Onyshchenko K. Identification of genetic and epigenetic changes in human clear cell carcinoma for the development of non-invasive diagnostics

Cancer is associated with various molecular-genetic aberrations that lead to the formation and progression of tumors, but they also offer the potential for predicting the course of the disease. The development of clear cell renal cell carcinoma is linked to alterations in numerous tumor suppressor genes. These genes may undergo inactivation due to mutations, allelic deletions, CpG island methylation in promoter regions, or regulation by various microRNAs. Literature analysis provides grounds to believe that significant progress in early cancer diagnosis can be achieved through the identification of genetic and epigenetic abnormalities in tumor DNA, which can be detected both in solid tumors and in "liquid biopsies." The use of circulating cell-free DNA (cfDNA) as a potential diagnostic marker for tumors has been subsequently confirmed by various groups for different types of malignancies. Therefore, the objective of the dissertation was to identify molecular-genetic changes both in kidney tumor biopsies and in extracellular nucleic acids in blood plasma that could serve as markers for the clear cell subtype of kidney cancer. To achieve this, a comprehensive molecular-genetic investigation was conducted on 122 samples from patients with kidney tumors, predominantly in stages I and II. It has been demonstrated that the expression levels of microRNAs exhibit promising diagnostic potential (AUC = 0.7-0.9) and can be utilized as additional diagnostic markers for clear cell renal cell carcinoma. This study identified genetic (somatic reorganizations) disruptions in genes using short tandem repeat (STR) markers at loci: D3S966 and D3S1568 for the RASSF1 gene, D3S1038, D3S1317, and D3S1038, VHL2, D3S1317 for VHL gene, and D9S916 and D9S974 for CDKN2A. Loss of heterozygosity of the RASSF1 gene was detected in 68.3%, VHL in 48.2%, and CDKN2A in 32.8% of informative samples from kidney cancer patients. Our studies on determining the concentrations cfDNA in the blood of kidney cancer patients revealed elevated cfDNA levels in patients with tumors compared to healthy donors. The use of two diagnostic methods showed that real-time quantitative PCR is more accurate for disease diagnosis (AUC=0.8049) compared to the fluorescence intercalating dye method (AUC=0.7679). An increase in cfDNA concentration in the blood plasma of patients with kidney cancer was also established. The obtained data suggest the potential application of this method as an additional marker in the primary diagnosis of kidney cancer. Previous studies indicate that the "integrity index," along with the level of circulating cell-free DNA (cfDNA) concentration, can serve as markers for early cancer diagnosis and for monitoring the presence of metastatic cells not removed during surgery. In our studies, we assessed the presence of high-molecular-weight cfDNA both for the integrity of the beta-actin gene and using primers simultaneously specific to short and long fragments of the GAPDH gene. Analysis of the results revealed significantly different data for cancer patients and the control group (AUC=0.8613). In the control group, cfDNA was moderately fragmented (median for ACTB384/ACTB106=0.685), while in patients with renal cell carcinoma (RCC), a significantly higher degree of fragmentation was observed (median for ACTB384/ACTB106=1.126). These research methods validated both the increased content of high-molecular-weight DNA in the plasma of kidney cancer patients and the potential use of this method for tumor diagnostics. In the presented study, the methylation of CpG islands in the promoters of 11 tumor suppressor genes (RASSF1A, RASSF1C, LRRC3B, GPX3, PCDH8, RUNX3, APC, CDKN2A (p14ARF), CDKN2A (p16INK4a)) was determined on DNA extracted from tumors and blood plasma of kidney cancer 15 patients in comparison with control samples. It was identified that simultaneous detection of the methylation status of CpG islands in the promoters of RASSF1A, GPX3, APC, and CDKN2A (p14ARF) genes on cfDNA in blood plasma allows for highly reliable identification of kidney tumors (sensitivity – 98%, specificity – 96%). Therefore, in the dissertation, genetic (somatic reorganizations) and epigenetic (gene methylation, microRNA level changes) alterations were identified in patients with RCC. Blood plasma markers characteristic of this type of cancer were also determined. The obtained results can be utilized as markers for the development of a comprehensive system for early diagnosis of kidney cancer.