Orsolya Galamb

Inflammation, adenoma and cancer: objective classification of colon biopsy specimens with gene expression signature.

Gene expression analysis of colon biopsies using high-density oligonucleotide microarrays can contribute to the understanding of local pathophysiological alterations and to functional classification of adenoma (15 samples), colorectal carcinomas (CRC) (15) and inflammatory bowel diseases (IBD) (14). Total RNA was extracted, amplified and biotinylated from frozen colonic biopsies. Genome-wide gene expression profile was evaluated by HGU133plus2 microarrays and verified by RT-PCR. We applied two independent methods for data normalization and used PAM for feature selection. Leave one-out stepwise discriminant analysis was performed. Top validated genes included collagenIVα1, lipocalin-2, calumenin, aquaporin-8 genes in CRC; CD44, met proto-oncogene, chemokine ligand-12, ADAM-like decysin-1 and ATP-binding casette-A8 genes in adenoma; and lipocalin-2, ubiquitin D and IFITM2 genes in IBD. Best differentiating markers between Ulcerative colitis and Crohns disease were cyclin-G2; tripartite motif-containing-31; TNFR shedding aminopeptidase regulator-1 and AMICA. The discriminant analysis was able to classify the samples in overall 96.2% using 7 discriminatory genes (indoleamine-pyrrole-2,3-dioxygenase, ectodermal-neural cortex, TIMP3, fucosyltransferase-8, collectin sub-family member 12, carboxypeptidase D, and transglutaminase-2). Using routine biopsy samples we successfully performed whole genomic microarray analysis to identify discriminative signatures. Our results provide further insight into the pathophysiological background of colonic diseases. The results set up data warehouse which can be mined further.

Reversal of gene expression changes in the colorectal normal-adenoma pathway by NS398 selective COX2 inhibitor

Background and aims:Treatment of colorectal adenomas with selective cyclooxygenase-2 inhibitors can contribute to the chemoprevention of colorectal cancer (CRC), but the molecular background of their effect is not fully understood. We analysed the gene expression modulatory effect of N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS398) on HT29 cells to be correlated with expression data gained from biopsy samples.Methods:HT29 colon adenocarcinoma cells were treated with NS398, and global mRNA expression was analysed on HGU133Plus2.0 microarrays. Discriminatory transcripts between normal and adenoma and between adenoma and CRC biopsy samples were identified using HGU133Plus2.0 microarrays. The results were validated using RT–PCR and immunohistochemistry.Results:Between normal and adenoma samples, 20 classifiers were identified, including overexpressed cadherin 3, KIAA1199, and downregulated peptide YY, glucagon, claudin 8. Seventeen of them changed in a reverse manner in HT29 cells under NS398 treatment, 14 (including upregulated claudin 8, peptide YY, and downregulated cadherin 3, KIAA1199) at a significance of P<0.05. Normal and CRC could be distinguished using 38 genes, the expression of 12 of them was changed in a reverse manner under NS398 treatment.Conclusion:NS398 has a reversal effect on the expression of several genes that altered in colorectal adenoma–carcinoma sequence. NS398 more efficiently inverted the expression changes seen in the normal-adenoma than in the normal-carcinoma transition.

Diagnostic mRNA Expression Patterns of Inflamed, Benign, and Malignant Colorectal Biopsy Specimen and their Correlation with Peripheral Blood Results

Purpose: Gene expression profile (GEP)–based classification of colonic diseases is a new method for diagnostic purposes. Our aim was to develop diagnostic mRNA expression patterns that may establish the basis of a new molecular biological diagnostic method. Experimental Design: Total RNA was extracted, amplified, and biotinylated from frozen colonic biopsies of patients with colorectal cancer (n = 22), adenoma (n = 20), hyperplastic polyp (n = 11), inflammatory bowel disease (n = 21), and healthy normal controls (n = 11), as well as peripheral blood samples of 19 colorectal cancer and 11 healthy patients. Genome-wide gene expression profile was evaluated by HGU133plus2 microarrays. To identify the differentially expressed features, the significance analysis of microarrays and, for classification, the prediction analysis of microarrays were used. Expression patterns were validated by real-time PCR. Tissue microarray immunohistochemistries were done on tissue samples of 121 patients. Results: Adenoma samples could be distinguished from hyperplastic polyps by the expression levels of nine genes including ATP-binding cassette family A, member 8, insulin-like growth factor 1 and glucagon (sensitivity, 100%; specificity, 90.91%). Between low-grade and high-grade dysplastic adenomas, 65 classifier probesets such as aquaporin 1, CXCL10, and APOD (90.91/100) were identified; between colorectal cancer and adenoma, 61 classifier probesets including axin 2, von Willebrand factor, tensin 1, and gremlin 1 (90.91/100) were identified. Early- and advanced-stage colorectal carcinomas could be distinguished using 34 discriminatory transcripts (100/66.67). Conclusions: Whole genomic microarray analysis using routine biopsy samples is suitable for the identification of discriminative signatures for differential diagnostic purposes. Our results may be the basis for new GEP-based diagnostic methods. (Cancer Epidemiol Biomarkers Prev 2008;17(10):2835–45)

Molecular detection of circulating cancer cells. Role in diagnosis, prognosis and follow-up of colon cancer patients.

