Acid-induced demineralisation of human enamel as a function of time and pH observed using X-ray and polarised light imaging.

Acid-induced enamel demineralisation affects many individuals either by exposure to acidic diets, acidic gas pollution (dental erosion) or to dental plaque acids (dental caries). This study aimed to develop in situ X-ray and light imaging methods to determine progression of enamel demineralisation and the dynamic relationship between acid pH and mineral density. Hourly digital microradiograph time-lapse sequences showed the depth of enamel demineralisation in 500 µm thick sections progressed with time from the surface towards the dentine following a power-law function, which was 21% faster than the lateral demineralisation progression after exposure for 85 h to lactic acid (10%, pH 2.2). The minimum greyscale remaining (mineral content) within the induced enamel lesion followed an exponential decay, while the accumulated total greyscale loss with time was linear, which showed a constant anisotropic mineral release within the enamel architecture. This 85 h demineralisation method studied by polarised light microscopy time-lapse sequences showed that once the demineralisation front reached the enamel Hunter-Schreger bands, there was preferential demineralisation along those bands. Mineral density loss was linear with increasing pH acidity between pH 5.2 and pH 4.0 (with 0.4 pH increments) when incubated over a 3-week period exposed to 0.5% lactic acid. At pH 4.0, there was complete mineral loss in the centre of the demineralised area after the 3-week period and the linear function intercepted the x-axis at ~ pH 5.5, near the critical pH for hydroxyapatite (HAp). These observations showed how intrinsic enamel structure and pH affected the progression of demineralisation. STATEMENT OF SIGNIFICANCE: Hydroxyapatite crystallites (HAp) in human enamel dissolve when exposed to an acidic environment but little is known about how the intrinsic structures in enamel and pH influence the demineralisation kinetics. We have developed a time-lapse in situ microradiography method to quantify microscopic anisotropic mineral loss dynamics in response to an acid-only caries model. Correlation with polarised light microscopy time-lapse sequences showed that larger structures in enamel also influence demineralisation progression as demineralisation occurred preferentially along the Hunter-Schreger bands (decussating prismatic enamel). The pH-controlled enamel mineral release in a linear manner quantifying the relationship between HAp orientation and acid solubility. These findings should direct the development of improved anti-demineralisation/ remineralisation treatments to retain/ restore the natural intrinsic enamel structure.

Whole Genome Methylation Analysis of Nondysplastic Barrett Esophagus that Progresses to Invasive Cancer.

OBJECTIVE: To investigate differences in methylation between patients with nondysplastic Barrett esophagus who progress to invasive adenocarcinoma and those who do not. BACKGROUND: Identifying patients with nondysplastic Barrett esophagus who progress to invasive adenocarcinoma remains a challenge. Previous studies have demonstrated the potential utility of epigenetic markers for identifying this group. METHODS: A whole genome methylation interrogation using the Illumina HumanMethylation 450 array of patients with nondysplastic Barrett esophagus who either develop adenocarcinoma or remain static, with validation of findings by bisulfite pyrosequencing. RESULTS: In all, 12 patients with "progressive" versus 12 with "nonprogressive" nondysplastic Barrett esophagus were analyzed via methylation array. Forty-four methylation markers were identified that may be able to discriminate between nondysplastic Barrett esophagus that either progress to adenocarcinoma or remain static. Hypomethylation of the recently identified tumor suppressor OR3A4 (probe cg09890332) validated in a separate cohort of samples (median methylation in progressors 67.8% vs 96.7% in nonprogressors; P = 0.0001, z = 3.85, Wilcoxon rank-sum test) and was associated with the progression to adenocarcinoma. There were no differences in copy number between the 2 groups, but a global trend towards hypomethylation in the progressor group was observed. CONCLUSION: Hypomethylation of OR3A4 has the ability to risk stratify the patient with nondysplastic Barrett esophagus and may form the basis of a future surveillance program.

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).

Multiplex PCR and Next Generation Sequencing for the Non-Invasive Detection of Bladder Cancer.

BACKGROUND: Highly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA. METHODS: DNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free). RESULTS: Despite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity). CONCLUSION: This simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer.