Changes in Serum, Red Blood Cell, and Colonic Fatty Acids in a Personalized Omega-3 Fatty Acid Supplementation Trial.

This study evaluated changes in fatty acids from sera, red blood cells, and colonic biopsies from a phase Ib clinical trial of personalized ω-3 fatty acid dosing in 47 healthy volunteers. The trial aimed to reduce colonic prostaglandin E2 (PGE2), a pro-inflammatory product of arachidonic acid (AA) oxidation. The personalized doses ranged 2-10 grams/day (54% eicosapentaenoic acid, EPA, 24% other ω-3 fatty acids). In colon, increases in ω-3 highly unsaturated fatty acids (HUFA) and EPA:AA ratios each were correlated with decreases in PGE2. Changes in either colonic EPA:AA ratios or ω-3 HUFA were significantly correlated with changes in the same fatty acid measures in red blood cells or serum. The only blood-based measure significantly correlated with changes in colonic PGE2 was change in red blood cell ω-3 HUFA (ρ = -0.39), and the increase in red blood cell ω-3 HUFA was significantly greater in participants who had at least a median reduction in colonic PGE2 vs. those who did not. In summary, fatty acid changes in blood did reflect fatty acid changes in the colon, but additional factors will be needed for optimizing dosing models that seek to predict the anti-inflammatory effects of ω-3 fatty acids on the colon.

It's not 'just a tube of blood': principles of protocol development, sample collection, staffing and budget considerations for blood-based biomarkers in immunotherapy studies.

Immunotherapy for cancer is now a standard pillar in the armamentarium of treatments for many cancers. Immune checkpoint inhibitors, in particular, have resulted in significant therapeutic benefit and prolongation of survival in solid organ cancers, such as melanoma and lung cancer. However, the extent of benefit is not uniform. There are several groups studying predictors of benefit from these therapies. Recently, there has been a burgeoning interest in studying predictive biomarkers from the blood. These markers include circulating tumor DNA, circulating tumor cells, lymphocyte subpopulations, exosomes and metabolites to name a few. The logistics involved in such biomarker work are complex and rigorous with potential to impact a given study. Such pre-analytic components include development of a rigorous protocol, standard operating procedures for collection and storage of various blood components, ethics of patient consent, personnel involved as well as budget considerations. In this primer, we lay out representative aspects of each of the aforementioned components as a guide to blood-based biomarker research for immunotherapy studies in cancer.

An Adaptive Bayesian Design for Personalized Dosing in a Cancer Prevention Trial.

INTRODUCTION: In biomarker-driven clinical trials, translational strategies typically involve moving findings from animal experiments to human trials. Typically, the translation is static, using a fixed model derived from animal experiments for the duration of the trial. Bayesian designs, capable of incorporating information external to the experiment, provide a dynamic translational strategy. This article demonstrates an example of such a dynamic Bayesian strategy in a clinical trial. METHODS: This study explored the effect of a personalized dose of fish oil for reducing prostaglandin E2, an inflammatory marker linked to colorectal cancer. A Bayesian design was implemented for the dose-finding algorithm that adaptively updated a dose-response model derived from a previously completed animal study during the clinical trial. In the initial stages of the trial, the dose-response model parameters were estimated from the rodent data. The model was updated following a Bayesian algorithm after data on every 10‒15 subjects were obtained until the model stabilized. Subjects were enrolled in the study between 2013 and 2015, and the data analysis was carried out in 2016. RESULTS: The 3 dosing models were used for groups of 16, 15, and 15 subjects. The mean target dose significantly decreased from 6.63 g/day (Model 1) to 4.06 g/day (Model 3) (p=0.001). Compared with the static strategy of dosing with a single model, the dynamic modeling reduced the dose significantly by about 1.38 g/day on average. CONCLUSIONS: A Bayesian design was effective in adaptively revising the dosing algorithm, resulting in a lower pill burden. TRIAL REGISTRATION: This study is registered at www.clinicaltrials.gov NCT01860352.

How Does Obesity Drive Human Carcinogenesis? Challenges in Dissecting the Mechanisms of Adipose-Epithelial Signaling.