Background: The circulating tumor cells of solid tumors, especially in colorectal cancer has been of great interest in medical research in the last and new century. Methods: A literature review of recent data for the preparation of a proposal for clinical applications. Results: Starting with the detection of circulating cancer cells in histological sections, lymph nodes, bone marrow and peripheral blood have recently become the primary samples for these investigations. Following microscopic morphology, new methods were and are being developed continuously including rare cell enrichment and detection techniques like fluorescence microscopy, flow cytometry, RT-PCR and methylated DNA PCR. The sensitivity of these assays reaches down to the concentration of 1 tumor cell/ml blood. A lot of different epithelial markers were targeted with these techniques e.g. cytokeratins, EGFR, CEA, and EMA. Clinically, circulating tumor cells were found as independent prognostic factors in lymph nodes and bone marrow. In blood, their presence appears to be an early marker for recurrence and relapse. The change in their quantitative number is supposed to reflect the chemotherapeutic sensitivity and metastatic growth activity of the tumor. Conclusions: The detection of circulating tumor cells in bone marrow and lymph nodes is of clinical significance. Their presence in peripheral blood and therapeutic applications need further clarification.

Identification and Validation of Colorectal Neoplasia–Specific Methylation Markers for Accurate Classification of Disease

Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis. (Mol Cancer Res 2007;5(2):153–63)

Matrix Metalloproteinase-9 Expression in the Normal Mucosa–Adenoma–Dysplasia–Adenocarcinoma Sequence of the Colon

It has been proposed that matrix metalloproteinases (MMPs) play a role in tumor invasion. We determined protein expression of matrix metalloproteinase-9 (MMP-9) in colorectal cancer (CRC), corresponding normal mucosa and colorectal adenomas. For confirmation of immunohistochemical results MMP-9 TaqMan RT-PCR analysis was performed. Expression of MMP-9 was determined on paraffin embedded biopsy sections by immunohistochemistry in 31 CRC patients (from cancer tissue and corresponding normal mucosa) and in 30 patients with adenoma (nine adenomas with high grade of dysplasia). MMP-9 immunostaining was determined semi-quantitatively. For Taqman RT-PCR analyses normal mucosa (n = 5), adenoma without (n = 6) and with high grade dysplasia (n = 7) and CRC (n = 10) were investigated. Statistical analysis with ANOVA, LSD test and correlation analysis were performed. P value of <0.05 was considered significant. The MMP-9 expression in CRC was significantly higher compared to adenomas or the normal mucosa (P = 0.001). Significantly higher expression of MMP-9 has been observed in adenomas with high grade dysplasia compared to other adenomas or normal colon (P < 0.001). Diffuse strong MMP-9 expression was present in tumor as well as in stromal cells. In adenoma samples, dysplastic epithelial cells showed moderate intensive cytoplasmic MMP-9 expression, with a clear-cut differentiation between dysplastic and non-dysplastic areas. Staining intensity correlated with the grade of CRC. We demonstrate a significantly higher expression of MMP-9 in adenoma with high grade dysplasia—CRC sequence as compared to normal tissue. The over-expression of MMP-9 strongly suggests its association with colorectal carcinogenesis.

The Influence of Methylated Septin 9 Gene on RNA and Protein Level in Colorectal Cancer

