Clinical Implications of Molecular Heterogeneity of Gastric Cancer

Gastric cancer incidence has been steadily declining in countries with low frequencies of gastric carcinoma since early 1930s. In areas with higher incidences, the decline has been less obvious and slower. Nevertheless, gastric adenocarcinoma remains one of the most common causes of cancer‐related death worldwide. The poor outcome has been attributed to late detection of the condition, particularly in Americas and Europe, aggressive pathogenesis and lack of symptoms during early stages of the tumor development. In addition, sporadic stomach cancer mostly affects elderly individuals. In the majority of countries with low incidence, the average age at the disease presentation is above 65. Therefore, gastric adenocarcinoma, among other diseases associated with old age, raises health concerns in countries with changing demographic age profiles that show a trend of an increase in the proportion of the population aged over 60. The low 5‐year survival rate of patients underscores the critical need for the development of more accurate diagnostic tools and safe targeted chemotherapeutics. However, the heterogeneity of molecular changes represents one of the most pressing issues in the current research of gastric cancer, impeding the translation of genetic aberrations into novel applications for medical practice

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

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

Abstract 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

Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

<p>Abstract</p> <p>Background</p> <p>Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the <it>hMLH1 </it>gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all <it>MLH1 </it>alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, <it>in vivo </it>assays for functional characterization of <it>MLH1 </it>mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of <it>hMLH1 </it>mutations <it>in vivo</it>, based on co-expression of human MLH1 and PMS2 in yeast cells.</p> <p>Methods</p> <p>Yeast <it>MLH1 </it>and <it>PMS1 </it>genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested.</p> <p>Results</p> <p>The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related <it>MLH1 </it>variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic.</p> <p>Conclusion</p> <p>Results of our <it>in vivo </it>yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of <it>MLH1 </it>variants in cancer patients found throughout the entire coding region of the gene.</p

PKD1 and PKD2 mutations in Slovenian families with autosomal dominant polycystic kidney disease

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis. METHODS: We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 (KG8, AC2.5, CW3 and CW2) and five for PKD2 (D4S1534, D4S2929, D4S1542, D4S1563 and D4S423). Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31–46 and PKD2 . RESULTS: Lod scores indicated linkage to PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients (including 20 patients from the families where linkage analysis could not be performed). We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2. CONCLUSION: In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course

Challenges of deciphering gastric cancer heterogeneity

Gastric cancer is in decline in most developed countrieshowever, 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

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 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 paper summarizes the most common genetic alterations leading to instability in sporadic gastric cancers and its consequences