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Development and validation of a highly sensitive urine-based test to identify patients with colonic adenomatous polyps.

ABSTRACT: OBJECTIVES: Adenomatous polyps are precursors of colorectal cancer; their detection and removal is the goal of colon cancer screening programs. However, fecal-based methods identify patients with adenomatous polyps with low levels of sensitivity. The aim or this study was to develop a highly accurate, prototypic, proof-of-concept, spot urine-based diagnostic test using metabolomic technology to distinguish persons with adenomatous polyps from those without polyps. METHODS: Prospective urine and stool samples were collected from 876 participants undergoing colonoscopy examination in a colon cancer screening program, from April 2008 to October 2009 at the University of Alberta. Colonoscopy reference standard identified 633 participants with no colonic polyps and 243 with colonic adenomatous polyps. One-dimensional nuclear magnetic resonance spectra of urine metabolites were analyzed to define a diagnostic metabolomic profile for colonic adenomas. A urine metabolomic diagnostic test for colonic adenomatous polyps was established using 67% of the samples (un-blinded training set) and validated using the other 33% of the samples (blinded testing set). The urine metabolomic diagnostic test's specificity and sensitivity were compared with those of fecal-based tests. RESULTS: Using a two-component, orthogonal, partial least-squares model of the metabolomic profile, the un-blinded training set identified patients with colonic adenomatous polyps with 88.9% sensitivity and 50.2% specificity. Validation using the blinded testing set confirmed sensitivity and specificity values of 82.7% and 51.2%, respectively. Sensitivities of fecal-based tests to identify colonic adenomas ranged from 2.5 to 11.9%. CONCLUSIONS: We describe a proof-of-concept spot urine-based metabolomic diagnostic test that identifies patients with colonic adenomatous polyps with a greater level of sensitivity (83%) than fecal-based tests.

SUBMITTER: Wang H

PROVIDER: S-EPMC3940838 | biostudies-other | 2014

REPOSITORIES: biostudies-other

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Project description:BackgroundColorectal cancer is the second leading cause of cancer-related deaths. This study demonstrates the utility of a simple blood test with high sensitivity and specificity for colorectal adenomatous polyps and cancer. A simple blood test with high sensitivity and specificity for adenomas would help identify individuals for a follow-up colonoscopy during which any adenomatous polyps found could be removed, thus preventing colorectal cancer (CRC).MethodsWe determined the H-score by using immunohistochemical analyses of N-myristoyltransferase 2 (NMT2) in peripheral blood mononuclear cells (PBMC) isolated from the blood. We determined the sensitivity and specificity of the NMT2-based blood test in identifying colorectal adenomatous polyps and cancer.DesignAll experimental procedures were performed by research personnel blinded to the colonoscopy status of the participants.SettingIn this cohort study, participants were recruited from those coming for an outpatient colonoscopy at a referral center.ParticipantsPBMC were collected from 74 subjects at the Health Sciences Centre, Winnipeg, Canada. Samples were collected from colonoscopy patients prior to colonoscopy. All 74 subjects were included in CRC vs. non-CRC analysis, whereas only 70 subjects were analyzed for colorectal adenomatous polyps and cancer versus individuals with no evidence of disease and non-adenomatous polyps. NMT2 expression was tested in samples by immunohistochemistry.ResultsThe expression of NMT2 was significantly higher in PBMC of subjects with colorectal adenomatous polyps and cancer (n = 34) compared with individuals with non-adenomatous polyps or no evidence of disease (n = 36) (P < 0.0001). The test had an overall sensitivity of 91% (95% confidence intervals: 84.49-97.80) and specificity of 81% (95% confidence intervals: 71.28-89.83) in detecting colorectal adenomatous polyps and cancer (all stages).ConclusionsOur results suggest that the sensitivity of NMT2 in detecting adenomatous polyps is high (91%). A simple blood-based CRC screening test using NMT2 expression detects colorectal adenomatous polyps and cancer with high sensitivity and specificity has the potential of increasing the compliance for CRC screening as has been reported for other blood-based CRC screening tests.

Project description:BackgroundEnergy metabolism in carcinogenesis is poorly understood. It is widely accepted the majority of colorectal cancers (CRCs) arise from adenomatous polyps (APs). We aimed to characterize the bioenergetic alterations in APs and CRCs.MethodsFifty-six APs, 93 CRCs and adjacent normal mucosae were tested. Oxygen consumption rate (OCR) was measured representing mitochondrial oxidative phosphorylation (OxPhos), and extracellular acidification rate (ECAR)was measured representing glycolysis. Mitochondrial DNA (mtDNA) variants and mutations were studied. Over-expressed metabolic genes in APs were identified by microarray and validated by qRT-PCR, Western blots and immunohistochemistry. Identified genes were knocked down in WiDr and colo205 CRC cell lines, and their expression was analyzed in APs/CRCs with enhanced glycolysis.FindingsECAR, not OCR, was significantly increased in APs. While no difference of ECAR was found between CRCs and normal mucosae, OCR was significantly reduced in CRCs. OCR/ECAR ratio was decreased in APs over 1 cm, APs with a villous component and CRCs, indicating their glycolytic tendencies. The number of mtDNA mutations was increased in APs and CRCs, but not correlated with metabolic profiles. Two metabolic genes ALDOB and SLC16A4 were up-regulated in APs. Both ALDOB-knockdown and SLC16A4-knockdown CRC cell lines showed increased basal motichondrial OxPhos and decreased basal glycolysis. Moreover, the increase of mitochondrial ATP-linked respiration and the decrease of glycolytic capacity were showed in SLC16A4-knockdown cells. Finally, APs/CRCs with enhanced glycolysis had increased SLC16A4 expression.InterpretationATP production shifts from OxPhos to glycolysis in the process of AP enlargement and villous transformation. OxPhos defects are present in CRCs but not in APs. APs and CRCs tend to accumulate mtDNA mutations, but these are not correlated with bioenergetic profiles. Finally, the ALDOB and SLC16A4 may contribute to the glycolytic shift in APs/CRCs.

