Mutational Signatures in Gastric Cancer and Their Clinical Implications.

Gastric cancer is characterised by high inter- and intratumour heterogeneity. The majority of patients are older than 65 years and the global burden of this disease is increasing due to the aging of the population. The disease is usually diagnosed at advanced stages, which is a consequence of nonspecific symptoms. Few improvements have been made at the level of noninvasive molecular diagnosis of sporadic gastric cancer, and therefore the mortality rate remains high. A new field of mutational signatures has emerged in the past decade with advances in the genome sequencing technology. These distinct mutational patterns in the genome, caused by exogenous and endogenous mutational processes, can be associated with tumour aetiology and disease progression, and could provide novel perception on the treatment possibilities. This review assesses the mutational signatures found in gastric cancer and summarises their potential for use in clinical setting as diagnostic or prognostic biomarkers. Associated treatment options and biomarkers already implemented in clinical use are discussed, together with those that are still being explored or are in clinical studies.

PLK2 Single Nucleotide Variant in Gastric Cancer Patients Affects miR-23b-5p Binding.

PURPOSE: Chromosomal instability is a hallmark of gastric cancer (GC). It can be driven by single nucleotide variants (SNVs) in cell cycle genes. We investigated the associations between SNVs in candidate genes, PLK2, PLK3, and ATM, and GC risk and clinicopathological features. MATERIALS AND METHODS: The genotyping study included 542 patients with GC and healthy controls. Generalized linear models were used for the risk and clinicopathological association analyses. Survival analysis was performed using the Kaplan-Meier method. The binding of candidate miRs was analyzed using a luciferase reporter assay. RESULTS: The PLK2 Crs15009-Crs963615 haplotype was under-represented in the GC group compared to that in the control group (Pcorr=0.050). Male patients with the PLK2 rs963615 CT genotype had a lower risk of GC, whereas female patients had a higher risk (P=0.023; P=0.026). The PLK2 rs963615 CT genotype was associated with the absence of vascular invasion (P=0.012). The PLK3 rs12404160 AA genotype was associated with a higher risk of GC in the male population (P=0.015). The ATM Trs228589-Ars189037-Grs4585 haplotype was associated with a higher risk of GC (P<0.001). The ATM rs228589, rs189037, and rs4585 genotypes TA+AA, AG+GG, and TG+GG were associated with the absence of perineural invasion (P=0.034). In vitro analysis showed that the cancer-associated miR-23b-5p mimic specifically bound to the PLK2 rs15009 G allele (P=0.0097). Moreover, low miR-23b expression predicted longer 10-year survival (P=0.0066) in patients with GC. CONCLUSIONS: PLK2, PLK3, and ATM SNVs could potentially be helpful for the prediction of GC risk and clinicopathological features. PLK2 rs15009 affects the binding of miR-23b-5p. MiR-23b-5p expression status could serve as a prognostic marker for survival in patients with GC.

The Role of VHL in the Development of von Hippel-Lindau Disease and Erythrocytosis.

Von Hippel-Lindau disease (VHL disease or VHL syndrome) is a familial multisystem neoplastic syndrome stemming from germline disease-associated variants of the VHL tumor suppressor gene on chromosome 3. VHL is involved, through the EPO-VHL-HIF signaling axis, in oxygen sensing and adaptive response to hypoxia, as well as in numerous HIF-independent pathways. The diverse roles of VHL confirm its implication in several crucial cellular processes. VHL variations have been associated with the development of VHL disease and erythrocytosis. The association between genotypes and phenotypes still remains ambiguous for the majority of mutations. It appears that there is a distinction between erythrocytosis-causing VHL variations and VHL variations causing VHL disease with tumor development. Understanding the pathogenic effects of VHL variants might better predict the prognosis and optimize management of the patient.

