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The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2'-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.

Exosomes are small membrane vesicles that have important roles in transporting a great variety of bioactive molecules between epithelial compartment and their microenvironment during tumor formation including colorectal adenoma-carcinoma sequence. We tested the mRNA expression of the top 25 exosome-related markers based on ExoCharta database in healthy (n=49), adenoma (n=49) and colorectal carcinoma (n=49) patients using Affymetrix HGU133 Plus2.0 microarrays. Most related genes showed significantly elevated expression including PGK1, PKM, ANXA5, ENO1, HSP90AB1 and MSN during adenoma-carcinoma sequence. Surprisingly, the expression of ALIX (ALG 2-interacting protein X), involved in multivesicular body (MVB) and exosome formation, was significantly reduced in normal vs adenoma (P=5.02 × 10(-13)) and in normal vs colorectal carcinoma comparisons (P=1.51 × 10(-10)). ALIX also showed significant reduction (P<0.05) at the in situ protein level in the epithelial compartment of adenoma (n=35) and colorectal carcinoma (n=37) patients compared with 27 healthy individuals. Furthermore, significantly reduced ALIX protein levels were accompanied by their gradual transition from diffuse cytoplasmic expression to granular signals, which fell into the 0.6-2 μm diameter size range of MVBs. These ALIX-positive particles were seen in the tumor nests, including tumor-stroma border, which suggest their exosome function. MVB-like structures were also detected in tumor microenvironment including α-smooth muscle actin-positive stromal cells, budding off cancer cells in the tumor front as well as in cancer cells entrapped within lymphoid vessels. In conclusion, we determined the top aberrantly expressed exosome-associated markers and revealed the transition of diffuse ALIX protein signals into a MVB-like pattern during adenoma-carcinoma sequence. These tumor-associated particles seen both in the carcinoma and the surrounding microenvironment can potentially mediate epithelial-stromal interactions involved in the regulation of tumor growth, metastatic invasion and therapy response.

The vast majority of disease-associated single-nucleotide polymorphisms (SNPs) mapped by genome-wide association studies (GWASs) are located in the non-protein-coding genome, but establishing the functional and mechanistic roles of these sequence variants has proven challenging. Here we describe a general pipeline in which candidate functional SNPs are first evaluated by fine mapping, epigenomic profiling, and epigenome editing, and then interrogated for causal function by using genome editing to create isogenic cell lines followed by phenotypic characterization. To validate this approach, we analyzed the 6q22.1 prostate cancer risk locus and identified rs339331 as the top-scoring SNP. Epigenome editing confirmed that the rs339331 region possessed regulatory potential. By using transcription activator-like effector nuclease (TALEN)-mediated genome editing, we created a panel of isogenic 22Rv1 prostate cancer cell lines representing all three genotypes (TT, TC, CC) at rs339331. Introduction of the 'T' risk allele increased transcription of the regulatory factor 6 (RFX6) gene, increased homeobox B13 (HOXB13) binding at the rs339331 region, and increased deposition of the enhancer-associated H3K4me2 histone mark at the rs339331 region compared to lines homozygous for the 'C' protective allele. The cell lines also differed in cellular morphology and adhesion, and pathway analysis of differentially expressed genes suggested an influence of androgens. In summary, we have developed and validated a widely accessible approach that can be used to establish functional causality for noncoding sequence variants identified by GWASs.

Background: Colorectal cancer (CRC) development is accompanied by changes in expression for several genes; but the details of the underlying regulatory procesess remain unknown. Our aims were to assess the role of epigenetic processes in tumour formation and to identify characteristic DNA methylation and miRNA alterations in the colorectal adenoma-carcinoma sequence., Methods: Whole genome expression profiling was performed on colonic biopsy samples (49 healthy normal, 49 colorectal adenoma (AD), 49 CRC); on laser capture microdissected (LCM) epithelial and stromal cells from 6 CRC-normal adjacent tissue (NAT) samples pairs, and on demethylated human CRC cell lines using HGU133 Plus 2.0 microarrays (Affymetrix). Methylation status of genes with gradually altering expression along the AD-CRC sequence was further analysed on 10-10 macrodissected and 5-5 LCM samples from healthy colon, from adenoma and from CRC biopsy samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes was performed with miRWALK algorithm, miRNA expression was analysed on 3 CRC-NAT sample pairs and 3 adenoma tissue samples using the Human Panel I + II (Exiqon). SFRP1 immunohistochemistry experiments were performed., Results: A set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p < 0.01) in the biopsy samples along the adenoma-carcinoma sequence. Three of those (COL1A2, SFRP2, SOCS3) showed hypermethylation and THBS2 showed hypomethylation both in AD and in CRC samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p < 0.01) upregulated in adenoma and tumour samples compared to the healthy colonic tissue controls and could explain the altered expression of genes for which DNA methylation changes do not appear to play role (e.g. BCL2, MAL, PTGS2). Demethylation treatment could upregulate gene expression of genes that were found to be hypermethylated in human CRC tissue samples. Decreasing protein levels of SFRP1 was also observed along the adenoma-carcinoma sequence., Conclusion: Hypermethylation of the selected markers (MAL, PRIMA1, PTGDR and SFRP1) can result in reduced gene expression and may contribute to the formation of colorectal cancer.

Microarray analysis of promoter hypermethylation provides insight into the role and extent of DNA methylation in the development of colorectal cancer (CRC) and may be co-monitored with the appearance of driver mutations. Colonic biopsy samples were obtained endoscopically from 10 normal, 23 adenoma (17 low-grade (LGD) and 6 high-grade dysplasia (HGD)), and 8 ulcerative colitis (UC) patients (4 active and 4 inactive). CRC samples were obtained from 24 patients (17 primary, 7 metastatic (MCRC)), 7 of them with synchronous LGD. Field effects were analyzed in tissues 1 cm (n = 5) and 10 cm (n = 5) from the margin of CRC. Tissue materials were studied for DNA methylation status using a 96 gene panel and for KRAS and BRAF mutations. Expression levels were assayed using whole genomic mRNA arrays. SFRP1 was further examined by immunohistochemistry. HT29 cells were treated with 5-aza-2' deoxycytidine to analyze the reversal possibility of DNA methylation. More than 85% of tumor samples showed hypermethylation in 10 genes (SFRP1, SST, BNC1, MAL, SLIT2, SFRP2, SLIT3, ALDH1A3, TMEFF2, WIF1), whereas the frequency of examined mutations were below 25%. These genes distinguished precancerous and cancerous lesions from inflamed and healthy tissue. The mRNA alterations that might be caused by systematic methylation could be partly reversed by demethylation treatment. Systematic changes in methylation patterns were observed early in CRC carcinogenesis, occuring in precursor lesions and CRC. Thus we conclude that DNA hypermethylation is an early and systematic event in colorectal carcinogenesis, and it could be potentially reversed by systematic demethylation therapy, but it would need more in vitro and in vivo experiments to support this theory.

Background: A recently published transcript set is suitable for gene expression-based discrimination of normal colonic and colorectal cancer (CRC) biopsy samples. Our aim was to test the discriminatory power of the CRC-specific transcript set on independent biopsies and on formalin-fixed, paraffin-embedded (FFPE) tissue samples., Methods: Total RNA isolations were performed with the automated MagNA Pure 96 Cellular RNA Large Volume Kit (Roche) from fresh frozen biopsies stored in RNALater (CRC (n = 15) and healthy colonic (n = 15)), furthermore from FFPE specimens including CRC (n = 15) and normal adjacent tissue (NAT) (n = 15) specimens next to the tumor. After quality and quantity measurements, gene expression analysis of a colorectal cancer-specific marker set with 11 genes (CA7, COL12A1, CXCL1, CXCL2, CHI3L1, GREM1, IL1B, IL1RN, IL8, MMP3, SLC5A7) was performed with array real-time PCR using Transcriptor First Strand cDNA Synthesis Kit (Roche) and RealTime ready assays on LightCycler480 System (Roche). In situ hybridization for two selected transcripts (CA7, CXCL1) was performed on NAT (n = 3), adenoma (n = 3) and CRC (n = 3) FFPE samples., Results: Although analytical parameters of automatically isolated RNA samples showed differences between fresh frozen biopsy and FFPE samples, both quantity and the quality enabled their application in gene expression analyses. CRC and normal fresh frozen biopsy samples could be distinguished with 93.3% sensitivity and 86.7% specificity and FFPE samples with 96.7 and 70.0%, respectively. In situ hybridization could confirm the upregulation of CXCL1 and downregulation of CA7 in colorectal adenomas and tumors compared to healthy controls., Conclusion: According to our results, gene expression analysis of the analyzed colorectal cancer-specific marker set can also be performed from FFPE tissue material. With the addition of an automated workflow, this marker set may enhance the objective classification of colorectal neoplasias in the routine procedure in the future.

Background: Epithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions., Aims: To evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts., Materials and Methods: DNA isolated from normal and tumorous colonic epithelia of fresh frozen surgically removed tissue samples was used for 24 and 6 hour treatment of HT-29 colon carcinoma and HDF-α fibroblast cells. Whole genome mRNA expression analysis and qRT-PCR was performed for the elements/members of TLR9 signaling pathway. Immunocytochemistry was performed for epithelial markers (i.e. CK20 and E-cadherin), DNA methyltransferase 3a (DNMT3a) and NFκB (for treated HDFα cells)., Results: Administration of tumor derived DNA on HT29 cells resulted in significant (p<0.05) mRNA level alteration in 118 genes (logFc≥1, p≤0.05), including overexpression of metallothionein genes (i.e. MT1H, MT1X, MT1P2, MT2A), metastasis-associated genes (i.e. TACSTD2, MACC1, MALAT1), tumor biomarker (CEACAM5), metabolic genes (i.e. INSIG1, LIPG), messenger molecule genes (i.e. DAPP, CREB3L2). Increased protein levels of CK20, E-cadherin, and DNMT3a was observed after tumor DNA treatment in HT-29 cells. Healthy DNA treatment affected mRNA expression of 613 genes (logFc≥1, p≤0.05), including increased expression of key adaptor molecules of TLR9 pathway (e.g. MYD88, IRAK2, NFκB, IL8, IL-1β), STING pathway (ADAR, IRF7, CXCL10, CASP1) and the FGF2 gene., Conclusions: DNA from tumorous colon epithelium, but not from the normal epithelial cells acts as a pro-metastatic factor to HT-29 cells through the overexpression of pro-metastatic genes through TLR9/MYD88 independent pathway. In contrast, DNA derived from healthy colonic epithelium induced TLR9 and STING signaling pathway in normal fibroblasts.

