Search

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. [ABSTRACT FROM AUTHOR]

Plasmids carrying high-risk resistance mechanisms in pathogenic E. coli have gained particular attention in veterinary medicine, especially since the discovery of the colistin resistance gene, mcr-1 . Here, we provide the first evidence of its emergence and describe the complete mcr-1 plasmid sequence of a multi-resistant avian pathogenic E. coli (APEC) strain from waterfowl in Hungary. Whole-genome sequencing analysis and core-genome MLST were performed to characterize the genome structure of the mcr-1 plasmid and to reveal the phylogenetic relation between the Hungarian duck strain Ec45-2020 and the internationally circulating mcr-1 -positive E. coli strains from poultry and humans. Results showed that plasmid pEc45-2020-33kb displayed a high level of genome identity with mcr-1 plasmids of IncX4 type widespread among human, animal and food reservoirs of enteric bacteria of public health. The mcr-1 -positive E. coli strain Ec45-2020 belongs to the ST162 genotype, considered as one of the globally disseminated zoonotic genotypes of MDR E. coli . In accordance with international findings, our results underline the importance of continuous surveillance of enteric bacteria with high-risk antimicrobial resistance genotypes, including neglected animals, such as waterfowls, as possible reservoirs for the colistin resistance gene mcr-1 .

MYC is one of the most deregulated oncogenes on multiple levels in cancer. As a node transcription factor, MYC plays a diverse regulatory role in many cellular processes, including cell cycle and metabolism, both in physiological and pathological conditions. The relentless growth and proliferation of tumor cells lead to an insatiable demand for energy and nutrients, which requires the rewiring of cellular metabolism. As MYC can orchestrate all aspects of cellular metabolism, its altered regulation plays a central role in these processes, such as the Warburg effect, and is a well-established hallmark of cancer development. However, our current knowledge of MYC suggests that its spatial- and concentration-dependent contribution to tumorigenesis depends more on changes in the global or relative expression of target genes. As the direct targeting of MYC is proven to be challenging due to its relatively high toxicity, understanding its underlying regulatory mechanisms is essential for the development of tumor-selective targeted therapies. The aim of this review is to comprehensively summarize the diverse forms of MYC oncogenic deregulation, including DNA-, transcriptional- and post-translational level alterations, and their consequences for cellular metabolism. Furthermore, we also review the currently available and potentially attractive therapeutic options that exploit the vulnerability arising from the metabolic rearrangement of MYC-driven tumors.

The basis of the conventional gene-centric view on tumor evolution is that vertically inherited mutations largely define the properties of tumor cells. In recent years, however, accumulating evidence shows that both the tumor cells and their microenvironment may acquire external, non-vertically inherited genetic properties via horizontal gene transfer (HGT), particularly through small extracellular vesicles (sEVs). Many phases of sEV-mediated HGT have been described, such as DNA packaging into small vesicles, their release, uptake by recipient cells, and incorporation of sEV-DNA into the recipient genome to modify the phenotype and properties of cells. Recent techniques in sEV separation, genome sequencing and editing, as well as the identification of new secretion mechanisms, shed light on a number of additional details of this phenomenon. Here, we discuss the key features of this form of gene transfer and make an attempt to draw relevant conclusions on the contribution of HGT to tumor evolution., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Valcz, Újvári, Buzás, Krenács, Spisák, Kittel, Tulassay, Igaz, Takács and Molnár.)

Even though people worldwide tend to consume probiotic products for their beneficial health effects on a daily basis, recently, concerns were outlined regarding the uptake and potential intestinal colonisation of the bacteria that they carry. These bacteria are capable of executing horizontal gene transfer (HGT) which facilitates the movement of various genes, including antimicrobial resistance genes (ARGs), among the donor and recipient bacterial populations. Within our study, 47 shotgun sequencing datasets deriving from various probiotic samples (isolated strains and metagenomes) were bioinformatically analysed. We detected more than 70 ARGs, out of which rpoB mutants conferring resistance to rifampicin, tet(W/N/W) and potentially extended-spectrum beta-lactamase (ESBL) coding TEM-116 were the most common. Numerous ARGs were associated with integrated mobile genetic elements, plasmids or phages promoting the HGT. Our findings raise clinical and public health concerns as the consumption of probiotic products may lead to the transfer of ARGs to human gut bacteria.