Genetic variations in AURORA cell cycle kinases are associated with glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most frequent type of primary astrocytomas. We examined the association between single nucleotide polymorphisms (SNPs) in Aurora kinase A (AURKA), Aurora kinase B (AURKB), Aurora kinase C (AURKC) and Polo-like kinase 1 (PLK1) mitotic checkpoint genes and GBM risk by qPCR genotyping. In silico analysis was performed to evaluate effects of polymorphic biological sequences on protein binding motifs. Chi-square and Fisher statistics revealed a significant difference in genotypes frequencies between GBM patients and controls for AURKB rs2289590 variant (p = 0.038). Association with decreased GBM risk was demonstrated for AURKB rs2289590 AC genotype (OR = 0.54; 95% CI = 0.33-0.88; p = 0.015). Furthermore, AURKC rs11084490 CG genotype was associated with lower GBM risk (OR = 0.57; 95% CI = 0.34-0.95; p = 0.031). Bioinformatic analysis of rs2289590 polymorphic region identified additional binding site for the Yin-Yang 1 (YY1) transcription factor in the presence of C allele. Our results indicated that rs2289590 in AURKB and rs11084490 in AURKC were associated with a reduced GBM risk. The present study was performed on a less numerous but ethnically homogeneous population. Hence, future investigations in larger and multiethnic groups are needed to strengthen these results.

Characterization and risk association of polymorphisms in Aurora kinases A, B and C with genetic susceptibility to gastric cancer development.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in genes encoding mitotic kinases could influence development and progression of gastric cancer (GC). METHODS: Case-control study of nine SNPs in mitotic genes was conducted using qPCR. The study included 116 GC patients and 203 controls. In silico analysis was performed to evaluate the effects of polymorphisms on transcription factors binding sites. RESULTS: The AURKA rs1047972 genotypes (CT vs. CC: OR, 1.96; 95% CI, 1.05-3.65; p = 0.033; CC + TT vs. CT: OR, 1.94; 95% CI, 1.04-3.60; p = 0.036) and rs911160 (CC vs. GG: OR, 5.56; 95% CI, 1.24-24.81; p = 0.025; GG + CG vs. CC: OR, 5.26; 95% CI, 1.19-23.22; p = 0.028), were associated with increased GC risk, whereas certain rs8173 genotypes (CG vs. CC: OR, 0.60; 95% CI, 0.36-0.99; p = 0.049; GG vs. CC: OR, 0.38; 95% CI, 0.18-0.79; p = 0.010; CC + CG vs. GG: OR, 0.49; 95% CI, 0.25-0.98; p = 0.043) were protective. Association with increased GC risk was demonstrated for AURKB rs2241909 (GG + AG vs. AA: OR, 1.61; 95% CI, 1.01-2.56; p = 0.041) and rs2289590 (AC vs. AA: OR, 2.41; 95% CI, 1.47-3.98; p = 0.001; CC vs. AA: OR, 6.77; 95% CI, 2.24-20.47; p = 0.001; AA+AC vs. CC: OR, 4.23; 95% CI, 1.44-12.40; p = 0.009). Furthermore, AURKC rs11084490 (GG + CG vs. CC: OR, 1.71; 95% CI, 1.04-2.81; p = 0.033) was associated with increased GC risk. A combined analysis of five SNPs, associated with an increased GC risk, detected polymorphism profiles where all the combinations contribute to the higher GC risk, with an OR increased 1.51-fold for the rs1047972(CT)/rs11084490(CG + GG) to 2.29-fold for the rs1047972(CT)/rs911160(CC) combinations. In silico analysis for rs911160 and rs2289590 demonstrated that different transcription factors preferentially bind to polymorphic sites, indicating that AURKA and AURKB could be regulated differently depending on the presence of particular allele. CONCLUSIONS: Our results revealed that AURKA (rs1047972 and rs911160), AURKB (rs2241909 and rs2289590) and AURKC (rs11084490) are associated with a higher risk of GC susceptibility. Our findings also showed that the combined effect of these SNPs may influence GC risk, thus indicating the significance of assessing multiple polymorphisms, jointly. The study was conducted on a less numerous but ethnically homogeneous Bosnian population, therefore further investigations in larger and multiethnic groups and the assessment of functional impact of the results are needed to strengthen the findings.

Methylation of the first exon in the erythropoietin receptor gene does not correlate with its mRNA and protein level in cancer cells.