Background: Diabetes mellitus is linked to pancreatic cancer. We hypothesized a role for pancreatic stellate cells (PSC) in the hyperglycemia induced deterioration of pancreatic cancer and therefore studied two human cell lines (RLT-PSC, T3M4) in hyperglycemic environment., Methodology/principal Findings: The effect of chronic hyperglycemia (CHG) on PSCs was studied using mRNA expression array with real-time PCR validation and bioinformatic pathway analysis, and confirmatory protein studies. The stress fiber formation (IC: αSMA) indicated that PSCs tend to transdifferentiate to a myofibroblast-like state after exposure to CHG. The phosphorylation of p38 and ERK1/2 was increased with a consecutive upregulation of CDC25, SP1, cFOS and p21, and with downregulation of PPARγ after PSCs were exposed to chronic hyperglycemia. CXCL12 levels increased significantly in PSC supernatant after CHG exposure independently from TGF-β1 treatment (3.09-fold with a 2.73-fold without TGF-β1, p<0.05). The upregualtion of the SP1 transcription factor in PSCs after CHG exposure may be implicated in the increased CXCL12 and IGFBP2 production. In cancer cells, hyperglycemia induced an increased expression of CXCR4, a CXCL12 receptor that was also induced by PSC's conditioned medium. The receptor-ligand interaction increased the phosphorylation of ERK1/2 and p38 resulting in activation of MAP kinase pathway, one of the most powerful stimuli for cell proliferation. Certainly, conditioned medium of PSC increased pancreatic cancer cell proliferation and this effect could be partially inhibited by a CXCR4 inhibitor. As the PSC conditioned medium (normal glucose concentration) increased the ERK1/2 and p38 phosphorylation, we concluded that PSCs produce other factor(s) that influence(s) pancreatic cancer behaviour., Conclusions: Hyperglycemia induces increased CXCL12 production by the PSCs, and its receptor, CXCR4 on cancer cells. The ligand-receptor interaction activates MAP kinase signaling that causes increased cancer cell proliferation and migration.

Adenoid cystic carcinoma (ACC) is a malignant tumor of the salivary glands but identical tumors can also arise from the breast. Despite their similar histomorphological appearance the salivary gland- and the breast-derived forms differ in their clinical features: while ACC of the salivary glands (sACC) have an aggressive clinical course, the breast-derived form (bACC) shows a very favourable clinical outcome. To date no exact molecular alterations have yet been identified which would explain the diverse clinical features of the ACCs of different origin. In our pilot experiment we investigated the post-transcriptional features of ACC cases by performing microRNA-profiling on 2-2 bACC and sACC tissues and on 1-1 normal breast and salivary gland tissue. By comparing the microRNA-profiles of the investigated samples we identified microRNAs which were expressed differently in bACC and sACC cases according to their normal controls: 7 microRNAs were overexpressed in sACC cases and downexpressed in bACC tumors (let-7b, let-7c, miR-17, miR-20a, miR-24, miR-195, miR-768-3) while 9 microRNAs were downexpressed in sACC cases and overexpressed in bACC tissues (let-7e, miR-23b, miR-27b, miR-193b, miR-320a, miR-320c, miR-768-5p, miR-1280 and miR-1826) relative to their controls. We also identified 8 microRNAs which were only expressed in sACCs and one microRNA (miR-1234) which was only absent in sACC cases. By target predictor online databases potential targets of the these microRNAs were detected to identify genes that may play central role in the diverse clinical outcome of bACC and sACC cases.

