Serum proteomic profiling at diagnosis predicts clinical course, and need for intensification of treatment in inflammatory bowel disease.

BACKGROUND: Success in personalized medicine in complex disease is critically dependent on biomarker discovery. We profiled serum proteins using a novel proximity extension assay [PEA] to identify diagnostic and prognostic biomarkers in inflammatory bowel disease [IBD]. METHODS: We conducted a prospective case-control study in an inception cohort of 552 patients [328 IBD, 224 non-IBD], profiling proteins recruited across six centres. Treatment escalation was characterized by the need for biological agents or surgery after initial disease remission. Nested leave-one-out cross-validation was used to examine the performance of diagnostic and prognostic proteins. RESULTS: A total of 66 serum proteins differentiated IBD from symptomatic non-IBD controls, including matrix metallopeptidase-12 [MMP-12; Holm-adjusted p = 4.1 × 10-23] and oncostatin-M [OSM; p = 3.7 × 10-16]. Nine of these proteins are associated with cis-germline variation [59 independent single nucleotide polymorphisms]. Fifteen proteins, all members of tumour necrosis factor-independent pathways including interleukin-1 (IL-1) and OSM, predicted escalation, over a median follow-up of 518 [interquartile range 224-756] days. Nested cross-validation of the entire data set allowed characterization of five-protein models [96% comprising five core proteins ITGAV, EpCAM, IL18, SLAMF7 and IL8], which define a high-risk subgroup in IBD [hazard ratio 3.90, confidence interval: 2.43-6.26], or allowed distinct two- and three-protein models for ulcerative colitis and Crohn's disease respectively. CONCLUSION: We have characterized a simple oligo-protein panel that has the potential to identify IBD from symptomatic controls and to predict future disease course. Further prospective work is required to validate our findings.

Whole Blood Profiling of T-cell-Derived microRNA Allows the Development of Prognostic models in Inflammatory Bowel Disease.

BACKGROUND: MicroRNAs [miRNAs] are cell-specific small non-coding RNAs that can regulate gene expression and have been implicated in inflammatory bowel disease [IBD] pathogenesis. Here we define the cell-specific miRNA profiles and investigate its biomarker potential in IBD. METHODS: In a two-stage prospective multi-centre case control study, next generation sequencing was performed on a discovery cohort of immunomagnetically separated leukocytes from 32 patients (nine Crohn's disease [CD], 14 ulcerative colitis [UC], eight healthy controls) and differentially expressed signals were validated in whole blood in 294 patients [97 UC, 98 CD, 98 non-IBD, 1 IBDU] using quantitative PCR. Correlations were analysed with phenotype, including need for early treatment escalation as a marker of progressive disease using Cox proportional hazards. RESULTS: In stage 1, each leukocyte subset [CD4+ and CD8+ T-cells and CD14+ monocytes] was analysed in IBD and controls. Three specific miRNAs differentiated IBD from controls in CD4+ T-cells, including miR-1307-3p [p = 0.01], miR-3615 [p = 0.02] and miR-4792 [p = 0.01]. In the extension cohort, in stage 2, miR-1307-3p was able to predict disease progression in IBD (hazard ratio [HR] 1.98, interquartile range [IQR]: 1.20-3.27; logrank p = 1.80 × 10-3), in particular CD [HR 2.81; IQR: 1.11-3.53, p = 6.50 × 10-4]. Using blood-based multimarker miRNA models, the estimated chance of escalation in CD was 83% if two or more criteria were met and 90% for UC if three or more criteria are met. INTERPRETATION: We have identified and validated unique CD4+ T-cell miRNAs that are differentially regulated in IBD. These miRNAs may be able to predict treatment escalation and have the potential for clinical translation; further prospective evaluation is now indicated.

Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease.

Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8+ T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression.

A cell line, ROSE 199, derived from normal rat ovarian surface epithelium.

