Chronic lymphocytic leukemia (CLL), characterized by the progressive and uncontrolled accumulation of CD19+ B cells, currently remains as an incurable malignancy. The difficulties of eliciting curative measures in CLL are partly driven by the adaptability of the transcriptional response mediated by epigenetic mechanisms. In this study, we sought to better characterize the complexities of the CLL transcriptional profile by conducting an integrative analysis between the B cell enhancer and super enhancer signatures defined from 3 B cell H3K27Ac ChIPseq samples (CD19+ B cell, GM12878, and MEC1), the DNA methylation signatures defined from reduced-representation bisulfite sequencing (RRBS) of 42 CLL patient and 8 healthy donor samples, and the mRNA expression signatures defined from RNA sequencing of 47 CLL patient and 5 healthy donor samples. From our analysis, we identified super enhancers (SEs) in each of the ChIPseq profiles (approximately 4% of called enhancers) and discovered 741 SEs in GM12878, 374 SEs in MEC1, and 523 SEs in the CD19+ B cell profiles, respectively. Based on MSigDB gene ontology analysis, many of the genes corresponding with SEs were involved in pathways regulating immune signaling activation (e.g. TNFA_SIGNALING_VIA_NFKB, INFLAMMATORY RESPONSE) or metabolic homeostasis (e.g. MTORC1_SIGNALING, FATTY_ACID_METABOLISM). By further analyzing the corresponding expression level of SE-associated genes in CLL patients, we identified 190 transcripts associated with SEs that were significantly overexpressed in CLL patient B cells (Student’s t-test p<0.05), and this overexpressed subset of SE-associated transcripts was enriched in genes involved in either immune signaling (e.g. LCK, FCER2) or metabolic regulation (e.g. LSR, ENO2). Based on the differential expression of genes associated with enhancers occurring between CLL patient and healthy donor B cells, we then wanted to determine whether differential DNA methylation within enhancers corresponded with upregulation of CLL transcripts. Based on differential DNA methylation (DM) analysis (methylation difference +/- .25; Student’s t-test p-value<0.05) from our RRBS samples, we discovered 744 DM CpG sites that overlapped within our identified B cell enhancers, and most of the DM CpG sites in CLL were significantly hypomethylated (avg. DM GpG difference: enhancer = -0.40; non-enhancer = -0.08). Examples of hypomethylated enhancers included super enhancers corresponding with overexpressed transcripts ENO2, SEPT9, RXRA, and CCR7 as well as a typical enhancer that corresponded with the overexpressed transcript PDCD1. Based on the derived information from our integrative analysis of B cell enhancers, we then compared the effects of preferentially targeting enhancer-mediated expression with either the BET bromodomain inhibitor JQ1 or the cyclin dependent kinase-7 (CDK7) inhibitor THZ1. Based on in vitro assays and RNAseq expression analysis comparing THZ1 and JQ1-treated CLL cell lines MEC1 and MEC2, we saw that JQ1 could inhibit CLL cell line proliferation, suppress IgM-mediated primary CLL proliferation, and differentially disrupt transcription of genes involved in immune signaling cascades. Contrastingly, we saw that THZ1 elicited a different response in CLL cell lines and primary cells by disrupting cell viability, inducing apoptosis, and differentially downregulating genes involved in metabolic homeostasis. The specific enhancer-associated genes disrupted by the respective treatments further highlight the dichotomy of JQ1 and THZ1-mediated effects, as JQ1 selectively suppressed the B cell activation marker gene FCER2 and the PD-1 receptor gene PDCD1, whereas THZ1 selectively suppressed the glycolytic enolase gene ENO2 and the proto-oncogene FGR. Collectively, these results reveal how CLL DNA hypomethylation within B cell enhancers can mediate immune signaling and metabolic expression signatures in CLL and can differentially be disrupted by BET bromodomain or CDK7 inhibition.