BACKGROUND: Erythropoietin receptor (EPOR) is a functional membrane-bound cytokine receptor. Erythropoietin (EPO) represents an important hematopoietic factor for production, maturation and differentiation of erythroid progenitors. In non-hematopoietic tissue, EPO/EPOR signalization could also play cytoprotective and anti-apoptotic role. Several studies identified pro-stimulating EPO/EPOR effects in tumor cells; however, numerous studies opposed this fact due to the usage of unspecific EPOR antibodies and thus potential absence or very low levels of EPOR in tumor cells. It seems that this problem is more complex and therefore we have decided to focus on EPOR expression at several levels such as the role of methylation in the regulation of EPOR expression, identification of possible EPOR transcripts and the presence of EPOR protein in selected tumor cells. METHODS: Methylation status was analysed by bisulfite conversion reaction, PCR and sequencing. The expression of EPOR was monitored by quantitative RT-PCR and western blot analysis. RESULTS: In this study we investigated the methylation status of exon 1 of EPOR gene in selected human cancer cell lines. Our results indicated that CpGs methylation in exon 1 do not play a significant role in the regulation of EPOR transcription. However, methylation status of EPOR exon 1 was cell type dependent. We also observed the existence of two EPOR splice variants in human ovarian adenocarcinoma cell line - A2780 and confirmed the expression of EPOR protein in these cells using specific A82 anti-EPOR antibody. CONCLUSION: We outlined the methylation status of all selected cancer cell lines in exon 1 of EPOR gene and these results could benefit future investigations. Moreover, A82 antibody confirmed our previous results demonstrating the presence of functional EPOR in human ovarian adenocarcinoma A2780 cells.

Outlook on Epigenetic Therapeutic Approaches for Treatment of Gastric Cancer.

The incidence of gastric cancer has been declining globally in the last decades. Despite the improvements in the diagnostic procedures, most cases are still detected at advanced stages due to lack of specific symptoms associated with early phases of tumour development. Consequently, gastric cancer poses a major health burden worldwide due to high mortality rates. Continuing advances in high-throughput technologies are revealing an intricate network of genetic and epigenetic changes associated with carcinogenesis. In addition, several risk factors, both environmental and genetic, have been recognized, which promote accumulation of diverse alterations affecting the expression of oncogenes, tumour suppressor genes, DNA repair genes, and other genes, implicated in normal gastric cell functions. A plethora of aberrant molecular events found in patients with this disease and intragenic heterogeneity of tumours from individuals are delaying the development of targeted biological therapies. Frequent occurrence of characteristic CpG island methylator phenotypes (CIMP phenotypes) in gastric cancers, particularly in association with Helicobacter pylori or EBV infection, could lead to introduction of epigenetic modulators into standard treatment regimens used against early and advanced forms of adenocarcinomas. This review highlights aberrant DNA methylation events in the development of gastric tumours and addresses the different aspects associated with the application of therapeutic epigenetic modulation in the management of the disease.

Single nucleotide polymorphisms rs911160 in AURKA and rs2289590 in AURKB mitotic checkpoint genes contribute to gastric cancer susceptibility.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in mitotic checkpoint genes could confer increased susceptibility to gastric cancer (GC). We investigated the association of Aurora kinase A (AURKA), Aurora kinase B (AURKB), Aurora kinase C (AURKC), Polo-like kinase 1 (PLK1) and Budding uninhibited by benzimidazol 3, yeast (BUB3) gene polymorphisms with GC risk. MATERIALS AND METHODS: Genotyping of 6 SNPs in AURKA (rs911160 and rs8173), AURKB (rs2289590), AURKC (rs11084490), PLK1 (rs42873), and BUB3 (rs7897156) was performed using TaqMan genotyping assays. RESULTS: Our study demonstrated that rs911160 (AURKA) heterozygous genotype was associated with an increased GC risk (OR = 1.50, 95% CI = 1.01-2.22, P = 0.043). Analysis of rs911160 (AURKA) showed significant association with an increased risk for intestinal type GC (OR = 1.80, 95%CI = 1.01-3.21, P = 0.040) and the risk was significantly higher in women than men (OR = 2.65, 95%CI = 1.02-6.87, P = 0.033). SNP rs2289590 in AURKB might contribute to susceptibility for the development of gastric cancer, particularly in women (OR = 2.08, 95% CI = 1.05-4.09, P = 0.032). CONCLUSION: Our findings suggested that AURKA (rs911160) and AURKB (rs2289590) polymorphisms could affect GC risk. Further validation studies in larger and multi-ethnical populations are needed to elucidate their functional impact on the development of GC. Environ. Mol. Mutagen. 58:701-711, 2017. © 2017 Wiley Periodicals, Inc.

