Clinical impact of copy number variation changes in bladder cancer samples.

The aim of the present study was to detect copy number variations (CNVs) related to tumour progression and metastasis of urothelial carcinoma through whole-genome scanning. A total of 30 bladder cancer samples staged from pTa to pT4 were included in the study. DNA was extracted from freshly frozen tissue via standard phenol-chloroform extraction and CNV analysis was performed on two alternative platforms (CytoChip Oligo aCGH, 4x44K and Infinium OncoArray-500K BeadChip; Illumina, Inc.). Data were analysed with BlueFuse Multi software and Karyostudio, respectively. The results highlight the role of genomic imbalances in regions containing genes with metastatic and proliferative potential for tumour invasion. A high level of genomic instability in uroepithelial tumours was observed and a total of 524 aberrations, including 175 losses and 349 gains, were identified. The most prevalent genetic imbalances affected the following regions: 1p, 1q, 2q, 4p, 4q, 5p, 5q, 6p, 6q, 7q, 8q, 9p, 9q, 10p, 10q, 11q, 13q and 17q. High-grade tumours more frequently harboured genomic imbalances (n=227) than low-grade tumours (n=103). A total of 36 CNVs in high-grade bladder tumours were detected in chromosomes 1-5, 8-11, 14, 17, 19 and 20. Furthermore, five loss of heterozygosity variants containing 176 genes were observed in high-grade bladder cancer and may be used as potential targets for precision therapy. Revealing specific chromosomal regions related to the metastatic potential of uroepithelial tumours may lay a foundation for implementing molecular CNV profiling of bladder tumours as part of a routine progression risk estimation strategy, thus expanding the personalized therapeutic approach.

Unbalanced human embryos secrete more hyperglycosylated human chorionic gonadotrophin (hCG-H) than balanced ones.

PURPOSE: The aim of this study was to compare the levels of hyperglycosylated human chorionic gonadotropin (hCG-H) secreted from balanced and unbalanced human embryos. METHODS: Single-step culture media samples from 155 good quality embryos, derived from 90 good prognosis patients undergoing intracytoplasmic sperm injection (ICSI), were collected on the fifth day of embryo cultivation. All embryos were tested by next-generation sequencing (NGS) technique. The hCG-H levels in the culture media were evaluated by ELISA kit (Cusabio Biotech, CBS-E15803h) according to the manufacturer's instructions. Statistical analysis was performed using SPSS v.21 (IBM Corp., Armonk, NY, USA). RESULTS: The NGS analysis revealed that 36% of the embryos (n = 56) were balanced, and 64% of the embryos were unbalanced (n = 99). The presence of hCG-H was confirmed in all embryo culture media samples but was absent in the negative control. In addition, hCG-H concentration was significantly higher in the culture media from unbalanced embryos compared with the balanced ones (0.72 ± 0.30 mIU/ml vs. 0.62 ± 0.12 mIU/ml, p = 0.02, respectively). Furthermore, the mean levels of hCG-H were significantly increased in the samples from embryos with multiple abnormalities. Finally, the highest levels of hCG-H were expressed from embryos with monosomy of chromosome 11 (1.28 ± 0.04 mIU/ml) and those with trisomies of chromosomes 21 (2.23 mIU/ml) and 4 (1.02 ± 0.35 mIU/ml). CONCLUSION: Our results suggest that chromosomal aberrations in human embryos are associated with an increased secretion of hCG-H. However, hCG-H concentration in embryo culture media as a single biomarker is not sufficient for an accurate selection of balanced embryos.

Centenarian Exomes as a Tool for Evaluating the Clinical Relevance of Germline Tumor Suppressor Mutations.

OBJECTIVES: The aim of the present study was to evaluate the clinical relevance of mutations in tumor suppressor genes using whole-exome sequencing data from centenarians and young healthy individuals. METHODS: Two pools, one of centenarians and one of young individuals, were constructed and whole-exome sequencing was performed. We examined the whole-exome sequencing data of Bulgarian individuals for carriership of tumor suppressor gene variants. RESULTS: Of all variants annotated in both pools, 5080 (0.06%) are variants in tumor suppressor genes but only 46 show significant difference in allele frequencies between the two studied groups. Four variants (0.004%) are pathogenic/risk factors according to single nucleotide polymorphism database: rs1566734 in PTPRJ, rs861539 in XRCC3, rs203462 in AKAP10, and rs486907 in RNASEL. DISCUSSION: Based on their high minor allele frequencies and presence in the centenarian group, we could reclassify them from pathogenic/risk factors to benign. Our study shows that centenarian exomes can be used for re-evaluating the clinically uncertain variants.