Project description:Colorectal tumors mostly arise from sporadic adenomatous polyps. Polyps are defined as a mass of cells that protrudes into the lumen of the colon. Adenomatous polyps are benign neoplasms that, by definition display some characteristics of dysplasia. It has been shown that polyps were benign tumors which may undergo malignant transformation. Adenomatous polyps have been classified into three histologic types; tubular, tubulovillous, and villous with increasing malignant potential. The ability to differentially diagnose these colorectal adenomatous polyps is important for therapeutic intervention. To date, little efforts have been directed to identifying genetic changes involved in adenomatous polyps. This study was designed to examine the relevance of mitochondrial genome alterations in the three adenomatous polyps. Using high resolution restriction endonucleases and PCR-based sequencing, fifty-seven primary fresh frozen tissues of adenomatous polyps (37 tumors and 20 matched surrounding normal tissues) obtained from the southern regional Cooperative Human Tissue Network (CHTN) and Grady Memorial Hospital at Atlanta were screened with three mtDNA regional primer pairs that spanned 5.9 kbp. Results from our data analyses revealed the presence of forty-four variants in some of these mitochondrial genes that the primers spanned; COX I, II, III, ATP 6, 8, CYT b, ND 5, 6 and tRNAs. Based on the MITODAT database as a sequence reference, 25 of the 44 (57%) variants observed were unreported. Notably, a heteroplasmic variant C8515G/T in the MT-ATP 8 gene and a germline variant 8327delA in the tRNAlys was observed in all the tissue samples of the three adenomatous polyps in comparison to the referenced database sequence. A germline variant G9055A in the MT-ATP 6 gene had a frequency of 100% (17/17) in tubular and 57% (13/23) in villous adenomas; no corresponding variant was in tubulovillous adenomas. Furthermore, A9006G variant at MT-ATP 6 gene was observed at frequency of 57% (13/23) in villous adenomas only. Interestingly, variants A9006G and G9055A were absent in the villous tissue samples that were clinicopathological designated as "polyvillous adenomas". Our current data provide a basis for continued investigation of certain mtDNA variants as predictors of the three adenomatous polyps in a larger number of clinicopathological specimens.

Project description:BackgroundOral monosaccharides and disaccharides are used to measure in vivo human gut permeability through urinary excretion.AimsThe aims were as follows: (1) to obtain normative data on small intestinal and colonic permeability; (2) to assess variance on standard 16 g fiber diet performed twice; (3) to determine whether dietary fiber influences gut permeability measurements; and (4) to present pilot data using 2 selected probes in patients with diarrhea-predominant irritable bowel syndrome (IBS-D).MethodsSixty healthy female and male adults, age 18-70 years, participated in 3 randomized studies (2 studies on 16.25 g and 1 study on 32.5 g fiber) in otherwise standardized diets. At each test, the following sugars were ingested: 12C-mannitol, 13C-mannitol, rhamnose (monosaccharides), sucralose, and lactulose (disaccharides). Standardized meals were administered from 24 hours before and during 24 hours post-sugars with 3 urine collections: 0-2, 2-8, and 8-24 hours. Sugars were measured using high-performance liquid chromatography-tandem mass spectrometry. Eighteen patients with IBS-D underwent 24-hour excretion studies after oral 13C-mannitol and lactulose.ResultsBaseline sugars (>3-fold above lower limits of quantitation) were identified in the 3 studies: 12C-mannitol in all participants; sucralose in 4-8, and rhamnose in 1-3. Median excretions/24 h (percentage of administered dose) for 13C-mannitol, rhamnose, lactulose, and sucralose were ∼30%, ∼15%, 0.32%, and 2.3%, respectively. 13C-mannitol and rhamnose reflected mainly small intestinal permeability. Intraindividual saccharide excretions were consistent, with minor differences with 16.25 g vs 32.5 g fiber diets. Median interindividual coefficient of variation was 76.5% (10-90 percentile: 34.6-111.0). There were no significant effects of sex, age, or body mass index on permeability measurements in health. 13C-mannitol measurements are feasible in IBS-D.ConclusionsBaseline 12C-mannitol excretion precludes its use; 13C-mannitol is the preferred probe for small intestinal permeability.

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Development and validation of a highly sensitive urine-based test to identify patients with colonic adenomatous polyps.

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