Obesity is the second leading environmental association with cancer risk; yet, the mechanisms by which obesity drives carcinogenesis are poorly understood. The paper published in this issue of Cancer Prevention Research by Holowatyj and colleagues explores the mechanisms of human visceral adipose-epithelial signaling using samples collected at surgery in patients with invasive colorectal cancer. They identify pathway intermediates potentially involved in the regulation of fibrosis, inflammation, glycosis, and epithelial-mesenchymal transition in neoplastic tissue. 'Omics-based profiling of perioperative human biosamples has potential for inherent biases (preoperative and intraoperative drug therapies, hydration, dynamics, inflammatory response to surgical intervention) and appropriate control samples are difficult to identify and collect. Solutions to this dilemma may include strategies to identify patients undergoing similar surgical procedures but who are without neoplasms, for example, patients with gynecologic problems, abdominal exploration for suspected appendicitis, symptoms or resection of gall stone disease or undergoing bariatric surgery. As the field continues to grow, studies incorporating robust statistical analyses, validation of findings in diverse cohorts, and public data sharing will be essential to identify biological pathways linking obesity and carcinogenesis to be further interrogated using focused, hypothesis-driven approaches.See related article by Holowatyj et al., p. 817.

Dietary polyunsaturated fatty acids modulate adipose secretome and is associated with changes in mammary epithelial stem cell self-renewal.

Chronic low-grade adipose inflammation, characterized by aberrant adipokine production and pro-inflammatory macrophage activation/polarization is associated with increased risk of breast cancer. Adipocyte fatty acid composition is influenced by dietary availability and may regulate adipokine secretion and adipose inflammation. After feeding F344 rats for 20 weeks with a Western diet or a fish oil-supplemented diet, we cultured primary rat adipose tissue in a three-dimensional explant culture and collected the conditioned medium. The rat adipose tissue secretome was assayed using the Proteome Profiler Cytokine XL Array, and adipose tissue macrophage polarization (M1/M2 ratio) was assessed using the iNOS/ARG1 ratio. We then assessed the adipokine's effects upon stem cell self-renewal using primary human mammospheres from normal breast mammoplasty tissue. Adipose from rats fed the fish oil diet had an ω-3:ω-6 fatty acid ratio of 0.28 compared to 0.04 in Western diet rats. The adipokine profile from the fish oil-fed rats was shifted toward adipokines associated with reduced inflammation compared to the rats fed the Western diet. The M1/M2 macrophage ratio decreased by 50% in adipose of fish oil-fed rats compared to that from rats fed the Western diet. Conditioned media from rats fed the high ω-6 Western diet increased stem cell self-renewal by 62%±9% (X¯%±SD) above baseline compared to only an 11%±11% increase with the fish oil rat adipose. Modulating the adipokine secretome with dietary interventions therefore may alter stromal-epithelial signaling that plays a role in controlling mammary stem cell self-renewal.

Increases in Colonic Bacterial Diversity after ω-3 Fatty Acid Supplementation Predict Decreased Colonic Prostaglandin E2 Concentrations in Healthy Adults.

BACKGROUND: The intestinal microbiome is an important determinant of inflammatory balance in the colon that may affect response to dietary agents. OBJECTIVE: This is a secondary analysis of a clinical trial, the Fish Oil Study, to determine whether interindividual differences in colonic bacteria are associated with variability in the reduction of colonic prostaglandin E2 (PGE2) concentrations after personalized supplementation with ω-3 (n-3) fatty acids. METHODS: Forty-seven healthy adults (17 men, 30 women, ages 26-75 y) provided biopsy samples of colonic mucosa and luminal stool brushings before and after personalized ω-3 fatty acid supplementation that was based on blood fatty acid responses. Samples were analyzed using 16S ribosomal RNA sequencing. The data analyses focused on changes in bacterial community diversity. Linear regression was used to evaluate factors that predict a reduction in colonic PGE2. RESULTS: At baseline, increased bacterial diversity, as measured by the Shannon and Inverse Simpson indexes in both biopsy and luminal brushing samples, was positively correlated with dietary fiber intakes and negatively correlated with fat intakes. Dietary supplementation with ω-3 fatty acids increased the Yue and Clayton community dis-similarity index between the microbiome in luminal brushings and colon biopsy samples post-supplementation (P = 0.015). In addition, there was a small group of individuals with relatively high Prevotella abundance who were resistant to the anti-inflammatory effects of ω-3 fatty acid supplementation. In linear regression analyses, increases in diversity of the bacteria in the luminal brushing samples, but not in the biopsy samples, were significant predictors of lower colonic PGE2 concentrations post-supplementation in models that included baseline PGE2, baseline body mass index, and changes in colonic eicosapentaenoic acid-to-arachidonic acid ratios. The changes in bacterial diversity contributed to 6-8% of the interindividual variance in change in colonic PGE2 (P = 0.001). CONCLUSIONS: Dietary supplementation with ω-3 fatty acids had little effect on intestinal bacteria in healthy humans; however, an increase in diversity in the luminal brushings significantly predicted reductions in colonic PGE2. This trial was registered at www.clinicaltrials.gov as NCT01860352.