Purpose: Cisplatin-based chemotherapy is a first-line treatment for muscle-invasive and metastatic urothelial cancer. Approximately 10% of bladder urothelial tumors have a somatic missense mutation in the nucleotide excision repair (NER) gene, ERCC2 , which confers increased sensitivity to cisplatin-based chemotherapy. However, a significant subset of patients is ineligible to receive cisplatin-based therapy due to medical contraindications, and no NER-targeted approaches are available for platinum-ineligible or platinum-refractory ERCC2 -mutant cases., Experimental Design: We used a series of NER-proficient and NER-deficient preclinical tumor models to test sensitivity to irofulven, an abandoned anticancer agent. In addition, we used available clinical and sequencing data from multiple urothelial tumor cohorts to develop and validate a composite mutational signature of ERCC2 deficiency and cisplatin sensitivity., Results: We identified a novel synthetic lethal relationship between tumor NER deficiency and sensitivity to irofulven. Irofulven specifically targets cells with inactivation of the transcription-coupled NER (TC-NER) pathway and leads to robust responses in vitro and in vivo , including in models with acquired cisplatin resistance, while having minimal effect on cells with intact NER. We also found that a composite mutational signature of ERCC2 deficiency was strongly associated with cisplatin response in patients and was also associated with cisplatin and irofulven sensitivity in preclinical models., Conclusions: Tumor NER deficiency confers sensitivity to irofulven, a previously abandoned anticancer agent, with minimal activity in NER-proficient cells. A composite mutational signature of NER deficiency may be useful in identifying patients likely to respond to NER-targeting agents, including cisplatin and irofulven. See related commentary by Jiang and Greenberg, p. 1833 ., (©2020 American Association for Cancer Research.)

Equine grass sickness (also known as dysautonomia) is a life-threatening polyneuropathic disease affecting horses with approx. 80% mortality. Since its first description over a century ago, several factors, such as the phenotype, intestinal microbiome, environment, management and climate, have been supposed to be associated with the increased risk of dysautonomia. In this retrospective study, we examined the possible involvement of genetic factors. Medical and pedigree datasets regarding 1,233 horses with 49 affected animals born during a 23-year period were used in the analysis. Among the descendants of some stallions, the proportion of animals diagnosed with dysautonomia was unexpectedly high. Among males, the odds of dysautonomia were found to be higher, albeit not significantly, than among females. Significant familial clustering (genealogical index of familiality, P = 0.001) was observed among the affected animals. Further subgroups were identified with significant (P < 0.001) aggregation among close relatives using kinship-based methods. Our analysis, along with the slightly higher disease frequency in males, suggests that dysautonomia may have a genetic causal factor with an X-linked recessive inheritance pattern. This is the first study providing ancestry data and suggesting a heritable component in the likely multifactorial aetiology of the disease.

Background: Homologous recombination (HR) repair deficiency arising from defects in BRCA1 or BRCA2 is associated with characteristic patterns of somatic mutations. In this genetic study, we ask whether inactivating mutations in further genes of the HR pathway or the DNA damage checkpoint also give rise to somatic mutation patterns that can be used for treatment prediction., Results: Using whole genome sequencing of an isogenic knockout cell line panel, we find a universal HR deficiency-specific base substitution signature that is similar to COSMIC signature 3. In contrast, we detect different deletion phenotypes corresponding to specific HR mutants. The inactivation of BRCA2 or PALB2 leads to larger deletions, typically with microhomology, when compared to the disruption of BRCA1, RAD51 paralogs, or RAD54. Comparison with the deletion spectrum of Cas9 cut sites suggests that most spontaneously arising genomic deletions are not the consequence of double-strand breaks. Surprisingly, the inactivation of checkpoint kinases ATM and CHK2 has no mutagenic consequences. Analysis of tumor exomes with biallelic inactivating mutations in the investigated genes confirms the validity of the cell line models. We present a comprehensive analysis of sensitivity of the investigated mutants to 13 therapeutic agents for the purpose of correlating genomic mutagenic phenotypes with drug sensitivity., Conclusion: Our results suggest that no single genomic mutational class shows perfect correlation with sensitivity to common treatments, but the contribution of COSMIC signature 3 to base substitutions, or a combined measure of different features, may be reasonably good at predicting platinum and PARP inhibitor sensitivity.