Background: Presence of cell-free-circulating DNA (fcDNA) sequences in sera of patients with inflammatory bowel diseases (IBD) is a well-established phenomenon. Potential roles of fcDNA in diagnosis, prognosis and therapy monitoring of chronic inflammatory colonic disorders have already been examined, albeit its actual biological function still remains unclear., Aims and Methods: In the present experiment, we studied the immunobiological effects of isolated fcDNA of normal and inflammatory origin administered intravenously to mice prior to induction of dextran sulfate sodium (DSS)-colitis. In addition to evaluate the current disease and histological activity, changes of the gene expression profile in isolated lamina propria cells upon TLR9 ligation were assayed., Results: A single intravenous dose of fcDNA pretreatment with colitic fcDNA exhibited beneficial response concerning the clinical and histological severity of DSS-colitis as compared to effects of normal fcDNA. Pretreatment with colitic fcDNA substantially altered the expression of several TLR9-related and inflammatory cytokine genes in a clinically favorable manner., Conclusions: During the process of acute colitis, the subsequent inflammatory environment presumably results in changes of fcDNA with the potential to facilitate the downregulation of inflammation and improvement of regeneration. Thus, preconditioning of mice with colitis-derived fcDNA via TLR9 signaling could exert a tissue-protective effect and influence beneficially the course of DSS-colitis. Elucidating mechanisms of immune response alterations by nucleic acids may provide further insight into the etiology of IBD and develop the basis of novel immunotherapies.

Epigenetic changes of stromal-epithelial interactions are of key importance in the regulation of colorectal carcinoma (CRC) cells and morphologically normal, but genetically and epigenetically altered epithelium in normal adjacent tumor (NAT) areas. Here we demonstrated retained protein expression of well-known Wnt inhibitor, secreted frizzled-related protein 1 (SFRP1) in stromal myofibroblasts and decreasing epithelial expression from NAT tissues towards the tumor. SFRP1 was unmethylated in laser microdissected myofibroblasts and partially hypermethylated in epithelial cells in these areas. In contrast, we found epigenetically silenced myofibroblast-derived SFRP1 in CRC stroma. Our results suggest that the myofibroblast-derived SFRP1 protein might be a paracrine inhibitor of epithelial proliferation in NAT areas and loss of this signal may support tumor proliferation in CRC.

In inflammatory bowel diseases the presence of free-circulating DNA (fcDNA) sequences in the sera is an established phenomenon, albeit its real biological function still remains unclear. In our study the immunobiologic effects of a single-dose, intravenously administered fcDNA of normal and colitic origin were assayed in DSS-colitic and control mice. In parallel with disease and histological activity evaluations changes of the TLR9 and inflammatory cytokine signaling gene expression profiles were assayed in isolated cells of the lamina propria. Intravenously administered colitis-derived fcDNA displayed a more prominent beneficial action regarding the clinical and histological severity of DSS-colitis than that of fcDNA of normal origin. Systemic administration of colitis-derived fcDNA significantly altered the expression of certain TLR9-related and proinflammatory cytokine genes in a clinically favorable manner. Presumably due to induction of severe colitis, the subsequent marked inflammatory environment may result changes in fcDNA with a potential to promote the downregulation of inflammation and improvement of tissue regeneration. Elucidating mechanisms of innate immune alterations by nucleic acids may provide further insight into the etiology of inflammatory bowel diseases, and develop the basis of novel nucleic acid-based immunotherapies.

Early molecular detection of the colorectal dysplasia-carcinoma transition may augment the accuracy of diagnosis in case of biopsy orientation errors. The combination of high-throughput microarray-based biomarker screening with tissue microarray-based prospective protein biomarker expression analysis could represent an additional test in routine automated diagnostic procedures. Our aim was to test and select protein markers to identify protein expression profile alterations, focusing on the dysplasia-carcinoma transition in sporadic colorectal tumors. Dysplasia-carcinoma transition-specific transcript sets were previously identified using HGU133plus2 microarrays and Taqman RT-PCR cards. Here, 26 potential dysplasia-carcinoma transition-specific markers were tested by immunohistochemistry at the protein level using tissue microarrays in a total of 168 independent colonic biopsy samples. A set of 26 transcripts [including matrix metalloproteinase-3 (MMP3) and chemokine (C-X-C motif) ligand 1 (CXCL1)] has been determined recently, indicating a linear expression correlation with the adenoma-dysplasia-carcinoma sequence, thereby having the potential to discriminate between dysplasia and early malignancy. Currently, we find that high-grade dysplastic sessile adenomatous-stage and early-stage colorectal cancer conditions can be differentiated correctly by the stromal expression of MMP3 and CXCL1, respectively, on tissue microarray-based analysis. Furthermore, in cases of sporadic colorectal tumors, MMP3 protein expression in the lamina propria itself seems to be highly specific for the detection of tumorous transition. Our current and recent results indicate that appropriate antibody marker combinations are highly suitable for tissue microarray-based and digital microscopy-based, automated, high-capacity diagnostic application in tumorous colonic diseases.

