Ferenc Sipos

Detection of Methylated SEPT9 in Plasma Is a Reliable Screening Method for Both Left- and Right-Sided Colon Cancers

Background Methylated Septin 9 (SEPT9) is a sensitive biomarker for colorectal cancer (CRC) from peripheral blood. However, its relationship to cancer localization, guaiac-based fecal occult blood test (gFOBT) and carcinoembryonic antigen (CEA) have not been described. Methodology/Principal Findings Plasma samples were collected for SEPT9 analysis from patients with no evidence of disease (NED) (n = 92) before colonoscopy and CRC (n = 92) before surgical treatment. DNA was isolated and bisulfite-converted using Epi proColon kit 2.0. Qualitative determination was performed using Epi proColon 2.0 RT-PCR assay. Samples for gFOBT and CEA analysis were collected from NED (n = 17 and 27, respectively) and CRC (n = 22 and 27, respectively). SEPT9 test was positive in 15.2% (14/92) of NED and 95.6% (88/92) of CRC, including 100% (67/67) from stage II to stage IV CRC and 84% (21/25) of stage I CRC when a sample was called positive if 1 out of 3 PCR replicates was positive. In a second analysis (2 out of 3 PCR replicates) specificity improved to 99% (91/92) of NEDs, at a sensitivity of 79.3% (73/92) of SEPT9 positives in CRC. gFOBT was positive in 29.4% (5/17) of NED and 68.2% (15/22) of CRC and elevated CEA levels were detected in 14.8% (4/27) of NED and 51.8% (14/27) of CRC. Both SEPT9 (84.8%) and CEA (85.2%) showed higher specificity than gFOBT (70.6%). SEPT9 was positive in 96.4% (54/56) of left-sided colon cancer (LSCC) cases and 94.4% (34/36) of right-sided colon cancer (RSCC) cases. gFOBT was positive in 83.3% (10/12) of cases with LSCC and 50% (5/10) of cases with RSCC, elevated CEA was detected 60% (9/15) of LSCC and 41.7% (5/12) of RSCC. Conclusions/Significance The high degree of sensitivity and specificity of SEPT9 in plasma makes it a better method to detect CRC than gFOBT and CEA, even for the more difficult to detect RSCC.

Changes of the cytokine profile in inflammatory bowel diseases

Cytokines are indispensable signals of the mucosa-associated immune system for maintaining normal gut homeostasis. An imbalance of their profile in favour of inflammation initiation may lead to disease states, such as that is observed in inflammatory bowel diseases (IBD). Although Crohns disease (CD) is often described as a prototype of T-helper 1-type diseases, and ulcerative colitis (UC) is traditionally viewed as a T-helper 2-mediated condition, the classic paradigm, which categorises cytokines into pro- and anti-inflammatory groups, has recently been changed. The inflammation regulatory pathways may not be mutually exclusive as individual cytokines can have diverse and even opposing functions in various clinical and immunological settings. None the less there are many common immunological responses in IBD that are mediated by cytokines. Although they regulate and influence the development, course and recurrence of the inflammatory process, the concrete pathogenic role of these small signaling molecules is sometimes not unambiguous in the subtypes of the disease. Our aim is to review the current information about pro- and anti-inflammatory effects of traditionally studied and recently discovered cytokines in the pathogenesis of UC and CD. The better understanding of their production and functional activity may lead to the development of new therapeutic modalities.

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)

Detection of Methylated Septin 9 in Tissue and Plasma of Colorectal Patients with Neoplasia and the Relationship to the Amount of Circulating Cell-Free DNA

