. The proposed method for the quantitative assessment of aberrantly methylated DNA is simple, implemented in the closed-tube format, does not require normalization and usage of standard curves. The possibility of optimization through the use of a multiplex variant with simultaneous analysis of several markers is assumed.

Keywords

• liquid biopsy
• colorectal cancer
• sept9 gene
• dna melting analysis