To understand the biologic role of self-DNA bound to Toll-like Receptor 9 (TLR9), we assayed its effect on gene and methyltransferase expressions and cell differentiation in HT29 cells. HT29 cells were incubated separately with type-1 (normally methylated/nonfragmented), type-2 (normally methylated/fragmented), type-3 (hypermethylated/nonfragmented), or type-4 (hypermethylated/fragmented) self-DNAs. Expression levels of TLR9-signaling and proinflammatory cytokine-related genes were assayed by qRT-PCR. Methyltransferase activity and cell differentiation were examined by using DNA methyltransferase (DNMT1, -3A, -3B) and cytokeratin (CK) antibodies. Treatment with type-1 DNA resulted in significant increase in TLR9 expression. Type-2 treatment resulted in the overexpression of TLR9-related signaling molecules (MYD88A, TRAF6) and the IL8 gene. In the case of type-3 treatment, significant overexpression of NFkB, IRAK2, and IL8 as well as downregulation of TRAF6 was detected. Using type-4 DNA, TRAF6 and MYD88A gene expression was upregulated, while MYD88B, IRAK2, IL8, and TNFSF10 were all underexpressed. CK expression was significantly higher only after type-1 DNA treatment. DNMT3A expression could also be induced by type-1 DNA treatment. DNA structure may play a significant role in activation of the TLR9-dependent and even independent proinflammatory pathways. There may be a molecular link between TLR9 signaling and DNMT3A. The mode of self-DNA treatment may influence HT29 cell differentiation.

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.

Introduction: Adenoid cystic carcinoma is a salivary gland-derived malignant tumor, but rarely it can originate from the breast, too. The salivary gland-derived form shows a very aggressive clinical outcome, while adenoid cystic carcinoma of the breast has mostly a very good prognosis., Aim: The aim of the authors was to compare the miRNA-expression profile of breast- and salivary gland-derived cases., Method: The miRNA-profiles of two salivary gland derived and two breast-derived adenoid cystic carcinoma tissues as well as one normal breast and one salivary gland tissues were analysed using the Affymetrix® Gene Chip., Results: The expression of some miRNAs differed in the tumor tissues compared to their controls: the let-7b was overexpressed in salivary gland-derived adenoid cystic carcinoma, while decreased in breast-derived adenoid cystic carcinoma. In addition, the miR-24 was decreased in salivary gland-derived but overexpressed in breast-derived adenoid cystic carcinomas. The miR-181a-2* was only detected in salivary gland-derived adenoid cystic carcinomas., Conclusions: Through post-transcriptional regulation of the genes, the diverse expression of some miRNAs may partially explain the diverse clinical outcome of salivary gland-derived and breast-derived adenoid cystic carcinomas.

Although RNA isolation is a routine process in gene expression analysis studies, the applicability of most widely available formalin-fixed, paraffin-embedded (FFPE) samples is still limited compared to fresh frozen tissue samples due to the lower quality of the isolated RNA. Recently, novel automated isolation methods were developed in order to reduce manual sample handling and increase RNA quality and quantity. Here we present a comparison of the performance of fresh frozen and matched FFPE tissue samples obtained from the same surgically removed colonic specimens (10 normal, 10 CRC) in RT-PCR experiments. RNA isolations were performed with the automated MagNA Pure 96 Cellular RNA Large Volume Kit (Roche) compared to the manual RNeasy FFPE Mini Kit (Qiagen). Gene expression analysis of a colorectal cancer-specific marker set (with 7 genes: COL12A1, CXCL1, CXCL2, GREM1, IL1B, IL8, SLC7A5) was performed with array real-time PCR using Transcriptor First Strand cDNA Synthesis Kit (Roche) and RealTime ready assays on LightCycler® 480 System (Roche). On the basis of the gene expression of the analyzed markers, fresh frozen tumorous and normal samples could be distinguished with 100% sensitivity and 100% specificity after both isolation methods. The FFPE samples could be distinguished by similarly high specificity and sensitivity with the MagNA Pure 96 isolated samples (sensitivity: 90,0%; specificity: 90,0%) and the samples isolated with manual Qiagen method (sensitivity: 85,0%; specificity: 70,0%). According to these results, FFPE samples isolated by automated methods can serve as valuable source for retrospective gene expression studies in the field of biomarker discovery and development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Tumorigenesis is accompanied by changes in the DNA methylation pattern. Our aim was to test a novel approach for identification of transcripts at whole transcript level which are regulated by DNA methylation. Our approach is based on comparison of data obtained from transcriptome profiling of primary human samples and in vitro cell culture models. Epithelial cells were collected by LCM from normal, adenoma, and tumorous colonic samples. Using gene expression analysis, we identified downregulated genes in the tumors compared to normal tissues. In parallel 3000 upregulated genes were determined in HT-29 colon adenocarcinoma cell culture model after DNA demethylation treatment. Of the 2533 transcripts showing reduced expression in the tumorous samples, 154 had increased expression as a result of DNA demethylation treatment. Approximately 2/3 of these genes had decreased expression already in the adenoma samples. Expression of five genes (GCG, NMES-1, LRMP, FAM161B and PTGDR), was validated using RT-PCR. PTGDR showed ambiguous results, therefore it was further studied to verify the extent of DNA methylation and its effect on the protein level. Results confirmed that our approach is suitable for genome-wide screening of genes which are regulated or inactivated by DNA methylation. Activity of these genes possibly interferes with tumor progression, therefore genes identified can be key factors in the formation and in the progression of the disease.