Background Determination of methylated Septin 9 (mSEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, the relationship between methylated DNA in plasma and colon tissue of the same subjects has not been reported. Methods Plasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 patients with adenoma and 34 patients with CRC. Following bisulfite conversion of DNA a commercial RT-PCR assay was used to determine the total amount of DNA in each sample and the fraction of mSEPT9 DNA. The Septin-9 protein was assessed using immunohistochemistry. Results The percent of methylated reference (PMR) values for SEPT9 above a PMR threshold of 1% were detected in 4.2% (1/24) of NED, 100% (26/26) of adenoma and 97.1% (33/34) of CRC tissues. PMR differences between NED vs. adenoma and NED vs. CRC comparisons were significant (p<0.001). In matching plasma samples using a PMR cut-off level of 0.01%, SEPT9 methylation was 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC. Significant PMR differences were observed between NED vs. CRC (p<0.01) and adenoma vs. CRC (p<0.01). Significant differences (p<0.01) were found in the amount of cfDNA (circulating cell-free DNA) between NED and CRC, and a modest correlation was observed between mSEPT9 concentration and cfDNA of cancer (R2 = 0.48). The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with abundant expression in normals, and diminished expression in adenomas and tumors. Conclusions Methylated SEPT9 was detected in all tissue samples. In plasma samples, elevated mSEPT9 values were detected in CRC, but not in adenomas. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Additional parameters including the amount of cfDNA in plasma appear to also play a role.

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)

Growth in epithelial cell proliferation and apoptosis correlates specifically to the inflammation activity of inflammatory bowel diseases: ulcerative colitis shows specific p53- and EGFR expression alterations.

PURPOSEEpithelial cell turnover related differences between ulcerative colitis, Crohn’s colitis, and aspecific colitis are not known yet.METHODSTotally 345 formalin-fixed, paraffin-embedded biopsy specimens from 33 ulcerative colitis, 26 Crohn’s colitis, 30 aspecific colitis, and 10 healthy patients were observed with the TdT-mediated dUTP nick end labeling method and proliferating cell nuclear antigen-, p53-, and epithelial growth factor receptor immunohistochemistry. Because of epithelial growth factor receptor positivity of subepithelial cells epithelial growth factor receptor and CD45, CD68, or CD83 double fluorescence immunohistochemistry were performed on 16 freshly frozen samples from 8 severely active ulcerative colitis and 8 severely active Crohn’s colitis patients to describe lamina propria’s mononuclear cells, respectively.RESULTSThe epithelial growth factor receptor expression was significantly lower in each inflammatory group compared with normal (P < 0.005) and decreased significantly in mild ulcerative colitis compared with mild Crohn’s colitis or aspecific colitis (P < 0.005). Numerous epithelial growth factor receptor and CD45 double-positive submucosal mononuclear cells were observed in moderate-severe inflammations. The p53-expression was significantly higher in each inflammatory group compared with normal (P < 0.05). Significant differences were found between mildly, moderately, and severely inflamed samples in ulcerative colitis (P < 0.05) compared with Crohn’s colitis or aspecific colitis. Apoptotic/proliferative rates increased significantly in line with the inflammatory process (P < 0.0001/0.05), but the TdT-mediated dUTP nick end labeling and proliferating cell nuclear antigen-labeling characteristics did not show disease type specificity.CONCLUSIONSBased on our results, the alterations of epithelial growth factor receptor and p53 expression show ulcerative colitis specificity, whereas the rate of epithelial apoptosis and proliferation are determined by the histologic activity of the inflammation. The increased epithelial growth factor receptor expression by the lamina propria’s mononuclear cells in inflammation may suggest its role as an autoantigen.

Serrated pathway: Alternative route to colorectal cancer

Serrated polyps have been an area of intense focus for gastroenterologists over the past several years. Contrary to what was thought before, a growing body of literature indicates that these polyps can be precursors of colorectal cancer (CRC). Most of these lesions, particularly those in the proximal colon, have so far been under-recognized and missed during colonoscopy, qualifying these lesions to be the main cause of interval cancers. It is estimated that 10%-20% of CRCs evolve through this alternative, serrated pathway, with a distinct genetic and epigenetic profile. Aberrant DNA methylation plays a central role in the development of this CRC subtype. This characteristic molecular background is reflected in a unique pathological and clinical manifestation different from cancers arising via the traditional pathway. In this review we would like to highlight morphological, molecular and clinical features of this emerging pathway that are essential for gastroenterologists and may influence their everyday practice.