Association between polymorphisms in segregation genes BUB1B and TTK and gastric cancer risk.

BACKGROUND: Malignant transformation of normal gastric cells is a complex and multistep process, resulting in development of heterogeneous tumours. Susceptible genetic background, accumulation of genetic changes, and environmental factors play an important role in gastric carcinogenesis. Single nucleotide polymorphisms (SNPs) in mitotic segregation genes could be responsible for inducing the slow process of accumulation of genetic changes, leading to genome instability. PATIENTS AND METHODS: We performed a case-control study of polymorphisms in mitotic kinases TTK rs151658 and BUB1B rs1031963 and rs1801376 to assess their effects on gastric cancer risk. We examined the TTK abundance in gastric cancer tissues using immunoblot analysis. RESULTS: C/G genotype of rs151658 was more frequent in patients with diffuse type of gastric cancer and G/G genotype was more common in intestinal types of gastric cancers (p = 0.049). Polymorphic genotype A/A of rs1801376 was associated with higher risk for developing diffuse type of gastric cancer in female population (p = 0.007), whereas A/A frequencies were increased in male patients with subserosa tumour cell infiltration (p = 0.009). T/T genotype of rs1031963 was associated with well differentiated tumours (p = 0.035). TT+CT genotypes of rs1031963 and GG+AG genotypes of rs1801376 were significantly associated with gastric cancer risk (dominant model; OR = 2,929, 95% CI: 1.281-6.700; p = 0.017 and dominant model; OR = 0,364, 95% CI: 0.192-0.691; p = 0.003 respectively). CONCLUSIONS: Our results suggest that polymorphisms in mitotic kinases TTK and BUB1B may contribute to gastric tumorigenesis and risk of tumour development. Further investigations on large populations and populations of different ethnicity are needed to determine their clinical utility.

Association of the AURKA and AURKC gene polymorphisms with an increased risk of gastric cancer.

Single nucleotide polymorphisms (SNPs) in mitotic checkpoint genes can contribute to susceptibility of human cancer, including gastric cancer (GC). We aimed to investigate the effects of Aurora kinase A (AURKA), Aurora kinase B (AURKB), and Aurora kinase C (AURKC) gene polymorphisms on GC risk in Slovenian population. We genotyped four SNPs in AURKA (rs2273535 and rs1047972), AURKB (rs2241909), and AURKC (rs758099) in a total of 128 GC patients and 372 healthy controls using TaqMan allelic discrimination assays to evaluate their effects on GC risk. Our results showed that genotype frequencies between cases and controls were significantly different for rs1047972 and rs758099 (P < 0.05). Our study demonstrated that AURKA rs1047972 TT and (CC + CT) genotypes were significantly associated with an increased risk of gastric cancer. Our results additionally revealed that AURKC rs758099 TT and (CC + CT) genotypes were also associated with increased GC risk. In stratified analysis, genotypes TT and (CC + CT) of AURKA rs1047972 SNP were associated with increased risk of both, intestinal and diffuse, types of GC. In addition, AURKC rs758099 TT and (CC + CT) genotypes were positively associated with increased intestinal type GC risk, but not with an increased diffuse type GC risk. Based on these results, we can conclude that AURKA rs1047972 and AURKC rs758099 polymorphisms could affect the risk of GC development. Further larger studies are needed to confirm these findings. © 2016 IUBMB Life, 68(8):634-644, 2016.

Challenges of deciphering gastric cancer heterogeneity.

Gastric cancer is in decline in most developed countries; however, it still accounts for a notable fraction of global mortality and morbidity related to cancer. High-throughput methods are rapidly changing our view and understanding of the molecular basis of gastric carcinogenesis. Today, it is widely accepted that the molecular complexity and heterogeneity, both inter- and intra-tumour, of gastric adenocarcinomas present significant obstacles in elucidating specific biomarkers for early detection of the disease. Although genome-wide sequencing and gene expression studies have revealed the intricate nature of the molecular changes that occur in tumour landscapes, the collected data and results are complex and sometimes contradictory. Several aberrant molecules have already been tested in clinical trials, although their diagnostic and prognostic utilities have not been confirmed thus far. The gold standard for the detection of sporadic gastric cancer is still the gastric endoscopy, which is considered invasive. In addition, genome-wide association studies have confirmed that genetic variations are important contributors to increased cancer risk and could participate in the initiation of malignant transformation. This hypothesis could in part explain the late onset of sporadic gastric cancers. The elaborate interplay of polymorphic low penetrance genes and lifestyle and environmental risk factors requires additional research to decipher their relative impacts on tumorigenesis. The purpose of this article is to present details of the molecular heterogeneity of sporadic gastric cancers at the DNA, RNA, and proteome levels and to discuss issues relevant to the translation of basic research data to clinically valuable tools. The focus of this work is the identification of relevant molecular changes that could be detected non-invasively.