Abstract Chronic lymphocytic leukemia (CLL) is a cancer in which B-lymphocytes proliferate in an unchecked manner and escape normal apoptotic death. It is one of the most common leukemias in the Western world, and currently, there is no cure for CLL due to the heterogeneous ways in which it can proliferate. Due to the dynamics of how CLL can develop, it is very critical to investigate the oncogenic mechanisms that allow particular clinical CLL subtypes to expand, as well as discovering potential underlying mechanisms that are common among all CLL patients. With this comprehensive goal in mind, we investigated the gene expression landscape in CLL by performing whole transcriptome analysis via RNA sequencing in 47 CLL patients and 5 normal CD19+ B-cell donors to determine differential expression and isoform patterns in CLL. Between CLL and normal CD19+ B-cells, there were 725 differentially expressed genes (FDR p<.01), with 217 of these genes being upregulated in CLL. Several of these upregulated genes include CTLA4, CD276, CLNK, and PDCD1, all of which are involved in T-cell interactions. We also discovered significant upregulation in the kinase suppressor of Ras 2 (KSR2) gene, an event previously not reported in CLL. We also see clear clinical separation of IGHV unmutated vs. IGHV mutated CLL patients based solely on the expression signature created by hierarchical clustering. Examples of prognostic genes that correspond to IGHV status include previously identified genes LPL, LDOC1, PTCH1, and CRY1, as well as potentially novel prognostic expression markers SPG20, ARSD, KLK2, and MTSS1. We also utilized the SpliceSeq algorithm software and investigated the differential isoform patterns that take place between CLL patients and normal B-cell donors. Based on the results, we discovered 300 alternative promoter, 135 alternative terminator, 287 exon skipping, and 30 premature stop events that differ between CLL and normal B-cells. From these results, we validated two isoform switching events that take place in the splicing machinery genes SF3B1 and SNRNP70. For SF3B1, a short transcript devoid of the critical protein domains is overexpressed in CLL compared to the normal B-cell counterpart. For SNRNP70, a particular transcript transcribed by an alternative promoter is overexpressed in CLL patients compared to normal CD19+ B-cells. This overexpressed SNRNP70 isoform does not contain the RNA recognition motif crucial for 5′ RNA binding and splicing initiation, thus theoretically rendering its canonical action nonfunctional. Overall, transcriptome profiling of CLL patients using RNA sequencing allowed for discovery of differential expression changes previously not reported in CLL, as well as discovering novel splicing events that provide insight into potentially new oncogenic mechanisms in CLL proliferation. Citation Format: Austin Y. Shull, Junfeng Luo, Jeong-Hyeon Choi, Lirong Pei, Farrukh T. Awan, Eun-Joon Lee, Jimei Liu, Phillip J. Buckhaults, Xiao-Jie Yan, Nicholas Chiorazzi, Huidong Shi. Identifying differential gene expression and splicing events in chronic lymphocytic leukemia patients through whole transcriptome profiling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5182. doi:10.1158/1538-7445.AM2014-5182

Abstract NOV (Nephroblastoma OVerexpressed) C-ter (NOV C-ter) is the carboxy-terminal sequence (170aa) of NOV/CCN3 protein. NOV/CCN3 (357aa), a member of CCN family, is secreted by quiescent endothelial cells and is involved in the regulation of various cellular functions including angiogenesis, proliferation, differentiation, survival, adhesion and migration. Although NOV C-ter does not have a direct cytotoxic effect when tested on a large panel of human cell lines including the NCI 60 cell lines (NCI Drug Screening Program), our data demonstrates significant anti-angiogenic and anti-tumor effects in a variety of experimental models. NOV C-ter inhibits in vitro endothelial cell tube formation in a dose-dependent fashion, using endothelial cells derived from porcine aorta, human microvasculature, or human umbilical vein (HUVECs). In addition, NOV C-ter impairs HUVEC proliferation, migration, invasion and adhesion. Interestingly, inhibition of HUVEC proliferation occurs via interference with growth-promoting signals involving Apelin-13 and adenomedullin. Furthermore, NOV C-ter specifically reduced phosphorylation of PI3K/Akt in endothelial cells, with no significant effect on the p44/42 (ERK1/2) pathway. In vivo, NOV C-ter had significant anti-tumor effect as a single agent in a murine glioblastoma xenograft model using human X-12 culture-adapted cells and in a nude mouse xenograft model using human non-small-cell lung cancer cells (A549). In these two systems, NOV C-ter provided superior survival benefit compared to treatment with bevacizumab or vehicle (PBS). Similarly, NOV C-ter significantly increased overall survival versus vehicle (PBS), an effect which was comparable to temozolomide treatment, in a murine syngeneic orthotopic glioblastoma model using the GL-261 cell line. To directly study the effect of NOV C-ter on angiogenesis, we treated zebrafish embryos prior to the onset of angiogenesis, and found a considerable reduction in vessel formation compared to bevacizumab-treated or untreated zebrafish. NOV C-ter also reduced tumor neovascularization in human breast cancer (T47D cell line) implanted in zebrafish. In summary, NOV C-ter demonstrates anti-angiogenic and tumoricidal effects via a novel mechanism of action. Mechanistic studies of NOV C-ter are underway to evaluate its precise effects on key signaling pathways in endothelial cells. Preclinical pharmacology and toxicology studies are also underway with a plan to advance to early-phase clinical trials. Citation Format: Junfeng Luo, Yong Teng, Minghui Li, Theodore S. Johnson, Franck Cuttitta, Zhengtao Chu, Xiaoyang Qi, John K. Cowell, Olivier Rixe. NOV C-ter: A novel preclinical anti-angiogenic agent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1023. doi:10.1158/1538-7445.AM2014-1023