Higher baseline expression of the PTGS2 gene and greater decreases in total colonic fatty acid content predict greater decreases in colonic prostaglandin-E2 concentrations after dietary supplementation with ω-3 fatty acids.

This study evaluated whether mRNA expression of major genes regulating formation of prostaglandin (PG)E2 in the colon and colonic fatty acid concentrations are associated with the reduction in colonic mucosal PGE2 after dietary supplementation with omega-3 (ω-3) fatty acids. Supplementation with ω-3 fatty acids was done for 12 weeks using personalized dosing that was expected to reduce colonic PGE2 by 50%. In stepwise linear regression models, the ω-3 fatty acid dose and baseline BMI explained 16.1% of the inter-individual variability in the fold change of colonic PGE2 post-supplementation. Increases in mRNA gene expression after supplementation were, however, modest and were not associated with changes in PGE2. When baseline expression of PTGS1, PTGS2 and HPGD genes was included in the linear regression model containing dose and BMI, only PTGS2, the gene coding for the inducible form cyclooxygenase, was a significant predictor. Higher relative expression of PTGS2 predicted greater decreases in colonic PGE2, accounting for an additional 13.6% of the inter-individual variance. In the final step of the regression model, greater decreases in total colonic fatty acid concentrations predicted greater decreases in colonic PGE2, contributing to an additional 18.7% of the variance. Overall, baseline BMI, baseline expression of PTGS2 and changes in colonic total fatty acids together accounted for 48% of the inter-individual variability in the change in colonic PGE2. This is consistent with biochemical data showing that fatty acids which are not substrates for cyclooxygenases can activate cyclooxygenase-2 allosterically. Further clinical trials are needed to elucidate the factors that regulate the fatty acid milieu of the human colon and how this interacts with key lipid metabolizing enzymes. Given the central role of PGE2 in colon carcinogenesis, these pathways may also impact on colon cancer prevention by other dietary and pharmacological approaches.

Performance of multitarget stool DNA testing in African American patients.

BACKGROUND: Multitarget stool DNA (mt-sDNA) is an approved method for colon cancer screening that is especially relevant for patients who cannot undergo colonoscopy. Although the test performance has been evaluated in a large clinical trial, it was limited to a predominantly white population. Given differences in the epidemiology and biology of colon cancer in African American individuals, the authors sought to compare the performance of mt-sDNA between racial groups. METHODS: The authors prospectively identified patients aged ≥40 years who were referred for colonoscopy at an academic medical center and 2 satellite facilities. Prior to the colonoscopy, the authors collected stool for mt-sDNA and fecal immunochemical testing (FIT). They compared the sensitivity, specificity, and receiver operating characteristic curve between African American and white patients for the detection of advanced lesions or any adenoma. RESULTS: A total of 760 patients were included, 34.9% of whom were African American. The prevalence of any adenoma (38.9% for African American patients and 33.9% for white patients) and that for advanced lesions (6.8% and 6.7%, respectively) were similar between groups. The overall sensitivities of mt-sDNA for the detection of advanced lesions and any adenoma were 43% and 19%, respectively, and the specificities were 91% and 93%, respectively. In general, mt-sDNA was more sensitive and less specific than FIT. When stratified by race, the sensitivity, specificity, and receiver operating characteristic curve area were similar between African American and white patients for both mt-sDNA and FIT. CONCLUSIONS: Test performance characteristics of mt-sDNA were comparable in African American and white patients. Given the lower uptake of colonoscopy in African American individuals, mt-sDNA may offer a promising screening alternative in this patient population.

Evaluation of Patients with an Apparent False Positive Stool DNA Test: The Role of Repeat Stool DNA Testing.