Aberrant methylation patterns in cancer: a clinical view.

Epigenetic mechanisms, such as DNA methylation, DNA hydroxymethylation, post-translational modifications (PTMs) of histone proteins affecting nucleosome remodelling, and regulation by small and large non-coding RNAs (ncRNAs) work in concert with cis and trans acting elements to drive appropriate gene expression. Advances in detection methods and development of dedicated platforms and methylation arrays resulted in an explosion of information on aberrantly methylated sequences linking deviations in epigenetic landscape with the initiation and progression of complex diseases. Here, we consider how DNA methylation changes in malignancies, such as breast, pancreatic, colorectal, and gastric cancer could be exploited for the purpose of developing specific diagnostic tools. DNA methylation changes can be applicable as biomarkers for detection of malignant disease in easily accessible tissues. Methylation signatures are already proving to be an important marker for determination of drug sensitivity. Even more, promoter methylation patterns of some genes, such as MGMT, SHOX2, and SEPT9, have already been translated into commercial clinical assays aiding in patient assessment as adjunct diagnostic tools. In conclusion, the changes in DNA methylation patterns in tumour cells are slowly gaining entrance into routine diagnostic tests as promising biomarkers and as potential therapeutic targets.

Contemporary proteomic strategies for clinical epigenetic research and potential impact for the clinic.

Novel proteomic methods are revealing the intricacy of the epigenetic landscape affecting gene regulation and improving our knowledge of the pathogenesis of complex diseases. Despite the enormous amount of data regarding epigenetic modifications present in DNA and histones, deciphering their biological relevance in the context of the disease and health is currently still an ongoing process. Here, we consider the relationship between epigenetic research in tumorigenesis and the prospect of knowledge transfer to clinical use, focusing primarily on the epigenetic histone post-translational modifications, which could be used as biomarkers. We additionally focus on the use of proteomic techniques in research and evaluate their usefulness in clinical setting.

Proteomic approaches in biomarker discovery: new perspectives in cancer diagnostics.

Despite remarkable progress in proteomic methods, including improved detection limits and sensitivity, these methods have not yet been established in routine clinical practice. The main limitations, which prevent their integration into clinics, are high cost of equipment, the need for highly trained personnel, and last, but not least, the establishment of reliable and accurate protein biomarkers or panels of protein biomarkers for detection of neoplasms. Furthermore, the complexity and heterogeneity of most solid tumours present obstacles in the discovery of specific protein signatures, which could be used for early detection of cancers, for prediction of disease outcome, and for determining the response to specific therapies. However, cancer proteome, as the end-point of pathological processes that underlie cancer development and progression, could represent an important source for the discovery of new biomarkers and molecular targets for tailored therapies.

De novo mutation in DMD gene in a patient with combined hemophilia A and Duchenne muscular dystrophy.

We report an unusual case of a patient with two combined X-linked diseases, severe hemophilia A (HA) and Duchenne muscular dystrophy (DMD), of which only HA was hereditary. There was no family history of muscular dystrophy. Genetic analysis revealed that HA was caused by the hereditary coagulation factor VIII (F8) intron 22 inversion (distal/type I inversion), whereas DMD was caused by a de novo deletion in the dystrophin gene. This is the first report of a patient with two severe X-linked diseases, of which only HA was hereditary. Despite the fact that the probability of acquiring two X-linked abnormalities, one hereditary and one de novo, is extremely low, the emergence of such cases indicates that genetic testing for distinct X-linked diseases could be of importance in patients with hereditary hemophilia.

The progress of proteomic approaches in searching for cancer biomarkers.