Small extracellular vesicles (EVs) are membrane enclosed structures that are usually released from cells upon exocytosis of multivesicular bodies (MVBs) as a collection of separate, free EVs. In this study, we analysed paraffin embedded sections of archived human colorectal cancer samples. We studied 3D reconstructions of confocal microscopic images complemented by HyVolution and STED imaging. Unexpectedly, we found evidence that large, MVB-like aggregates of ALIX/CD63 positive EV clusters were released en bloc by migrating tumour cells. These structures were often captured with partial or complete extra-cytoplasmic localization at the interface of the plasma membrane of the tumour cell and the stroma. Their diameter ranged between 0.62 and 1.94 μm (mean±S.D.: 1.17 ± 0.34 μm). High-resolution 3D reconstruction showed that these extracellular MVB-like EV clusters were composed of distinguishable internal particles of small EV size (mean±S.D.: 128.96 ± 16.73 nm). In vitro , HT29 colorectal cancer cells also showed the release of similar structures as confirmed by immunohistochemistry and immune electron microscopy. Our results provide evidence for an en bloc transmission of MVB-like EV clusters through the plasma membrane. Immunofluorescent-based detection of the MVB like small EV clusters in archived pathological samples may represent a novel and unique opportunity which enables analysis of EV release in situ in human tissues.