Colorectal cancer is one of the leading death causes in the world. Specificity and sensitivity of the present screening methods are unsuitable and their compliance is too low. Nowadays the most effective method is the colonoscopy, because it gives not only macroscopic diagnosis but therapeutic possibility as well, however the compliance of the patients is very low. Hence development of new diagnostic methods is needed. Altered expression of septin 9 was found in several tumor types including colorectal cancer. The aim of this study was to detect the methylation related mRNA and protein expression changes of septin 9 in colorectal adenoma-dysplasia-carcinoma sequence and to analyze its reversibility by demethylation treatment. Septin 9 protein expression showed significant difference between normal and colorectal cancer (CRC) samples (p < 0,001). According to biopsy microarray results, septin 9 mRNA expression decreased in the progression of colon neoplastic disease (p < 0,001). In laser microdissected epithelial cells, septin 9 significantly underexpressed in CRC compared to healthy controls (p < 0,001). The expression of septin9_v1 region was higher in the healthy samples, while septin9_v2, v4, v4*, v5 overexpression were detected in cancer epithelial cells compared to normal. The septin 9 mRNA and protein levels of HT29 cells increased after demethylation treatment. The increasing methylation of septin 9 gene during colorectal adenoma-dysplasia-carcinoma sequence progression is reflected in the decreasing mRNA and protein expression, especially in the epithelium. These changes can be reversed by demethylation agents converting this screening marker gene into therapeutic target.

Potential biomarkers of colorectal adenoma-dysplasia-carcinoma progression: mRNA expression profiling and in situ protein detection on TMAs reveal 15 sequentially upregulated and 2 downregulated genes.

Background: As most colorectal cancers (CRC) develop from villous adenomas, studying alterations in gene expression profiles across the colorectal adenoma–dysplasia–carcinoma sequence may yield potential biomarkers of disease progression. Methods: Total RNA was extracted, amplified, and biotinylated from colonic biopsies of 15 patients with CRC, 15 with villous adenoma and 8 normal controls. Gene expression profiles were evaluated using HGU133Plus2.0 microarrays and disease progression associated data were validated with RT-PCR. The potential biomarkers were also tested at the protein level using tissue microarray samples of 103 independent and 16 overlapping patients. Results: 17 genes were validated to show sequentially altered expression at mRNA level through the normal–adenoma–dysplasia–carcinoma progression. Prostaglandin-D2 receptor (PTGDR) and amnionless homolog (AMN) genes revealed gradually decreasing expression while the rest of 15 genes including osteonectin, osteopontin, collagen IV–alpha 1, biglycan, matrix GLAprotein, and von Willebrand factor demonstrated progressively increasing expression. Similar trends of expression were confirmed at protein level for PTGDR, AMN, osteopontin and osteonectin. Conclusion: Downregulated AMN and PTGDR and upregulated osteopontin and osteonectin were found as potential biomarkers of colorectal carcinogenesis and disease progression to be utilized for prospective biopsy screening both at mRNA and protein levels. Gene alterations identified here may also add to our understanding of CRC progression.

Molecular pathogenesis of helicobacter pylori infection: The role of bacterial virulence factors

Helicobacter pylori is one of the most common pathogens affecting humankind, infecting approximately 50% of the world’s population. Of those infected, many will develop asymptomatic gastritis, but 10% develop gastric or duodenal ulcers. The clinical outcome of the infection may involve a combination of bacterial factors, host factors and environmental factors. In the process of development of gastritis, ulceration and cancer, several cellular and molecular steps follow each other. Infection, acid survival, adhesion, cytotoxicity, epithelial cell turnover changes, inflammation, regeneration or pathological alteration towards erosions, ulceration, and cancer can be observed on the cellular level. Bacterial factors like urease, AmiE, AmiF, hydrogenase and arginase are needed for survival in the acidic gastric environment. The bacterial flagellae are essential to move the bacteria towards the epithelial surface. Adhesive factors like BabA, SabA and ureaseA are necessary for adhesion against MHC-II complexes and Le antigens. The bacteria VacA and CagA are cytotoxic factors. The Cag type IV secretion system delivers these proteins inside the epithelial cells. After disruption of epithelial cell junctions, the bacteria can pass through the gastric wall facing direct immune response from neutrophils, lymphocytes, mast cells and dendritic cells. This review describes and summarizes our present molecular biological information and knowledge about the Helicobacter infective component, cell functions and processes. The possible role of host counter responses and interactions with gastric epithelia and immune cells are also detailed.

Complete Genes May Pass from Food to Human Blood

Our bloodstream is considered to be an environment well separated from the outside world and the digestive tract. According to the standard paradigm large macromolecules consumed with food cannot pass directly to the circulatory system. During digestion proteins and DNA are thought to be degraded into small constituents, amino acids and nucleic acids, respectively, and then absorbed by a complex active process and distributed to various parts of the body through the circulation system. Here, based on the analysis of over 1000 human samples from four independent studies, we report evidence that meal-derived DNA fragments which are large enough to carry complete genes can avoid degradation and through an unknown mechanism enter the human circulation system. In one of the blood samples the relative concentration of plant DNA is higher than the human DNA. The plant DNA concentration shows a surprisingly precise log-normal distribution in the plasma samples while non-plasma (cord blood) control sample was found to be free of plant DNA.