Biomarkers are indicators of a specific biological state. Their detection in pathological conditions, such as cancer, is important for clinical disease management. One of their greatest values could be in early diagnosis and detection of neoplasms when the cancer is more manageable. Protein biomarkers are expected to be reliable predictors of pathological conditions, as they represent the endpoint of biological processes. The proteomic methodology has rapidly evolved in the past ten years, thus enabling discovery of a vast amount of potential biomarker candidates. However, the majority of novel candidates have not yet reached the integration into clinical environment. To do that, well-constructed large population validation studies are necessary as well as development of new algorithms for deciphering complex biological interactions and their involvement in pathological processes. This review focuses on advances in classical proteomic approaches and emerging high-throughput proteomic technologies for identifying cancer biomarkers.

Genetic aspects of gastric cancer instability.

Unravelling the molecular mechanisms underlying gastric carcinogenesis is one of the major challenges in cancer genomics. Gastric cancer is a very complex and heterogeneous disease, and although much has been learned about the different genetic changes that eventually lead to its development, the detailed mechanisms still remain unclear. Malignant transformation of gastric cells is the consequence of a multistep process involving different genetic and epigenetic changes in numerous genes in combination with host genetic background and environmental factors. The majority of gastric adenocarcinomas are characterized by genetic instability, either microsatellite instability (MSI) or chromosomal instability (CIN). It is believed that chromosome destabilizations occur early in tumour progression. This review summarizes the most common genetic alterations leading to instability in sporadic gastric cancers and its consequences.

Gene expression profiling of rat fetuses exposed to 2-dimensional ultrasound.

OBJECTIVES: This study evaluated the possible effects of ultrasound (US) on gene expression in brain tissue of rat embryos. METHODS: Four groups (n = 5 each) of pregnant Wistar Han rats were exposed to US for different durations (55, 100, 145, and 195 seconds) via a multifrequency transducer in the 2-dimensional imaging mode with a pulse duration of 1.29 microseconds, a pulse repetition frequency of 1 kHz, and a derated spatial-peak pulse-average intensity of 222.4 W/cm(2) on day 5, 9, 7, or 13 of gestation. Gene expression profiling was performed in fetal brain tissue (n = 5 per group) by quantitative reverse transcription-polymerase chain reaction arrays. RESULTS: The results indicated substantial alterations in gene expression. The most differentially expressed genes were Adamts5, Gadd45a, Npy2r, and Chrna1, which are implicated in important developmental signaling pathways. CONCLUSIONS: On the basis of our findings, routine short US examinations for monitoring fetal development are not contraindicated, but prolonged exposures should be used only when needed to obtain important diagnostic information.

A genomic approach to investigate expression profiles in Slovenian patients with gastric cancer.

Despite its decreasing frequency in developed countries, gastric cancer remains a significant health burden. The aim of the present study was to construct cDNA libraries and analyze differentially expressed genes related to this disease. Gene expression profiles were generated with suppressive subtraction hybridization (SSH). We constructed eight SSH libraries, four representing up-regulated genes and four representing down-regulated genes in tumor tissues. Our approach revealed that several genes are abnormally expressed in gastric cancer. We also identified global deregulation of several pathways involved in the maintenance of normal gastric homeostasis. The results of this study support the view that, as a result of complex pathogenesis, diversity of genomic aberrations and multiplicity of carcinogenic causes, gastric cancer cannot be reduced to a single molecule. Our results may contribute new insight into molecular aspects of the disease and may prove advantageous for future development of therapeutic targets and diagnostic molecular markers.

Proteomic strategies and challenges in tumor metastasis research.

The rapidly evolving field of proteomics offers new approaches to understanding the pathogenesis of cancer and metastatic disease. Although numerous tumor markers have been identified with different genomic methods in the past, most are either not specific or sensitive enough to be used in routine clinical setting. The rationale for proteomic profiling is based on the fact that proteins represent the dynamic state of the cells, reflecting pathophysiological changes in the disease more accurately than genomic and epigenetic alterations. Emerging proteomic techniques allow simultaneous assessment of a large number of proteins at one time. The study of protein profiles in complex systems, such as plasma, serum or tissues of cancer patients is likely to become valuable for monitoring the response of patients during treatment or for detecting recurrence of the disease.