Background: The early molecular detection of the dysplasia-carcinoma transition may enhance the strength of diagnosis in the case of colonic biopsies. Our aims were to identify characteristic transcript sets in order to develop diagnostic mRNA expression patterns for objective classification of benign and malignant colorectal diseases and to test the classificatory power of these markers on an independent sample set., Methodology/principal Findings: Colorectal cancer (CRC) and adenoma specific transcript sets were identified using HGU133plus2 microarrays and 53 biopsies (22 CRC, 20 adenoma and 11 normal). Ninety-four independent biopsies (27 CRC, 29 adenoma and 38 normal) were analyzed on microarrays for testing the classificatory power of the discriminatory genes. Array real-time PCR validation was done on 68 independent samples (24 CRC, 24 adenoma and 20 normal). A set of 11 transcripts (including CXCL1, CHI3L1 and GREM1) was determined which could correctly discriminate between high-grade dysplastic adenoma and CRC samples by 100% sensitivity and 88.9% specificity. The discriminatory power of the marker set was proved to be high on independent samples in both microarray and RT-PCR analyses. 95.6% of original and 94.1% of cross-validated samples was correctly classified in discriminant analysis., Conclusions/significance: The identified transcripts could correctly characterize the dysplasia-carcinoma transition in biopsy samples, also on a large independent sample set. These markers can establish the basis of gene expression based diagnostic classification of colorectal cancer. Diagnostic RT-PCR cards can become part of the automated routine procedure.

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&#95;v1 region was higher in the healthy samples, while septin9&#95;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.

Unlabelled: Changes of the DNA methylation pattern are proven to be an important process during tumorigenesis. This event can occur in several manners in the tumor microenvironment and there are still not any effective and high-throughput methods for genome-wide analysis of this phenomenon., Aims: Our aim was to identify colorectal cancer development and progression specific marker genes regulated by DNA methylation using gene expression analysis. In this study we present a gene expression-based method combined with a cell culture model, which can be used for a genome-wide analysis of the methylation events during the colorectal tumorigenesis., Materials and Methods: Genes, which expression increased after the demethylation were determined in HT-29 colon adenocarcinoma cells treated with 10 microM 5-aza-2'-deoxycitidine. In parallel, 5000 epithelial cells were collected with laser microdissection (LCM) from normal, adenoma and tumorous colonic samples. The genes with gradually decreasing expression along the adenoma-carcinoma sequence were identified. By comparing the two groups, the transcripts, which are supposed to be regulated by methylation, could be determined. Finally, the identified gene set was validated on independent samples using RT-PCR., Conclusion: The regulation of the identified genes showing decreased expression during the adenoma-carcinoma sequence, can be associated with DNA methylation. On the basis of our results, the set of genes including tumorsuppressors can be determined genome-widely, which can be key factors in the formation and the prognosis of the disease. The identified genes showing colorectal cancer specific methylation pattern can be potential therapeutic targets in the future.

The continuous reformation and rapid repair of the colonic mucosa is essential for avoiding the aggregation of pernicious mutations induced by bacterial, toxic, or mitogenic factors. Gut-associated lymphoid tissue is supposed to play a central role in the organization of the repair mechanisms. In inflammatory conditions, the number, the diameter and the density of isolated lymphoid follicles (ILFs) are increasing. They are involved not just in immune surveillance, but their presence is also indispensable in normal mucosal regeneration of the colon. The relation of ILFs to the components of mucosal renewal such as bone marrow derived stem cells, follicular dendritic cells, subepithelial myofibroblasts or crypt formation has not been directly studied, and data about their putative organizer role are scattered in scientific literature. Whether they act as a regenerative pool containing stem cells in case of mucosal damage, or they are responsible only for the optimal cytokine milieu for the differentiation of immigrating stem cells is a question under debate. Our aim is to review the relation of ILFs to the different elements of colonic mucosal repair.

The exact molecular background and the connection between protein and mRNA expression in colorectal cancer (CRC) development and progression are not completely elucidated. Our purposes were the identification of protein markers of colorectal carcinogenesis and progression using protein arrays and validation on tissue microarrays. The connection between antibody and mRNA expression array results was also examined. Using cancerous and adjacent normal samples from 10 patients with early and 6 with advanced CRC, 67 differentially expressed genes were identified between normal and cancerous samples. A marker set containing 6 proteins (CCNA1, AR, TOP1, TGFB, HSP60, ERK1) was developed which could differentiate normal specimen, early and late stage CRC with high sensitivity and specificity. Dukes D stage samples were analyzed on HGU133plus2.0 microarrays. In these samples, mRNA and protein expression of 143 genes showed strong positive correlations (R2>0.8), while a negative correlation (R2>0.9) was found in case of 95 genes. Based on our results a correlation could be established between transcriptome and antibody array results, hence the former may be used as a high-capacity screening method before applying antibody arrays containing already planned targets. Antibody microarrays may have a fundamental importance in testing of marker combinations and future application in diagnostics of tumorous diseases.

