The search for molecular biomarkers of early-onset colorectal cancer (CRC) is an important but still quite challenging and unsolved task. Detection of CpG methylation in human DNA obtained from blood or stool has been proposed as a promising approach to a noninvasive early diagnosis of CRC. Thousands of abnormally methylated CpG positions in CRC genomes are often located in non-coding parts of genes. Novel bioinformatic methods are thus urgently needed for multi-omics data analysis to reveal causative biomarkers with a potential driver role in early stages of cancer. We have developed a method for finding potential causal relationships between epigenetic changes (DNA methylations) in gene regulatory regions that affect transcription factor binding sites (TFBS) and gene expression changes. This method also considers the topology of the involved signal transduction pathways and searches for positive feedback loops that may cause the carcinogenic aberrations in gene expression. We call this method “Walking pathways”, since it searches for potential rewiring mechanisms in cancer pathways due to dynamic changes in the DNA methylation status of important gene regulatory regions (“epigenomic walking”). In this paper, we analysed an extensive collection of full genome gene-expression data (RNA-seq) and DNA methylation data of genomic CpG islands (using Illumina methylation arrays) generated from a sample of tumor and normal gut epithelial tissues of 300 patients with colorectal cancer (at different stages of the disease) (data generated in the EU-supported SysCol project). Identification of potential epigenetic biomarkers of DNA methylation was performed using the fully automatic multi-omics analysis web service “My Genome Enhancer” (MGE) (my-genome-enhancer.com). MGE uses the database on gene regulation TRANSFAC®, the signal transduction pathways database TRANSPATH®, and software that employs AI (artificial intelligence) methods for the analysis of cancer-specific enhancers. The identified biomarkers underwent experimental testing on an independent set of blood samples from patients with colorectal cancer. As a result, using advanced methods of statistics and machine learning, a minimum set of 6 biomarkers was selected, which together achieve the best cancer detection potential. The markers include hypermethylated positions in regulatory regions of the following genes: CALCA, ENO1, MYC, PDX1, TCF7, ZNF43.

Objective: Mutations in TP53 lead to loss of function (LOF) or gain of function (GOF) of the corresponding protein p53 and produce a different effect on the tumor. Our goal was to determine the spectrum of somatic TP53 variants in BRCA1/2 associated high-grade serous ovarian cancer (HGSOC). Methods: The population under study comprised of HGSOCs with pathogenic variants in BRCA1 (n = 78) or BRCA2 (n = 21). Only chemo-naive and platinum-sensitive patients were included in this study. The case group of the IARC database (n = 1249) with HGSOC not stratified by BRCA status was used as a reference. A custom NGS panel was used for sequencing TP53 and mutational hot-spots of other genes, and p53 expression was evaluated by immunohistochemistry for 68 cases of HGSOCs. Results: Somatic TP53 variants (95) or inhibition of wild-type p53 expression (3) were observed in 98 cases. The sample with normal p53 had CDKNA1 variants. The frequency of truncating variants was significantly higher than in the reference cohort (30.3 vs. 21.0%, p = 0.01). Most of the samples (41/68) demonstrated low (or absent) expression of p53, and 17 samples overexpressed p53. LOH was typical for TP53 nonsense variants (14/15). In total, 68/95 samples were LOH positive and showed LOH in all tumorous cells, thus indicating the driver effect of TP53 mutations. Three specimens had KRAS, BAX, APC, and CTNNB1 subclones variants. Conclusion: High frequency of TP53 truncating variants, the low expression of mutant p53, and low incidence of oncogene mutations show potential GOF properties of p53 to be poorly represented in BRCA1/2 associated HGSOC.

Mutations in the BRCA1 and BRCA2 genes are known risk factors and drivers of breast and ovarian cancers. So far, few studies have been focused on understanding the differences in transcriptome and functional landscapes associated with the disease (breast vs. ovarian cancers), gene (BRCA1 vs. BRCA2), and mutation type (germline vs. somatic). In this study, we were aimed at systemic evaluation of the association of BRCA1 and BRCA2 germline and somatic mutations with gene expression, disease clinical features, outcome, and treatment. We performed BRCA1/2 mutation centered RNA-seq data analysis of breast and ovarian cancers from the TCGA repository using transcriptome and phenotype “portrayal” with multi-layer self-organizing maps and functional annotation. The results revealed considerable differences in BRCA1- and BRCA2-dependent transcriptome landscapes in the studied cancers. Furthermore, our data indicated that somatic and germline mutations for both genes are characterized by deregulation of different biological functions and differential associations with phenotype characteristics and poly(ADP-ribose) polymerase (PARP)-inhibitor gene signatures. Overall, this study demonstrates considerable variation in transcriptomic landscapes of breast and ovarian cancers associated with the affected gene (BRCA1 vs. BRCA2), as well as the mutation type (somatic vs. germline). These results warrant further investigations with larger groups of mutation carriers aimed at refining the understanding of molecular mechanisms of breast and ovarian cancers.

Non-small-cell lung cancer (NSCLC), a subtype of lung cancer, remains one of the most common tumors with a high mortality and morbidity rate. Numerous targeted drugs were implemented or are now developed for the treatment of NSCLC. Two genes, HER2 and MET, are among targets for these specific therapeutic agents. Alterations in HER2 and MET could lead to primary or acquired resistance to commonly used anti-EGFR drugs. Using current methods for detecting HER2 and MET amplifications is time and labor-consuming; alternative methods are required for HER2 and MET testing. We developed the first multiplex droplet digital PCR assay for the simultaneous detection of MET and HER2 amplification in NSCLC samples. The suitability of qPCR was assessed for the optimization of multiplex ddPCR. The optimal elongation temperature, reference genes for DNA quantification, and amplicon length were selected. The developed ddPCR was validated on control samples with various DNA concentrations and ratios of MET and HER2 genes. Using ddPCR, 436 EGFR-negative NSCLC samples were analyzed. Among the tested samples, five specimens (1.15%) showed a higher ratio of MET, and six samples (1.38%) showed a higher ratio of HER2. The reported multiplex ddPCR assay could be used for the routine screening of MET and HER2 amplification in NSCLC samples.

Significant effort has been devoted to developing effective cancer vaccines based on dendritic cells (DCs) loaded with various tumour antigens, including DNA constructs that carry sequences of tumour‐associated antigens (TAAs). Such vaccines efficiently and selectively activate the T cell immune response. In this study, we describe a method to induce an antitumour immune response in mononuclear cell (MNC) cultures from colorectal cancer patients using DNA‐transfected DCs encoding TAA epitopes of carcinoembryonic antigen, epithelial cell adhesion molecule and mucin 4. DCs were obtained from peripheral blood monocytes of colorectal cancer patients. Magnetic‐assisted transfection was used to deliver the genetic constructs to DCs. To assess the potency of the immune response, the antitumour cytotoxic response was assessed by lymphocyte intracellular perforin and the MNC cytotoxic activity against autologous tumour cells. We showed that polyepitope DNA‐transfected DCs enhanced MNC antitumour activity, increasing tumour cell death and the percentage of perforin‐positive lymphocytes. In addition, DNA‐transfected DCs elicited a cytotoxic response that was as efficient as that of tumour lysate‐loaded DCs. Taken together, the data suggest that it is feasible to induce an antitumour immune response in colorectal MNCs using transfected DCs. Thus, the DNA construct reported in this study may potentially be used in therapeutic and prophylactic DC‐based vaccines.