BACKGROUND: There is uncertainty as to the appropriate follow-up of patients who test positive on multimarker stool DNA (sDNA) testing and have a colonoscopy without neoplasia. AIMS: To determine the prevalence of missed colonic or occult upper gastrointestinal neoplasia in patients with an apparent false positive sDNA. METHODS: We prospectively identified 30 patients who tested positive with a commercially available sDNA followed by colonoscopy without neoplastic lesions. Patients were invited to undergo repeat sDNA at 11-29 months after the initial test followed by repeat colonoscopy and upper endoscopy. We determined the presence of neoplastic lesions on repeat evaluation stratified by results of repeat sDNA. RESULTS: Twelve patients were restudied. Seven patients had a negative second sDNA test and a normal second colonoscopy and upper endoscopy. In contrast, 5 of 12 subjects had a persistently positive second sDNA test, and 3 had positive findings, including a 3-cm sessile transverse colon adenoma with high-grade dysplasia, a 2-cm right colon sessile serrated adenoma with dysplasia, and a nonadvanced colon adenoma (p = 0.045). These corresponded to a positive predictive value of 0.60 (95% CI 0.17-1.00) and a negative predictive value of 1.00 (95% CI 1.00-1.00) for the second sDNA test. In addition, the medical records of all 30 subjects with apparent false positive testing were reviewed and no documented cases of malignant tumors were recorded. CONCLUSIONS: Repeat positive sDNA testing may identify a subset of patients with missed or occult colorectal neoplasia after negative colonoscopy for an initially positive sDNA. High-quality colonoscopy with careful attention to the right colon in patients with positive sDNA is critically important and may avoid false negative colonoscopy.

Identification, isolation and characterization of human LGR5-positive colon adenoma cells.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Protein and glycomic plasma markers for early detection of adenoma and colon cancer.

OBJECTIVE: To discover and confirm blood-based colon cancer early-detection markers. DESIGN: We created a high-density antibody microarray to detect differences in protein levels in plasma from individuals diagnosed with colon cancer <3 years after blood was drawn (ie, prediagnostic) and cancer-free, matched controls. Potential markers were tested on plasma samples from people diagnosed with adenoma or cancer, compared with controls. Components of an optimal 5-marker panel were tested via immunoblotting using a third sample set, Luminex assay in a large fourth sample set and immunohistochemistry (IHC) on tissue microarrays. RESULTS: In the prediagnostic samples, we found 78 significantly (t-test) increased proteins, 32 of which were confirmed in the diagnostic samples. From these 32, optimal 4-marker panels of BAG family molecular chaperone regulator 4 (BAG4), interleukin-6 receptor subunit beta (IL6ST), von Willebrand factor (VWF) and CD44 or epidermal growth factor receptor (EGFR) were established. Each panel member and the panels also showed increases in the diagnostic adenoma and cancer samples in independent third and fourth sample sets via immunoblot and Luminex, respectively. IHC results showed increased levels of BAG4, IL6ST and CD44 in adenoma and cancer tissues. Inclusion of EGFR and CD44 sialyl Lewis-A and Lewis-X content increased the panel performance. The protein/glycoprotein panel was statistically significantly higher in colon cancer samples, characterised by a range of area under the curves from 0.90 (95% CI 0.82 to 0.98) to 0.86 (95% CI 0.83 to 0.88), for the larger second and fourth sets, respectively. CONCLUSIONS: A panel including BAG4, IL6ST, VWF, EGFR and CD44 protein/glycomics performed well for detection of early stages of colon cancer and should be further examined in larger studies.

The Anti-inflammatory Effect of Personalized Omega-3 Fatty Acid Dosing for Reducing Prostaglandin E2 in the Colonic Mucosa Is Attenuated in Obesity.

