Validation of epigenetic markers to identify colitis associated cancer: Results of module 1 of the ENDCAP-C study.

BACKGROUND: Chronic inflammation caused by ulcerative colitis (UC) causes a pro-neoplastic drive in the inflamed colon, leading to a markedly greater risk of invasive malignancy compared to the general population. Despite surveillance protocols, 50% of cases proceed to cancer before neoplasia is detected. The Enhanced Neoplasia Detection and Cancer Prevention in Chronic Colitis (ENDCaP-C) trial is an observational multi-centre test accuracy study to ascertain the role of molecular markers in improving the detection of dysplasia. We aimed to validate previously identified biomarkers of neoplasia in a retrospective cohort and create predictive models for later validation in a prospective cohort. METHODS: A retrospective analysis using bisulphite pyrosequencing of an 11 marker panel (SFRP1, SFRP2, SRP4, SRP5, WIF1, TUBB6, SOX7, APC1A, APC2, MINT1, RUNX3) in samples from 35 patients with cancer, 78 with dysplasia and 343 without neoplasia undergoing surveillance for UC associated neoplasia across 6 medical centres. Predictive models for UC associated cancer/dysplasia were created in the setting of neoplastic and non-neoplastic mucosa. FINDINGS: For neoplastic mucosa a five marker panel (SFRP2, SFRP4, WIF1, APC1A, APC2) was accurate in detecting pre-cancerous and invasive neoplasia (AUC = 0.83; 95% CI: 0.79, 0.88), and dysplasia (AUC = 0.88; (0.84, 0.91). For non-neoplastic mucosa a four marker panel (APC1A, SFRP4, SFRP5, SOX7) had modest accuracy (AUC = 0.68; 95% CI: 0.62,0.73) in predicting associated bowel neoplasia through the methylation signature of distant non-neoplastic colonic mucosa. INTERPRETATION: This multiplex methylation marker panel is accurate in the detection of ulcerative colitis associated dysplasia and neoplasia and is currently being validated in a prospective clinical trial. FUNDING: The ENDCAP-C study was funded by the National Institute for Health Research Efficacy and Mechanism Evaluation (EME) Programme (11/100/29).

Cardiac lipoprotein lipase activity in the hypertrophied heart may be regulated by fatty acid flux.

Cardiac hypertrophy is characterised by an imbalance between lipid uptake and fatty acid ß-oxidation leading to an accumulation of lipids, particularly triacylglycerol (TAG). It is unclear whether uptake mechanisms such as lipoprotein lipase (LPL) can be attenuated to diminish this uptake. Rats were cold acclimated to induce cardiac hypertrophy and increase cardiac LPL. Lipid uptake and metabolism were altered by feeding a 'Western-style' high fat diet (WSD) or feeding oxfenicine (2g/L) in the drinking water. Diastolic stiffness (increased volume change/unit pressure change) was induced in hypertrophied hearts for rats fed WSD (P<0.05) or WSD+oxfenicine (P<0.01), although absolute performance of cardiac muscle, estimated from stress-strain calculations was unchanged. Cold acclimation increased cardiac endothelial LPL (P<0.05) but this was diminished following oxfenicine. Following WSD LPL was further decreased below WSD-fed control hearts (P<0.05) with no further decrease by oxfenicine supplementation. A negative correlation was noted between plasma TAG and endothelial LPL (correlation coefficient=-0.654; P<0.001) but not cardiac TAG concentration. Transcript levels of angiopoietin-like protein-4 (ANGPTL4) were increased 6-fold by WSD (P<0.05) and increased 15-fold following WSD+oxfenicine (P<0.001). For CA-hearts fed WSD or WSD+oxfenicine ANGPTL4 mRNA levels were preserved at chow-fed levels. VLDLR protein levels were increased 10-fold (P<0.01) by CA. ANGPTL4 protein levels were increased 2-fold (P<0.05) by WSD, but restored following oxfenicine. For CA-hearts WSD increased ANGPTL4 protein levels 3-fold (P<0.01) with WSD+oxfenicine increasing ANGPTL4 protein 4-fold (P<0.01). These data suggest that endothelial LPL levels in the heart are altered to maintain FA flux and may exploit ANGPTL4.

The Wnt antagonist sFRP1 in colorectal tumorigenesis.

Regions of the short arm of chromosome 8 are deleted frequently in a range of solid tumors, indicating that tumor suppressor genes reside at these loci. In this study, we have examined the properties of the Wnt signaling antagonist secreted frizzled-related protein (sFRP) 1 as a candidate for this role at c8p11.2. An initial survey of 10 colorectal tumors, selected by the presence of isolated short deletions of the 8p11.2 region, identified three chain-terminating mutations, all within the first exon, which encodes the cysteine-rich domain. None of these tumors exhibited microsatellite instability, indicating intact mismatch repair gene function. The preserved sFRP1 alleles in the remaining seven tumors each contained a polymorphic three-base insertion in the signal sequence, but in a broader study, no association was found between this and the development of colorectal cancer. Epigenetic inhibition of sFRP1 transcription was investigated, and increased methylation of the promotor region was demonstrated in an additional cohort of 51 locally advanced colorectal cancers. Hypermethylation was identified in 40 of 49 (82%) cancers and in only 11 of 36 (30%) matched normal mucosal samples (P < 0.001). Semiquantitative analysis, by real-time PCR, of mRNA expression in 37 of the same cohort of 51 cancers revealed that sFRP1 mRNA expression was down-regulated in 28 (76%) cases compared with matched normal large bowel mucosa. The 3' end of the sFRP1 mRNA also was found to be alternatively spliced, compared with the prototype liver and lung forms, in the colon and a number of other tissues, yielding an extended COOH terminus, which may influence its activity in a tissue-specific manner. The inactivation and down-regulation of sFRP1 observed are consistent with it acting as a tumor suppressor gene in colorectal carcinogenesis. Because beta-catenin is constitutively active in the majority of colorectal tumors, it is unlikely that sFRP1 can act in the canonical Wnt response pathway. Therefore, we propose that the reduced activity or absence of sFRP1 allows the transduction of noncanonical Wnt signals, which contribute to tumor progression.