Victoria Valinluck Lao

Epigenetics and colorectal cancer

Colorectal cancer (CRC) is a leading cause of cancer deaths worldwide. It results from an accumulation of genetic and epigenetic changes in colon epithelial cells, which transforms them into adenocarcinomas. Over the past decade, major advances have been made in understanding cancer epigenetics, particularly regarding aberrant DNA methylation. Assessment of the colon cancer epigenome has revealed that virtually all CRCs have aberrantly methylated genes and that the average CRC methylome has hundreds to thousands of abnormally methylated genes. As with gene mutations in the cancer genome, a subset of these methylated genes, called driver genes, is presumed to have a functional role in CRC. The assessment of methylated genes in CRCs has also revealed a unique molecular subgroup of CRCs called CpG island methylator phenotype (CIMP) cancers; these tumors have a particularly high frequency of methylated genes. These advances in our understanding of aberrant methylation in CRC have led to epigenetic alterations being developed as clinical biomarkers for diagnostic, prognostic and therapeutic applications. Progress in this field suggests that these epigenetic alterations will be commonly used in the near future to direct the prevention and treatment of CRC.

Impact of Base Analogues within a CpG Dinucleotide on the Binding of DNA by the Methyl-Binding Domain of MeCP2 and Methylation by DNMT1

The epigenetic control of transcription requires the selective recognition of methylated CpG dinucleotides by methylation-sensitive sequence-specific DNA binding proteins. In order to probe the mechanism of selective interaction of the methyl-binding protein with methylated DNA, we have prepared a series of oligonucleotides containing modified purines and pyrimidines at the recognition site, and we have examined the binding of these oligonucleotides to the methyl-binding domain (MBD) of the methyl-CpG-binding protein 2 (MeCP2). Our results suggest that pyrimidine 5-substituents similar in size to a methyl group facilitate protein binding; however, binding affinity does not correlate with the hydrophobicity of the substituent, and neither the 4-amino group of 5-methylcytosine (mC) nor Watson-Crick base pair geometry is essential for MBD binding. However, 5-substituted uracil analogues in one strand do not direct human DNA methyltransferase 1 (DNMT1) methylation of the opposing strand, as does mC. Important recognition elements do include the guanine O6 and N7 atoms present in the major groove. Unexpectedly, removal of the guanine 2-amino group from the minor groove substantially enhances MBD binding, likely resulting from DNA bending at the substitution site. The enhanced binding of the MBD to oligonucleotides containing several cytosine analogues observed here is better explained by a DNA-protein interface mediated by structured water as opposed to hydrophobic interactions.

Mechanisms of Base Selection by the Escherichia coli Mispaired Uracil Glycosylase

The repair of the multitude of single-base lesions formed daily in cells of all living organisms is accomplished primarily by the base excision repair pathway that initiates repair through a series of lesion-selective glycosylases. In this article, single-turnover kinetics have been measured on a series of oligonucleotide substrates containing both uracil and purine analogs for the Escherichia coli mispaired uracil glycosylase (MUG). The relative rates of glycosylase cleavage have been correlated with the free energy of helix formation and with the size and electronic inductive properties of a series of uracil 5-substituents. Data are presented that MUG can exploit the reduced thermodynamic stability of mispairs to distinguish U:A from U:G pairs. Discrimination against the removal of thymine results primarily from the electron-donating property of the thymine 5-methyl substituent, whereas the size of the methyl group relative to a hydrogen atom is a secondary factor. A series of parameters have been obtained that allow prediction of relative MUG cleavage rates that correlate well with observed relative rates that vary over 5 orders of magnitude for the series of base analogs examined. We propose that these parameters may be common among DNA glycosylases; however, specific glycosylases may focus more or less on each of the parameters identified. The presence of a series of glycosylases that focus on different lesion properties, all coexisting within the same cell, would provide a robust and partially redundant repair system necessary for the maintenance of the genome.

MRE11-Deficiency Associated with Improved Long-Term Disease Free Survival and Overall Survival in a Subset of Stage III Colon Cancer Patients in Randomized CALGB 89803 Trial

Purpose Colon cancers deficient in mismatch repair (MMR) may exhibit diminished expression of the DNA repair gene, MRE11, as a consequence of contraction of a T11 mononucleotide tract. This study investigated MRE11 status and its association with prognosis, survival and drug response in patients with stage III colon cancer. Patients and Methods Cancer and Leukemia Group B 89803 (Alliance) randomly assigned 1,264 patients with stage III colon cancer to postoperative weekly adjuvant bolus 5-fluorouracil/leucovorin (FU/LV) or irinotecan+FU/LV (IFL), with 8 year follow-up. Tumors from these patients were analyzed to determine stability of a T11 tract in the MRE11 gene. The primary endpoint was overall survival (OS), and a secondary endpoint was disease-free survival (DFS). Non-proportional hazards were addressed using time-dependent covariates in Cox analyses. Results Of 625 tumor cases examined, 70 (11.2%) exhibited contraction at the T11 tract in one or both MRE11 alleles and were thus predicted to be deficient in MRE11 (dMRE11). In pooled treatment analyses, dMRE11 patients showed initially reduced DFS and OS but improved long-term DFS and OS compared with patients with an intact MRE11 T11 tract. In the subgroup of dMRE11 patients treated with IFL, an unexplained early increase in mortality but better long-term DFS than IFL-treated pMRE11 patients was observed. Conclusions Analysis of this relatively small number of patients and events showed that the dMRE11 marker predicts better prognosis independent of treatment in the long-term. In subgroup analyses, dMRE11 patients treated with irinotecan exhibited unexplained short-term mortality. MRE11 status is readily assayed and may therefore prove to be a useful prognostic marker, provided that the results reported here for a relatively small number of patients can be generalized in independent analyses of larger numbers of samples. Trial Registration ClinicalTrials.gov NCT00003835

Laparoscopic Transperitoneal Repair of Pediatric Diaphragm Eventration Using an Endostapler Device

BACKGROUND Minimally invasive repairs of pediatric diaphragm eventration have been well described via a thoracoscopic approach, oftentimes requiring single-lung ventilation and tube thoracostomy, with the disadvantage of not being able to clearly visualize what lies beneath the diaphragm. We describe a novel pediatric diaphragm eventration repair using a laparoscopic transperitoneal approach and an endostapler device. We also describe our initial experience with this technique. PATIENTS AND METHODS Four pediatric diaphragmatic eventration patients underwent laparoscopic transperitoneal repair using an endostapler device. Repairs were performed in both male and female patients with right-sided eventrations. We approach the repair in a transperitoneal fashion using inverting sutures at the apex of the diaphragm to create tension toward the pelvis. Subsequently, an endostapler is used to remove the redundant portion of diaphragm, leaving a repaired, taut diaphragm. RESULTS The median age at operation was 10.5 months. The median operative time was 70 minutes. There was no mortality, surgical complications, or recurrence at a median follow-up of 17 months. CONCLUSIONS This laparoscopic approach allows for clear visualization of the intraabdominal organs and, at least in our early experience, a very simple, straightforward operation. Additionally, with the use of the endostapler, the redundant, often weakened diaphragm is removed, leaving the native, healthy diaphragm behind, resulting in a reliable and reproducible repair. This repair should be considered as a feasible alternative approach to the more traditional open and thoracoscopic repairs.

Postoperative Bowel Perforation due to Heterotopic Ossification (Myositis Ossificans Traumatica): A Case Report and Review of the Literature

Heterotopic ossification (HO) is the ectopic development of normal bone within soft tissue that can occur after traumatic injury. It is uncommon and may be missed or misdiagnosed, which can lead to complications. We report the case of an 84-year-old male with a previous history of a laparotomy who underwent resection of an intra-abdominal tumor through a midline incision. On postoperative day six, the patient was taken to the operating room, as succus was draining from the incision. Upon re-exploration, sharp bone-like material was found in the wound directly adjacent to an enterotomy. Pathology confirmed mature lamellar bone and the diagnosis of HO. This is the first report of postoperative intestinal perforation secondary to HO in a midline wound. We report this case to encourage accurate reporting of HO and its morbidity and complications for the benefit of appropriate surgical planning and epidemiologic tracking of outcomes.