Background: The exact molecular biological background of colorectal cancer development and progression are not hitherto known. Using microarray systems, hundreds or thousands of parameters could be examined simultaneously for answering the mentioned questions., Aim: To identify possible protein markers of colorectal cancer development and progression using antibody array, and the validation of these markers on tissue microarrays done with colorectal cancer samples. Furthermore, to determine colorectal cancer diagnostic marker combination in protein level., Materials and Methods: Surgically resected samples from ten Dukes B and six Dukes D stage patients containing both diseased and un-involved parts of the colon were freshly frozen. The samples were homogenized and the extracted proteins were used for Clontech AB500 arrays. Twelve selected genes were validated on tissue microarrays., Results: The expression of 67 proteins was altered (p < 0.05) between the normal colon and cancerous samples. These genes are related to apoptosis (n = 5), cell cycle regulation (n = 7), transcription (n = 4), DNA replication (n = 6) and other cell functions, such as transport and cell adhesion (n = 45). Twelve potential markers were immunohistochemically validated on morphological level by using tissue microarrays (CYCA1, HSP60, TOP1, APC, CBP, ERK, EGFR, C-myc, Cald, DARPP32, MRE11A, AR, EPS8)., Conclusion: Based on these results, validated colorectal cancer development related protein markers are involved in a wide spectrum of cell functions such as apoptosis, cell cycle regulation, and signal transduction. A set of six proteins has been determined, which helps to differentiate between normal specimen, early and late stage colorectal cancer with high sensitivity and specificity.

DNA methylation acts in early tumorigenesis. Its detection is possible either from tissue, stool or peripheral blood. Septin 9 is a sensitive methylation marker, which has been studied in several cancers such as breast and ovarian tumors and in neurological or hematological diseases. Septin proteins have an important role from cytoskeleton organisation to development of embryonal pattern. Nowadays intensive researches are going on about the relation between the septin 9 gene hypermethylation and colorectal cancer development.

There is rapid development in the field of protein microarray technology with the promise of important advancements in the near future. Protein microarrays have been reportedly successful in serum tumor marker profiling as well as in drug discovery and medicinal chemistry when the effect of small molecules in protein-protein interaction is studied. Some of the bottlenecks of the technology are protein instability, problems with immobilization and stabilization of proteins to the corresponding surface, as well as aspecific and /or not preferred interactions and the lack of protein amplification techniques to generate sufficient amounts of low abundance proteins. For the time being, the number of genes in RNA expression chips is significantly greater than the number of proteins available for microchip based analysis of gene expression at the protein level. The automation and standardization routinely used with nucleic acid microarrays is not yet available in their protein chip counterparts. One of the emerging applications of protein microchips is biomarker discovery via chromatographic surface-based protein array techniques, which is applicable to minute amounts of samples with excellent detection limits using mass spectrometry based interrogation. In this paper the advantages, technical limitations and main biomedical application of protein microarrays are reviewed.

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., Conclusions: 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.

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.

Background and Aims: Chronic Helicobacter pylori infection affects approximately half of the world, leads to chronic gastritis and peptic ulceration, and is linked to gastric carcinoma. Our aims were to compare the gene expression profile (GEP) of H. pylori-positive and H. pylori-negative gastric erosions and adjacent mucosa to explain the possible role and response to H. pylori infection and to get erosion-related mRNA expression patterns., Methods: Total RNA was extracted, amplified, and biotinylated from gastric biopsies of patients with H. pylori-positive and H. pylori-negative antrum erosions (ER) (8/8) and adjacent macroscopically normal mucosae (8/8). The GEP was evaluated using HGU133plus2.0 microarrays. Two independent normalizations (MAS5.0, RMA), PAM feature selection, hierarchical cluster analysis, and discriminant analysis were done. The expression of 14 genes was also measured by real-time-polymerase chain reaction. VCAM-1 and CXCL13 immunohistochemistry (IHC) was done., Results: In H. pylori infection, significant overexpression of MHC class II antigen-presenting genes, interleukin-7 receptor, ubiquitin-D, CXCR4, lactoferrin immune response-related genes, CXCL-2 and -13, CCL18 chemokine ligand, and VCAM-1 genes were established. In erosive gastritis, increased proliferation (MET) and transport (UCP2, SCFD1, KPNA4) were found, while genes associated with adhesion (SIGLEC11), transcription regulation (ESRRG), and electron and ion transport (ACADM, CLIC6) were down-regulated. Discriminant analysis successfully classified all samples into four groups (HP+ER-, HP+ER+, HP-ER+, HP-ER-) using a reduced gene set (20). Significant overexpression of VCAM-1 and CXC13 protein was detected by IHC in HP+ samples (p < .05)., Conclusions: Whole genomic microarray analysis yielded new H. pylori infection and erosion-related gene expression changes. Discriminative genes can be used in mRNA-based diagnostic classification of gastric biopsies.