Increased androgen receptor (AR) activity drives therapeutic resistance in advanced prostate cancer. The most common resistance mechanism is amplification of this locus presumably targeting the AR gene. Here, we identify and characterize a somatically acquired AR enhancer located 650 kb centromeric to the AR. Systematic perturbation of this enhancer using genome editing decreased proliferation by suppressing AR levels. Insertion of an additional copy of this region sufficed to increase proliferation under low androgen conditions and to decrease sensitivity to enzalutamide. Epigenetic data generated in localized prostate tumors and benign specimens support the notion that this region is a developmental enhancer. Collectively, these observations underscore the importance of epigenomic profiling in primary specimens and the value of deploying genome editing to functionally characterize noncoding elements. More broadly, this work identifies a therapeutic vulnerability for targeting the AR and emphasizes the importance of regulatory elements as highly recurrent oncogenic drivers., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Breast cancer 2 (BRCA2)-associated breast and ovarian cancers are sensitive to platinum-based chemotherapy. It is unknown whether BRCA2-associated prostate cancer responds favorably to such treatment., Methods: A retrospective analysis of a single-institution cohort of men with castration-resistant, metastatic prostate cancer was performed to determine the association between carrier status of pathogenic BRCA2 germline variants and prostate-specific antigen response to carboplatin-based chemotherapy. From 2001 through 2015, 8081 adult men with prostate cancer who had a consultation and/or underwent treatment at Dana-Farber Cancer Institute provided blood samples and consented to analyses of biologic material and clinical records. A subgroup of 141 men received at least 2 doses of carboplatin and docetaxel for castration-resistant disease (94% were also taxane refractory). These patients were categorized according to the absence or presence of pathogenic germline mutations in BRCA2 based on DNA sequencing from whole blood. The primary outcome was the response rate to carboplatin/docetaxel chemotherapy, defined according to a decline in prostate-specific antigen that exceeded 50% within 12 weeks of initiating this regimen. Associations between BRCA2 mutation status and response to carboplatin-based chemotherapy were tested using the Fisher exact test, with a 2-sided P value < .05 as the threshold for significance., Results: Pathogenic germline BRCA2 variants were observed in 8 of 141 men (5.7%; 95% confidence interval, 2.5%-10.9%). Six of 8 BRCA2 carriers (75%) experienced prostate-specific antigen declines >50% within 12 weeks, compared with 23 of 133 noncarriers (17%; absolute difference, 58%; 95% confidence interval, 27%-88%; P < .001). Prostate cancer cell lines functionally corroborated these clinical findings., Conclusions: BRCA2-associated, castration-resistant prostate cancer is associated with a higher likelihood of response to carboplatin-based chemotherapy than non-BRCA2-associated prostate cancer. Cancer 2017;123:3532-9. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Aberrant methylation is one of the most frequent epigenetic alterations that can contribute to tumor formation. Cell-free DNA can originate from tumor tissue; therefore, the evaluation of methylation markers in cell-free DNA can be a promising method for cancer screening. Our aim was to develop a panel of biomarkers with altered methylation along the colorectal adenoma-carcinoma sequence in both colonic tissue and plasma. Methylation of selected CpG sites in healthy colonic (n = 15), adenoma (n = 15), and colorectal cancer (n = 15) tissues was analyzed by pyrosequencing. MethyLight PCR was applied to study the DNA methylation of SFRP1, SFRP2, SDC2, and PRIMA1 gene promoters in 121 plasma and 32 biopsy samples. The effect of altered promoter methylation on protein expression was examined by immunohistochemistry. Significantly higher (P < 0.05) DNA methylation levels were detected in the promoter regions of all 4 markers, both in CRC and adenoma tissues compared with healthy controls. Methylation of SFRP1, SFRP2, SDC2, and PRIMA1 promoter sequences was observed in 85.1%, 72.3%, 89.4%, and 80.9% of plasma samples from patients with CRC and 89.2%, 83.8%, 81.1% and 70.3% from adenoma patients, respectively. When applied as a panel, CRC patients could be distinguished from controls with 91.5% sensitivity and 97.3% specificity [area under the curve (AUC) = 0.978], while adenoma samples could be differentiated with 89.2% sensitivity and 86.5% specificity (AUC = 0.937). Immunohistochemical analysis indicated decreasing protein levels of all 4 markers along the colorectal adenoma-carcinoma sequence. Our findings suggest that this methylation biomarker panel allows non-invasive detection of colorectal adenoma and cancer from plasma samples.

Background: MiRNA expression markers are well characterized in colorectal cancer (CRC), but less is known about miRNA expression profiles in colorectal adenomas. Genome-wide miRNA and mRNA expression analyses were conducted through the colorectal adenoma dysplasia sequence. Furthermore, analysis of the expression levels of miRNAs in matched plasma samples was performed, focusing on biomarker candidates; miRNA and mRNA expression analyses were performed on colorectal biopsies and plasma samples (20 normals; 11 tubular and 9 tubulovillous adenomas; 20 colorectal carcinomas) by miRNA 3.0 and Human Transcriptome Array (Affymetrix) and validated by RT-qPCR. Microarray data were analyzed using Expression Console and mRNA targets were predicted using miRWALK 2.0., Results: Based on microarray analysis, 447 miRNAs were expressed in tissue and 320 in plasma. Twelve were upregulated (miR-31, 8-fold p  < 0.001) and 11 were downregulated (miR-10b 3-fold p  < 0.001) in neoplastic lesions compared to normal group. Eleven miRNAs showed altered expression between adenoma subtypes (miR-183 2.8-fold change, p  < 0.007). Expression level of 24 miRNAs differed between adenoma and CRC groups (including miR-196a, 3.5-fold). Three miRNAs (miR-31, miR-4506, miR-452*) were differentially expressed in adenoma compared to normal both in tissue and plasma samples. miRNA expression data were confirmed by RT-PCR both in plasma and matched tissue samples., Conclusions: MiRNAs showed characteristic expression changes during CRC development in tissue. miRNAs were also presented in plasma and positively correlated with matched tissue expression levels. The identified miRNA expression changes could be verified RT-PCR methods facilitating routine application.

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.

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.

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.

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.

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.