Chemical Carcinogenesis and Epigenetics

Gene expression in higher eukaryotes is controlled in part by a complex series of chemical modifications to DNA and associated histone proteins that alter the condensation of chromatin and accessibility of genes for transcription. The transcriptional silencing of tumor suppressor genes or the inappropriate activation of transforming genes is a hallmark of human tumors and, in many cases, can be attributed to the perturbation of epigenetic signals. In this review, we provide a brief introduction to epigenetic gene control with a focus on molecular events and, in particular, the role of 5-methylcytosine (5mC) in DNA on epigenetic programming. The mechanisms by which enzymatic methylation alters DNA–protein interactions and methylase activity are described. The conversion of cytosine to 5mC changes the chemistry of the base and, in some cases, the surrounding DNA as well. We describe how carcinogens can modify epigenetic patterns through chemical interactions with cytosine and 5mC. Emerging evidence also suggests that chemical damage to DNA can alter interactions with methyltransferases and proteins containing a methyl-binding domain, resulting in heritable changes in epigenetic signals.

WRN Promoter CpG Island Hypermethylation Does Not Predict More Favorable Outcomes for Patients with Metastatic Colorectal Cancer Treated with Irinotecan-Based Therapy.

Purpose: WRN promoter CpG island hypermethylation in colorectal cancer has been reported to increase sensitivity to irinotecan-based therapies. We aimed to characterize methylation of the WRN promoter, determine the effect of WRN promoter hypermethylation upon expression, and validate a previous report that WRN promoter hypermethylation predicts improved outcomes for patients with metastatic colorectal cancer (mCRC) treated with irinotecan-based therapy. Experimental Design: WRN methylation status was assessed using methylation-specific PCR and bisulfite sequencing assays. WRN expression was determined using qRT-PCR and Western blotting. WRN methylation status was correlated with overall survival (OS) and progression-free survival (PFS) in 183 patients with mCRC. Among these patients, 90 received capecitabine monotherapy as first-line therapy, and 93 received capecitabine plus irinotecan (CAPIRI) therapy as part of the CAIRO phase III clinical trial. Results: WRN mRNA and WRN protein expression levels were low in colorectal cancer cell lines and in primary colorectal cancer and were largely independent of WRN methylation status. Patients with methylated WRN colorectal cancer had a shorter OS compared with patients who had unmethylated WRN colorectal cancer (HR = 1.6; 95% confidence interval [CI], 1.2–2.2; P = 0.003). Patients with unmethylated WRN showed a significantly longer PFS when treated with CAPIRI compared with capecitabine alone (HR = 0.48; 95% CI, 0.32–0.70; P = 0.0001). In contrast, patients did not benefit from adding irinotecan to capecitabine when WRN was methylated (HR = 1.1; 95% CI, 0.69–1.77; P = 0.7). Conclusions: WRN expression is largely independent of WRN promoter hypermethylation in colorectal cancer. Moreover, we could not validate the previous finding that WRN promoter hypermethylation predicts improved clinical outcomes of mCRC treated with irinotecan-based therapy and found instead the opposite result. Clin Cancer Res; 22(18); 4612–22. ©2016 AACR.

Abstract 1157: Increased expression of RecQ helicases in sporadic primary colorectal cancers

The RecQ helicase family of enzymes regulate nucleic acid metabolism through the major activity of unwinding double stranded nucleic acids. Heritable loss of RECQ helicase expression results in human syndromes associated with an elevated risk of cancers including colorectal cancer (CRC). In vitro studies have shown that loss of function of WRN and/or BLM increases sensitivity to killing by DNA damaging chemotherapeutic agents. Thus, over-expression of these helicases may mediate tumor resistance to DNA damaging chemotherapeutic agents, whereas under-expression may identify a population of tumors that may be more susceptible. Currently, the literature with regards to the expression status of these enzymes in CRC is sparse. In this study, we assess the expression of all five members of the RECQ helicase family (WRN, BLM, RECQL, RECQL4 and RECQL5) in 32 sporadic primary colorectal cancer cases with matched normal colonic mucosa using quantitative real-time PCR. The results show a significant increase in the fold change of RECQ helicase expression in tumor as compared to the matched normal mucosa, using the Wilcoxon Signed Rank test with a theoretical median of 1. The results are as follows: WRN median=1.191, mean=1.347, SD=0.6189, p-value=0.0043; BLM median=4.146, mean=12.88, SD=24.86, p-value Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1157. doi:1538-7445.AM2012-1157