Background: Overall mRNA expression analysis of colon biopsies can contribute to the understanding of molecular background of the local alterations and gene ontology-based functional classification of colonic biopsies into inflammatory and neoplastic diseases., Methods: Total RNA was extracted from frozen biopsies and amplified by T7-method. Expression profile was evaluated by Atlas Glass 1K microarrays. After microarray quality control, applicable data were available from 10 adenomas, 6 colorectal adenocarcinoma (CRC), and inflammatory bowel diseases (IBDs: 3-3 CD and UC). Multivariate statistical and cell functional analyses were performed. Real-time RT-PCR and immunohistochemistry were used for validation., Results: Discriminant analysis of selected genes could correctly reclassify all 22 samples using 4 parameters (heat shock transcription factor-1, bystin-like, calgranulin-A, TRAIL receptor 3). IBD samples were characterized by overregulated chemokine (C-X-C motif) ligand 13, replication protein A1, E74-like factor 2 and downregulated TNF receptor-associated factor 6, BCL2-interacting killer genes. In adenomas upregulation of TNF receptor-associated factor 6, replication protein A1, E74-like factor 2 and underexpression of BCL2-associated X protein, calgranulin-A genes were found. CRC cases had significantly increased epidermal growth factor receptor, topoisomerase-1, v-jun, TNF receptor-associated factor 6 and TRAIL receptor 3, and decreased RAD51 and RAD52 DNA repair gene, protein phosphatase-2A and BCL2-interacting killer mRNA levels. Epidermal growth factor receptor RT-PCR and immunohistochemistry, topoisomerase-1 RT-PCR confirmed the chip results., Conclusions: Different histological alterations can be objectively classified by functional, multivariate analysis using cDNA microarrays. Disease-specific gene expression patterns can assist to avoid the intermediate and nondescript cases in diagnostics, which do not belong to any of the conventional diagnostic groups.

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 collagenIValpha1, 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 Crohn's 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.

Background: Discrimination and classification of colorectal diseases (adenoma, colorectal cancer, inflammatory bowel disease) using biopsy samples and expression microarrays, has not been solved yet, nevertheless, it can contribute to the understanding of the colonic diseases., Methods: Total ribonucleic acid was extracted, amplified and biotinylated from frozen colonic biopsies of 15 patients with colorectal cancer, 15 with adenoma, 14 with inflammatory bowel disease and 8 normal controls. Genome-wide gene expression profile was evaluated by Human Genome U133 Plus 2.0 microarrays. Two independent methods were used for data normalization and "Prediction Analysis of Microarrays" was performed for feature selection. Leave one-out stepwise discriminant analysis was performed. The expression results were verified by real-time polymerase chain reaction., Results: Top validated genes included CD44 antigen, met proto-oncogene, chemokine ligand-12, ADAM-like decysin-1 and ATP-binding casette-A8 genes in adenoma; collagen IValpha1, lipocalin-2, calumenin, aquaporin-8 genes in colorectal cancer; and lipocalin-2, ubiquitin D and interferon induced transmembrane protein 2 genes in inflammatory bowel disease. The discriminant analysis was able to classify the samples in overall 96.2% using 7 discriminatory genes. The expression of 94% of the 52 genes measured by Taqman real-time polymerase chain reaction correlated with the results obtained using Affymetrix microarrays at a significance of p < 0.05., Conclusions: We successfully performed whole genomic microarray analysis to identify discriminative signatures using routine biopsy samples. The results set up data warehouse which can be further mined.

The confirmation of mRNA expression studies by protein chips is of high recent interest due to the widespread application of expression arrays. In this review the advantages, technical limitations, application fields and the first results of the protein arrays is described. The bottlenecks of the increasing protein array applications are the fast decomposition of proteins, the problem with aspecific binding and the lack of amplification techniques. Today glass slide based printed, SELDI (MS) based, electrophoresis based and tissue microarray based technologies are available. The advantage of the glass slide based chips are the simplicity of their application, and relatively low cost. The SELDI based protein chip technique is applicable to minute amounts of starting material (<1 microg) but it is the most expensive one. The electrophoresis based techniques are still under intensive development. The tissue microarrays can be used for the parallel testing of the sensitivity and specificity of single antibodies on a broad range of histological specimens on a single slide. Protein chips were successfully used for serum tumor marker detection, cancer research, cell physiology studies and for the verification of mRNA expression studies. Protein chips are envisioned to be available for routine diagnostic applications if the ongoing technology development will be successful in increase in sensitivity, specificity, costs reduction and for the reduction of the necessary sample volume.

The p53 gene and protein in 2005: new results, promising opportunities. The p53 gene is one of the most important genes which is involved in the regulation of cell division and tumor formation. The normal function of the p53 protein is to arrest the cell division and to turn the cell towards apoptosis in the case of cell stress and DNA damage, thereby to protect the integrity of the genome. Several p53 gene mutations that have function in carcinogenesis have been found in various tumors, including gastrointestinal carcinomas. The loss of p53 response plays an important role in the malignantly transformed cell proliferation. Promising experiments try to substitute the lost functions of the p53 gene. With their help new anti-tumor therapeutic methods should be developed.