This clinical trial developed a personalized dosing model for reducing prostaglandin E2 (PGE2) in colonic mucosa using ω-3 fatty acid supplementation. The model utilized serum eicosapentaenoic acid (EPA, ω-3):arachidonic acid (AA, ω-6) ratios as biomarkers of colonic mucosal PGE2 concentration. Normal human volunteers were given low and high ω-3 fatty acid test doses for 2 weeks. This established a slope and intercept of the line for dose versus serum EPA:AA ratio in each individual. The slope and intercept was utilized to calculate a personalized target dose that was given for 12 weeks. This target dose was calculated on the basis of a model, initially derived from lean rodents, showing a log-linear relationship between serum EPA:AA ratios and colonic mucosal PGE2 reduction. Bayesian methods allowed addition of human data to the rodent model as the trial progressed. The dosing model aimed to achieve a serum EPA:AA ratio that is associated with a 50% reduction in colonic PGE2 Mean colonic mucosal PGE2 concentrations were 6.55 ng/mg protein (SD, 5.78) before any supplementation and 3.59 ng/mg protein (SD, 3.29) after 12 weeks of target dosing. In secondary analyses, the decreases in PGE2 were significantly attenuated in overweight and obese participants. This occurred despite a higher target dose for the obese versus normal weight participants, as generated by the pharmacodynamic predictive model. Large decreases also were observed in 12-hydroxyicosatetraenoic acids, and PGE3 increased substantially. Future biomarker-driven dosing models for cancer prevention therefore should consider energy balance as well as overall eicosanoid homeostasis in normal tissue. Cancer Prev Res; 10(12); 729-37. ©2017 AACR.

Fatty acid and lipidomic data in normal and tumor colon tissues of rats fed diets with and without fish oil.

Data is provided to show the detailed fatty acid and lipidomic composition of normal and tumor rat colon tissues. Rats were fed either a Western fat diet or a fish oil diet, and half the rats from each diet group were treated with chemical carcinogens that induce colon cancer (azoxymethane and dextran sodium sulfate). The data show total fatty acid profiles of sera and of all the colon tissues, namely normal tissue from control rats and both normal and tumor tissues from carcinogen-treated rats, as obtained by gas chromatography with mass spectral detection. Data from lipidomic analyses of a representative subset of the colon tissue samples is also shown in heat maps generated from hierarchical cluster analysis. These data display the utility lipidomic analyses to enhance the interpretation of dietary feeding studies aimed at cancer prevention and support the findings published in the companion paper (Effects of fish oil supplementation on prostaglandins in normal and tumor colon tissue: modulation by the lipogenic phenotype of colon tumors, Djuric et al., 2017 [1]).

Effects of fish oil supplementation on prostaglandins in normal and tumor colon tissue: modulation by the lipogenic phenotype of colon tumors.

Dietary fish oils have potential for prevention of colon cancer, and yet the mechanisms of action in normal and tumor colon tissues are not well defined. Here we evaluated the impact of the colonic fatty acid milieu on the formation of prostaglandins and other eicosanoids. Distal tumors in rats were chemically induced to model inflammatory colonic carcinogenesis. After 21 weeks of feeding with either a fish oil diet containing an eicosapentaenoic acid/ω-6 fatty acid ratio of 0.4 or a Western fat diet, the relationships between colon fatty acids and prostaglandin E2 (PGE2) concentrations were evaluated. PGE2 is a key proinflammatory mediator in the colon tightly linked with the initiation and progression of colon cancer. The fish oil vs. the Western fat diet resulted in reduced total fatty acid concentrations in serum but not in colon. In the colon, the effects of the fish oil on fatty acids differed in normal and tumor tissue. There were distinct lipodomic patterns consistent with a lipogenic phenotype in tumors. In tumor tissue, the eicosapentaenoic acid/arachidonic acid ratio, cyclooxygenase-2 expression and the mole percent of saturated fatty acids were significant predictors of inter-animal variability in colon PGE2 after accounting for diet. In normal tissues from either control rats or carcinogen-treated rats, only diet was a significant predictor of colon PGE2. These results show that the fatty acid milieu can modulate the efficacy of dietary fish oils for colon cancer prevention, and this could extend to other preventive agents that function by reducing inflammatory stress.

A Multicenter Study of a Fluorescence In Situ Hybridization Probe Set for Diagnosing High-Grade Dysplasia and Adenocarcinoma in Barrett's Esophagus.