A cell line, ROSE 199, derived from rat ovarian surface epithelium (ROSE) formed papillary structures which resembled, histologically, serous papillary cystadenomas of borderline malignancy seen in the human ovary. Crowded cultures produced two layers of cells separated by a thick layer of collagen fibers. Such cultures shed viable cells into the growth medium, while no cells were shed by short-term ROSE cultures. The resemblance to ovarian tumors exhibited by ROSE 199 cells in culture, reinforces the hypothesis that the common epithelial tumors of the ovary are derived from the ovarian surface epithelium. ROSE 199 cells, while retaining their epithelial morphology and ultrastructural characteristics, express stromal activity such as abundant collagen production. Perhaps this ability to express epithelial and stromal behavior is a contributing factor to the ready neoplastic transformation of the ovarian surface epithelium.

Quantitative analysis of heterogeneity of estrogen binding in human breast tumor specimens.

A quantitative assessment was made of the heterogeneity of estrogen binding among histologically distinct regions and among nuclei within histologically uniform regions in ten human breast tumors. An autoradiographic method was used that employed sections from fixed tissues and required exposure times of one to two weeks. Grain counting of autoradiograms probably indicated both type I and type II estrogen binding. There were significant decreases in estrogen binding in all specimens after competition with excess diethylstilbestrol, suggesting that a component of binding was specific for estrogen. Significant differences in amount and distribution (nuclear vs cytoplasmic) of estrogen binding were seen not only among tissues with different histologies but also within histologically identical regions of a tissue. In histologically uniform regions, wide ranges of values for nuclear estrogen binding were seen. This method allows for the rapid evaluation of estrogen-binding profiles of tumor specimens and has potential for analyzing the process of neoplastic transformation in mammary epithelia as it affects, or is affected by estrogen binding.

Autoradiographic investigation of estrogen binding in cultured rat ovarian surface epithelial cells.

Autoradiographic evidence presented that the cultured rat ovarian surface epithelium exhibits estrogen receptor-like activity. Two autoradiographic techniques were used; one involved live cells that were labeled and freeze-dried, and the other the labeling of ethanol-fixed cells. Autoradiograms were prepared by dipping cells grown on plastic cover slips in liquid nuclear track emulsion. Exposure times were 1 to 4 weeks. Experiments using a pulse-chase technique in live cells and steroid competition tests in fixed and live cells gave evidence for translocation of tritiated estradiol from cytoplasm to nucleus in live cells and for a component of estrogen-specific binding in live and fixed cells. The techniques presented here for the investigation of estrogen receptors in cultured cells require little tissue, are simple, and relatively quick. Reports based on biochemical analyses of tissue homogenates claim the presence of estrogen receptors in human ovarian carcinomas of surface epithelial origin and in rat ovarian surface epithelium. The results of this study add further evidence that the ovarian surface epithelium has estrogen receptor activity and should be considered an estrogen target tissue.

Transformation of cultured rat ovarian surface epithelial cells by Kirsten murine sarcoma virus.

A method is described for the culture of rat ovarian surface epithelial cells, i.e., the cellular component thought to be the source of most ovarian cancers. These cells in culture have a characteristic epithelial morphology which distinguishes them from other ovarian cell types. Cultured surface epithelial cells are histochemically positive for 17 beta-hydroxysteroid dehydrogenase and negative for delta 5-3 beta-hydroxysteroid dehydrogenase, the same as in cryostat sections of whole rat ovary. Ultrastructurally, cultured surface epithelial cells have basement membranes, microvilli, and apical intercellular junctions. Kirsten murine sarcoma virus was used to produce three transformed cell lines from pure first-passage cultures of these cells. These three lines retained 17 beta-hydroxysteroid dehydrogenase activity and showed slight delta-3 beta-hydroxysteroid dehydrogenase activity. Tumors resulting when these cells were injected s.c. or i.p. into immunosuppressed female rats were highly malignant, resembling histologically human ovarian endometrioid stromal sarcoma. This is the first demonstration of the susceptibility of ovarian surface epithelium to an oncogenic virus.