Bisulfite conversion of genomic DNA combined with next-generation sequencing (NGS) has become a very effective approach for mapping the whole-genome and sub-genome wide DNA methylation landscapes. However, whole methylome shotgun bisulfite sequencing is still expensive and not suitable for analyzing large numbers of human cancer specimens. Recent advances in the development of targeted bisulfite sequencing approaches offer several attractive alternatives. The characteristics and applications of these methods are discussed in this review article. In addition, the bioinformatic tools that can be used for sequence capture probe design as well as downstream sequence analyses are also addressed.

Abstract Abstract 526 Chronic lymphocytic leukemia (CLL) is a biologically and clinically heterogeneous disease. The somatic hypermutation status of the immunoglobulin heavy chain variable (IGHV) genes has been identified as one of the most robust prognostic markers in CLL. Patients with unmutated IGHV status (U-CLL) typically experience an inferior outcome compared to those whose clones express mutated IGHV genes (M-CLL). We conducted a genome-wide DNA methylation analysis in CD19+ B-cells from a group of 43 CLL patients using reduced representation bisulfite sequencing (RRBS). Using base-pair resolution methylation sequencing, 2323 differentially methylated regions between CLL and normal B cells (CLL-specific DMRs) and 569 between M-CLL and U-CLL samples (IGHV-specific DMRs) were identified in the CLL genomes. The IGHV-specific DMRs are mostly unique when compared to the CLL-specific DMRs. Less than 10% of the IGHV-specific DMRs are located in promoter regions; however, more than half of these overlap with known DNase I hypersensitive sites, enhancer regions marked by histone modification (H3K4Me1 and H3K27Ac), and transcription factor binding sites in the ENCODE datasets, which indicates that these DMRs contain regulatory sequences. Distinctive DNA methylation patterns were observed in M-CLL and U-CLL samples. Overall, U-CLL was found to contain 50% more hypermethylated regions than M-CLL samples. The hypermethylated loci observed in the U-CLL samples also appear to be hypermethylated in normal naïve B cells as compared memory B cells, suggesting that M-CLL and U-CLL differ in differentiation status corresponding to normal B cell differentiation stages. RNA-seq analysis performed using matched samples (n=34), in which both DNA methylation and gene expression data were available, demonstrated excellent correlation between DNA methylation and gene expression. Several genes whose expression status was previously shown to be associated with CLL prognosis such as ZAP70, CRY1, LDOC1, SEPT10, LAG3, and LPL were differentially methylated in the promoter regions between M-CLL and U-CLL samples indicating that DNA methylation plays an important role in defining the gene expression patterns of these prognostic genes. We further validated 9 genes with IGHV-specific DMRs in the promoter regions using bisulfite pyrosequencing, and the results demonstrated excellent correlation between differential methylation and IGHV mutation status. These novel differentially methylated genes could be developed into biomarkers for CLL prognosis. In addition, DNA hypomethylation was observed in a significant number of genes involved in lymphocyte activation such as PDCD1, NFATc1, and CD5. DNA hypomethylation was observed in the proximal promoter and far up-stream enhancer regions of CD5, an important cell surface marker that uniquely identifies CLL. Overall, the DNA methylation landscape in CLL patients indicates that CLL B cells possess an active B-cell phenotype; at the same time, U-CLL and M-CLL are faithfully committed to their lineage resembling either naïve or memory B cells. In summary, this comprehensive DNA methylation analysis has identified a large number of novel epigenetic changes in CLL patients. The results from this study will further advance our understanding of the epigenetic contribution to molecular subtypes in CLL. Disclosures: No relevant conflicts of interest to declare.

We have compared here the nucleic acid hybridization abilities of three kinds of immobilized probes with different structures. Under the conventional structural design, we found that the share-stem hairpin structure probe (SHP) was more easily hybridized with the target than the linear probe and conventional hairpin shaped probe (HP). The HP probe had the lowest hybridization ability. However, it was shown a contrary result in the single-nucleotide mismatch discrimination ratio. Subsequently, we increased the Tm of the two hairpin-shaped probes and found both of the two probes were improved on the hybridization specificities even though the hybridization abilities were decreased. The result of the hybridization temperature optimization experiment showed that at 45 degrees C, the Dr Ratio (mismatch discrimination ratio) of HP-28T and SHP-32T were both as high as 8.5 while the ratios of linear probe were -0.3-0.7. The results suggested that the immobilized shared stem hairpin shaped probes also had the ability to discriminate single-nucleotide variation under proper design.

Abstract Abstract 3504 Programmed Death-1 (PD-1) is an inhibitory cell surface receptor of the immunoglobulin superfamily expressed on activated lymphocytes, monocytes and dendritic cells. Although PD-1 function is best characterized in T-cells, it is known that PD-1 also suppresses the immune response of B lymphocytes through protein phosphatase recruitment and dephosphorylation of signaling molecules downstream of the B-cell receptor (BCR). Recent studies have found that PD-1 expression is elevated at the mRNA as well as the protein levels in B cells obtained from chronic lymphocytic leukemia (CLL) patients compared to those from healthy controls. Using genome-wide DNA methylation sequencing, we identified PD-1 as one of the significantly hypomethylated genes in CLL compared to normal B-cell samples. Three differentially methylated regions (DMRs) were discovered in the first intron, proximal promoter and up-stream enhancer regions. We validated these DMRs in 43 CLL and 7 normal control samples using bisulfite pyrosequencing. The pyrosequencing analysis further confirmed that all three regions were significantly hypomethylated in CLL patient samples (p<0.001). These epigenetic changes resulted in the overexpression of PD-1 in primary CLL B cells, which was confirmed by real-time quantitative PCR. In B cells isolated from healthy controls, flow cytometry analysis showed that only approximately 1% expressed PD-1, whereas PD-1 positive B cells in CLL patients ranged from 5% to 64%. No correlation between PD-1 status and IGHV mutations or CD38 expression was observed. To elucidate the mechanisms of epigenetic regulation of PD-1 expression, we studied five non-Hodgkin's lymphoma cell lines including Mec-1, Granta 519, RL, Raji and DB. Bisulfite pyrosequencing results showed that only the up-stream enhancer is differentially methylated in these cell lines. Treatment of lymphoma cell lines with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors can up-regulate PD-1 expression in RL, Raji and DB cell lines in which the up-stream enhancer region is hypermethylated; however, the same treatments decreased the PD-1 expression in Mec-1 cells, which are demethylated in the PD-1 enhancer region and express PD-1 on the cell surface. Chromatin immunoprecipitation (ChIP) analysis revealed that H3K4me3 and H3K4me1 modifications were significantly enriched in the promoter and enhancer regions in PD-1 positive Mec-1 cells, respectively. However, H3K27me3 modification was enriched in both promoter and enhancer regions in PD-1 negative RL cells, while enrichment of H3K3me3 and H3K4me1 modification was significantly decreased. These results suggest that coordinated regulation of enhancer activity by DNA methylation and histone modification is crucial for PD-1 expression in CLL B cells. Furthermore, we mapped the nucleosome occupancy in the enhancer regions using a high-resolution, single-molecule approach. The nucleosome mapping results revealed nucleosome-depleted regions in Mec-1 cells, but not in RL cells. This novel finding demonstrates the complexity of epigenetic regulation of PD-1 expression. To determine the function of PD-1 in CLL, we co-cultured the Mec-1 cell line and primary CLL B-cells with a hepatocyte cell line Huh7.5 that overexpresses exogenous PD-1 ligand, PD-L1. Surprisingly, unlike PD-1 positive normal B cells, we did not observe increased apoptosis in the co-cultured CLL B cells, suggesting that PD-1 has a different functional role in CLL compared to normal B cells. In summary, we present here the novel finding that DNA hypomethylation in the enhancer region of PD-1 leads to aberrant overexpression of PD-1 on CLL B-cell surfaces and that in CLL PD-1 may have a different function than in normal B cells. Disclosures: No relevant conflicts of interest to declare.