BACKGROUND AND AIMS: Preliminary single-institution data suggest that fluorescence in situ hybridization (FISH) may be useful for detecting high-grade dysplasia (HGD) and esophageal adenocarcinoma (EA) in patients with Barrett's esophagus (BE). This multicenter study aims to validate the measurement of polysomy (gain of at least two loci) by FISH as a way to discriminate degrees of dysplasia in BE specimens. METHODS: Tissue specimens were collected from four different hospitals and read by both the local pathology department ("Site diagnosis") and a single central pathologist ("Review diagnosis") at a separate institution. The specimens then underwent FISH analysis using probes 8q24 (MYC), 9p21 (CDKN2A), 17q12 (ERBB2), and 20q13 (ZNF217) for comparison. A total of 46 non-BE, 42 non-dysplastic specialized intestinal metaplasia (SIM), 23 indefinite-grade dysplasia (IGD), 10 low-grade dysplasia (LGD), 29 HGD, and 42 EA specimens were analyzed. RESULTS: We found that polysomy, as detected by FISH, was the predominant chromosomal abnormality present as dysplasia increased. Polysomy was also the best predictor for the presence of dysplasia or EA when comparing its area under the curve to that of other FISH abnormalities. We observed that if at least 10% of cells had polysomy within a specimen, the FISH probe was able to differentiate between EA/HGD and the remaining pathologies with a sensitivity of 80% and a specificity of 88%. CONCLUSIONS: This study demonstrates that using FISH to determine the percentage of cells with polysomy can accurately and objectively aid in the diagnosis of HGD/EA in BE specimens.

Bringing Curcumin to the Clinic in Cancer Prevention: a Review of Strategies to Enhance Bioavailability and Efficacy.

Curcumin is widely available, inexpensive spice that has been used in ancient folk medicine for millennia, especially in India. Curcumin has the pharmacological properties that slow or reverse cellular proliferation and enhance apoptosis and differentiation associated with a diverse array of molecular effects. Despite its effective anticarcinogenesis properties, curcumin's poor solubility, instability, and extensive metabolism result in poor oral bioavailability. Strategies to enhance curcumin delivery include encapsulating or incorporating curcumin in a nanoparticle or microparticle drug delivery system, synthesizing more stable curcumin analogs that resist metabolism while retaining curcumin's pharmacological properties, and adding another natural product that has bioenhancing properties to curcumin or combination of two of these strategies. This review comprehensively explores curcumin's chemistry and pharmacology followed by comparing and contrasting a vast number of strategies designed to enhance curcumin's bioavailability and its therapeutic effects. The review provides insights into which curcumin formulation strategies have the greatest promise to reach clinical application.

Colonic Saturated Fatty Acid Concentrations and Expression of COX-1, but not Diet, Predict Prostaglandin E2 in Normal Human Colon Tissue.

Prostaglandin E2 (PGE2) in the colon is a pro-inflammatory mediator that is associated with increased risk of colon cancer. In this study, expression of genes in the PGE2 pathway were quantified in colon biopsies from a trial of a Mediterranean versus a Healthy Eating diet in 113 individuals at high risk for colon cancer. Colon biopsies were obtained before and after 6 months of intervention. Quantitative, real-time PCR was used to measure mRNA expression of prostaglandin H synthases (PTGS1 and 2), prostaglandin E synthases (PTGES1 and 3), prostaglandin dehydrogenase (HPGD), and PGE2 receptors (PTGER2, PTGER4). The most highly expressed genes were HPGD and PTGS1. In multivariate linear regression models of baseline data, both colon saturated fatty acid concentrations and PTGS1 expression were significant, positive predictors of colon PGE2 concentrations after controlling for nonsteroidal anti-inflammatory drug use, gender, age, and smoking status. The effects of dietary intervention on gene expression were minimal with small increases in expression noted for PTGES3 in both arms and in PTGER4 in the Mediterranean arm. These results indicate that short-term dietary change had little effect on enzymes in the prostaglandin pathway in the colon and other factors, such as differences in fatty acid metabolism, might be more influential.

Markers of systemic exposures to products of intestinal bacteria in a dietary intervention study.