MicroRNA Profiling in Patients with Upper Tract Urothelial Carcinoma Associated with Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a disease that affects people that live in the alluvial plains along the tributaries of the Danube River in the Balkan region. BEN is a chronic tubulointerstitial disease with a slow progression to terminal renal failure and has strong association with upper tract urothelial carcinoma (UTUC). There are several hypotheses about the etiology of BEN, but only the toxic effect of aristolochic acid has been confirmed as a risk factor in the occurrence of the disease. Aberrantly expressed miRNAs have been shown to be associated with many types of cancers. A number of studies have investigated the expression of microRNAs in urothelial carcinoma, mainly on urothelial bladder cancer, and only a few have included patients with UTUC. Here we present the first study of microRNA profiling in UTUC tissues from patients with BEN (BEN-UTUC) and patients with UTUC from nonendemic Balkan regions (non-BEN-UTUC) in comparison to normal kidney tissues. We found 10 miRNAs that were differentially expressed in patients with BEN-UTUC and 15 miRNAs in patients with non-BEN-UTUC. miRNA signature determined in BEN-UTUC patients differs from the non-BEN-UTUC patients; only miR-205-5p was mutual in both groups.

Changes in the gene expression profile of the bladder cancer cell lines after treatment with Helix lucorum and Rapana venosa hemocyanin.

PURPOSE: The purpose of this study was to elucidate the mechanism of action of the Helix lucorum hemocyanin (HlH), b-HlH-h, and RvH2-g hemocyanins as potential agents against bladder cancer. METHODS: We evaluated the viability of 647-V, T-24, and CAL-29 bladder cancer cell lines after treatment with the tested hemocyanins. The cell viability was measured at 72 hrs with MTT and WST-1 assays. Acridine orange/propidium iodide double staining was used to discriminate between apoptotic and necrotic cells. Gene expression profiling of the 168 genes from human inflammatory cytokines and signal transduction pathways were performed on the tumor cells before and after hemocyanins' treatment. RESULTS: The results showed decreased survival of cancer cells in the presence of HlH and two functional units: b-HlH-h and RvH2-g. Acridine orange/propidium iodide double staining revealed that the decreased viability was due to apoptosis. The gene expression data showed upregulation of genes involved in the apoptosis as well as of the immune system activation, and downregulation of the CCL2, CCL17, CCL21, CXCL1, and ABCF1 genes. CONCLUSIONS: The present study is the first to report gene expression in human cells under the influence of hemocyanins. The mechanism of antitumor activity of the HlH, b-HlH-h, and RvH2-g hemocyanins includes induction of apoptosis. In addition to the antiproliferative effect, downregulation of the genes with metastatic potential was observed. Together with the already known immunogenic effect, these findings support further studies on hemocyanins as potential therapeutic agents against bladder cancer.

Genetic, Genomic and Epigenomic Studies of Balkan Endemic Nephropathy (Ben).

BEN is a primary, chronic tubulointerstitial nephritis characterized with chronic anemia, absence of edema, xantoderma, normal blood pressure and normal findings on the fundus oculi. The disease is distributed in restricted areas in Bulgaria, Romania, Croatia, Bosnia, Former Yugoslavia. Despite numerous studies on genetic and environmental factors and their possible involvement in BEN, its etiopathogenesis still remains elusive. Our recent study aim to elucidate the possible epigenetic component in BEN development. Whole genome DNA array methylation analysis was applied to compare the methylation profiles of male and female BEN patients from endemic regions in Bulgaria and Serbia and healthy controls. All three most prominent candidate genes with aberrations in the epigenetic profile discovered with this study are involved in the inflammatory/immune processes and oncogenesis. These data are in concordance with the reported pathological alterations in BEN. This research supports the role of epigenetic changes in BEN pathology. Exome sequencing of 22.000 genes with Illumina Nextera Exome Enrichment Kit revealed three mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients which encode proteins involved in basement membrane/extracellular matrix and vascular tone, tightly connected to process of angiogenesis. We suggest that an abnormal process of angiogenesis plays a key role in the molecular pathogenesis of BEN.

Whole genome methylation array analysis reveals new aspects in Balkan endemic nephropathy etiology.

BACKGROUND: Balkan endemic nephropathy (BEN) represents a chronic progressive interstitial nephritis in striking correlation with uroepithelial tumours of the upper urinary tract. The disease has endemic distribution in the Danube river regions in several Balkan countries.DNA methylation is a primary epigenetic modification that is involved in major processes such as cancer, genomic imprinting, gene silencing, etc. The significance of CpG island methylation status in normal development, cell differentiation and gene expression is widely recognized, although still stays poorly understood. METHODS: We performed whole genome DNA methylation array analysis on DNA pool samples from peripheral blood from 159 affected individuals and 170 healthy individuals. This technique allowed us to determine the methylation status of 27 627 CpG islands throughout the whole genome in healthy controls and BEN patients. Thus we obtained the methylation profile of BEN patients from Bulgarian and Serbian endemic regions. RESULTS: Using specifically developed software we compared the methylation profiles of BEN patients and corresponding controls and revealed the differently methylated regions. We then compared the DMRs between all patient-control pairs to determine common changes in the epigenetic profiles.SEC61G, IL17RA, HDAC11 proved to be differently methylated throughout all patient-control pairs. The CpG islands of all 3 genes were hypomethylated compared to controls. This suggests that dysregulation of these genes involved in immunological response could be a common mechanism in BEN pathogenesis in both endemic regions and in both genders. CONCLUSION: Our data propose a new hypothesis that immunologic dysregulation has a place in BEN etiopathogenesis.