An improved technique for single nucleotide mismatch discrimination using immobilized double-stranded DNA probes with a shared-stem hairpin (SH) structure is developed. A hairpin-like double-stranded DNA probe without any chromophore was immobilized on an agarose film-coated slide. The base number of the stem area was increased to 9-19 nt and the entire shared-stem area was included in the hybridization area, in which a mutated nucleotide was introduced in the middle. For the perfect match SH probe, we introduced an inner mismatch in the middle positon of the complementary chain. After the introduction of the inner mismatch, the hybridization ability of the SHP probe with a long stem was enhanced significantly. On the other hand, the mismatch probe was not able to hybridize to the perfect matched target. The annealing properties, specificity and hybridization dynamics of this kind of double-stranded DNA probes immobilized on an agarose film are greatly improved in comparison with those for the linear ones and traditional hairpin-like ones. Collectively we demonstrated that this type of immobilized double-stranded DNA probes had an excellent discrimination ratio for single nucleotide mismatches.

Noise induced hearing loss (NIHL) is a complex occupational hazard caused by an interaction between genetic and environmental factors. Millions of Chinese industrial people are daily exposed to high level of noise. Although the environmental risk factors have been studied extensively, the nature of the genetic factors contributing to HIHL has not yet been clarified. In this study, we investigated 15 single nucleotide polymorphisms (SNPs) in 6 candidate genes influence susceptibility to noise in Chinese noise-exposed workers. Data from 3-dimensional polyacrylamide gel-based microarray platforms were analyzed. 103 blood samples were collected from noise-exposed laborers in Ningbo, Zhejiang, China. Subsequently, the interaction between noise exposure and genotypes and their effect on NIHL were analysed using logistic regression. Two interesting results were observed between noise exposure levels and genotypes of three SNPs, hence confirming that they are NIHL susceptibility genes in Chinese population.

In this paper we study the distribution of the cardinality of the intersection of a centrally symmetric convex lattice set with lines perpendicular to x − axis or y − axis. For some centrally symmetric convex lattice set, we prove that neither the section counter function f x nor f y can be strictly increasing functions.

Massively parallel genomic DNA fragments display on chip plays a key role in the new generation DNA sequencing. Here, we developed a new technology to display the parallel genomic DNA fragment massively based on two-step reaction with capital EF, Phi29 DNA polymerase. The genomic DNA fragments were firstly amplified by rolling-circle amplification (RCA) reaction in liquid phase, and then amplified further on the chip by the strand displacement of capital EF, Phi29 DNA polymerase. In our experiments, through DNA colonies produced by two-step amplification reaction T7 genomic DNA fragments are displayed massively and parallely on the chip, which has been verified through hybridizing the probe labeled with fluorescence or extension reaction with fluorescent-dNTP. The significant difference of fluourescence signals between background and displayed DNA fragments could be obtained. Our results show that the method has good reproducibility in experiments, which may be hopeful to serve the high-throughput sequencing.

A method for determining methylation density of target CpG islands has been established. In the method, DNA microarray was prepared by spotting a set of PCR products amplified from bisulfite-converted sample DNAs. The PCR products on the microarray were treated by SssI methyltransferase and labeled with TAMRA fluorescence. A recombinant, antibody-like methyl-CpG-binding protein labeled with Cy5 fluorescence was used to identify symmetrical methyl-CpG dinucleotide of the PCR products on the microarray. By use of a standard curve with control mixtures, the ratio of two fluorescence signals can be converted into percentage values to assess methylation density of targeted fragments. We obtained the methylation density of six CpG islands on the two tumor suppressor genes of CDK2A and CDK2B from seven cancer cell line samples and two normal blood samples. The validity of this method was tested by bisulfite sequencing. This method not only allows the quantitative analysis of regional methylation density of a set of given genes but also could provide information of methylation density for a large amount of clinical samples.