PURPOSE: Systemic exposures to intestinal bacteria may play a role in the etiology of the chronic, low-grade inflammation that is associated with western diets. Production of lipopolysaccharide-binding protein (LBP) is one biomarker of increased exposures to intestinal bacteria. This study evaluated whether changes in diet quality could affect serum LBP. METHODS: This was a randomized, controlled trial of Mediterranean and Healthy Eating diets over 6 months in 120 healthy subjects at increased risk of colon cancer. Blood samples obtained before and after intervention were analyzed for LBP, branched-chain fatty acids characteristic of intestinal bacteria, micronutrients and cytokines. Data were analyzed for changes in LBP over time and for predictors of LBP. RESULTS: Serum concentrations of branched-chain bacterial fatty acids declined significantly in both diet groups. However, there was no significant change in mean serum LBP concentrations with either diet intervention. In serum, LBP was positively associated with CRP and negatively associated with carotenoids both before and after intervention. After intervention, LBP was predicted positively by both CRP and bacterial fatty acid concentrations in serum, and negatively by serum carotenoids and the ω3/ω6 fatty acid ratio. This model accounted for 30 % of the inter-individual variation in serum LBP after intervention. CONCLUSIONS: These results indicate that dietary intervention over 6 months was insufficient to alter serum LBP. The relationships with inflammation-related markers, however, indicate that anti-inflammatory strategies other than changes in diet quality, such as weight loss or improved fitness, may have more potential for reducing systemic markers of LPS exposures in well-nourished populations.

Methylated B3GAT2 and ZNF793 Are Potential Detection Biomarkers for Barrett's Esophagus.

BACKGROUND: Barrett's esophagus (BE) is a preneoplastic condition in which normal esophageal squamous epithelium (SQ) is replaced by specialized intestinal metaplasia. It is the presumed precursor for esophageal adenocarcinoma (EAC) as well as the strongest risk factor for this cancer. Unfortunately, many patients with BE go undiagnosed under the current BE screening guidelines. The development of noninvasive and accurate BE detection assays could potentially identify many of these undiagnosed BE patients. METHODS: DNA methylation is a common epigenetic alteration in BE. Therefore, we conducted a genome-wide methylation screen to identify potential BE biomarkers. Samples from SQ (N = 12), stomach (N = 28), and BE (N = 29) were analyzed and methylation levels at over 485,000 CpG sites were compared. Pyrosequencing assays were used to validate the results and MethyLight assays were developed to detect the methylated alleles in endoscopic brushings. RESULTS: We discovered two genes, B3GAT2 and ZNF793, that are aberrantly methylated in BE. Clinical validation studies confirmed B3GAT2 and ZNF793 methylation levels were significantly higher in BE samples (median = 32.5% and 33.1%, respectively) than in control tissues (median = 2.29% and 2.52%, respectively; P < 0.0001 for both genes). Furthermore, gene-specific MethyLight assays could accurately detect BE (P < 0.0001 for both) in endoscopic brushing samples. CONCLUSION: B3GAT2 and ZNF793 are hypermethylated in BE, and the methylation status of these genes can be used to detect BE in tissue samples. IMPACT: These findings support the development of methylated B3GAT2 and ZNF793 as biomarkers for noninvasive assays for the detection of BE.

Tumor-selective proteotoxicity of verteporfin inhibits colon cancer progression independently of YAP1.

Yes-associated protein 1 (YAP1) is a transcriptional coactivator in the Hippo signaling pathway. Increased YAP1 activity promotes the growth of tumors, including that of colorectal cancer (CRC). Verteporfin, a drug that enhances phototherapy to treat neovascular macular degeneration, is an inhibitor of YAP1. We found that verteporfin inhibited tumor growth independently of its effects on YAP1 or the related protein TAZ in genetically or chemically induced mouse models of CRC, in patient-derived xenografts, and in enteroid models of CRC. Instead, verteporfin exhibited in vivo selectivity for killing tumor cells in part by impairing the global clearance of high-molecular weight oligomerized proteins, particularly p62 (a sequestrome involved in autophagy) and STAT3 (signal transducer and activator of transcription 3; a transcription factor). Verteporfin inhibited cytokine-induced STAT3 activity and cell proliferation and reduced the viability of cultured CRC cells. Although verteporfin accumulated to a greater extent in normal cells than in tumor cells in vivo, experiments with cultured cells indicated that the normal cells efficiently cleared verteporfin-induced protein oligomers through autophagic and proteasomal pathways. Culturing CRC cells under hypoxic or nutrient-deprived conditions (modeling a typical CRC microenvironment) impaired the clearance of protein oligomers and resulted in cell death, whereas culturing cells under normoxic or glucose-replete conditions protected cell viability and proliferation in the presence of verteporfin. Furthermore, verteporfin suppressed the proliferation of other cancer cell lines even in the absence of YAP1, suggesting that verteporfin may be effective against multiple types of solid cancers.