Aberrant DNA methylation of the CpG islands for cancer‐related genes is among the earliest and most frequent alterations in cancer and may be useful for diagnosing cancer or evaluating recurrent disease.

Background DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing. Results We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray), provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients. Conclusion This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

Aberrant DNA methylation of CpG site in the gene promoter region has been confirmed to be closely associated with carcinogenesis. In the present study, a microarray-based methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for parallel detecting changes of DNA methylation in cancer was developed. After modification by sodium sulfite, the unmethylated cytosine in the genomic DNA is converted to uracil while leaving the 5-methylcytosine unchanged, which can be detected by bifunctional primer carrying a unique sequence tag in addition to a locus-specific sequence. Because each locus has a distinct tag, the detecting reactions can be performed in a highly multiplexed fashion and the resulting product then be hybridized to the reverse complements of the sequence tags arrayed on a glass slide for methylation analysis. The calibration curves with the correlation coefficient >0.97 were established, which suggested that the method could be used in near-quantitative DNA methylation analysis. Two breast tumor-related genes (E-cad and p16) are successfully analyzed by two group primers (22 primers total), and the results are compatible with that of methylation-specific PCR (MSP). Our research proved that the method is simple and inexpensive, and could be applied as a high-throughput tool to quantitatively determine methylation status of the investigated genes.

Background: The emerging role of single nucleotide polymorphisms (SNPs) in clinical diagnostics and studies has created a need for simple and high-throughput genotyping methods. Previously, we developed a 3-dimensional polyacrylamide gel-based microarray (3-D microarray) of PCR-product. This method can detect single SNP locus from multiple DNA samples on one chip.Methods: Hyperbranched rolling circle amplification (HRCA) was used to recognize different SNP loci and amplify the fragments from genomic samples. Different HRCA products were used to fabricate the 3-D microarray. and dual-color fluorescent probes were used to detect signals.Results: This assay was applied to genotype 2 SNP loci from a set of 6 genomic DNA samples on one chip. Universal acryl-modified primer and one pair of dual-color fluorescent probes were used for all sample detection to reduce the cost. We demonstrate that this assay can detect 10 ng genomic DNA.Conclusions: Combination of HRCA and 3-D microarray allows parallel discrimination of different alleles from different samples on a single chip. It is a feasible method for high-throughput mutation analysis and disease diagnosis. (C) 2008 Published by Elsevier B.V.

Objectives: Molecular margin analysis is considered more sensitive in detecting preneoplastic lesions and residual cancer cells than conventional histological margin examination. Hence, we examined MGMT expression profile and methylation status in histologically negative margins of colorectal cancer patients.Design and methods: This study included 24 colorectal tumor tissues and corresponding negative surgical margin tissues. MGMT promoter methylation patterns were analyzed by using methylation-specific oligonucleotide microarray. In addition, MGMT protein expression was analyzed by immunohistochemistry. MGMT clinical significance was evaluated together with other well-known clinicopathological factors.Results: Extensive MGMT promoter methylation was observed in tumor tissues; a moderate methylation level was found in surgical margin tissues and little or no methylation was observed in the normal control. There was a trend towards longer overall survival for those patients with negative MGMT immunostaining in surgical margins.Conclusions: MGMT expression negative in surgical margin tissues indicates longer overall survival for colorectal tumor patients. (c) 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Aberrant DNA methylation of CpG site in the gene promoter region has been confirmed to be closely associated with carcinogenesis. The analysis of cancer-related gene promoters may be useful for cancer diagnosis or the detection of recurrence. In this present study, a microarray-based minisequencing for parallel detecting changes of DNA methylation in cancer was developed. After modification by the sodium sulfite, the unmethylated cytosine in the genomic DNA is converted to uracil while leaving the 5-methylcytosine unchanged, which can be detected by elongating one base on the site-specific primer immobilized on the microarray. Four CpG sites in the E-cadherin gene promoter were near- quantitative methylation analysis by this method. The experiments results demonstrated that this DNA microarray based method could be applied as a highthroughput tool for rapid quantitation of cytosine methylation suitable for a wide range of biological investigations.