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Background & Aims TP53 mutations underlie Barrett’s esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown. Methods Single-cell RNA sequencing of paired normal esophagus and BE tissues identified candidate E2s, further investigated in expression data from BE to esophageal adenocarcinoma (EAC) progression samples. Biochemical and cellular studies helped clarify the role of RNF128-E2 on mutant p53 stability. Results The UBE2D family member 2D3 (UBCH5C) is the most abundant E2 in normal esophagus. However, during BE to EAC progression, loss of UBE2D3 copy number and reduced expression of RNF128 Iso2 were noted, 2 known p53 degraders. In contrast, expression of UBE2D1 (UBCH5A) and RNF128 Iso1 in dysplastic BE and EAC forms an inactive E2–E3 complex, stabilizing mutant p53. To destabilize mutant p53, we targeted RNF128 Iso1 either by mutating asparagine (N48, 59, and 101) residues to block glycosylation to facilitate β-TrCP1–mediated degradation or by mutating proline (P54 and 105) residues to restore p53 polyubiquitinating ability. In addition, either loss of UBCH5A catalytic activity, or disruption of the Iso1-UBCH5A interaction promoted Iso1 loss. Consequently, overexpression of either catalytically dead or Iso1-binding–deficient UBCH5A mutants destabilized Iso1 to degrade mutant p53, thus compromising the clonogenic survival of mutant p53-dependent BE cells. Conclusions Loss of RNF128 Iso2–UBCH5C and persistence of the Iso1–UBCH5A complex favors mutant p53 stability to promote BE cell survival. Therefore, targeting of Iso1-UBCH5A may provide a novel therapeutic strategy to prevent BE progression., Graphical abstract

Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease (GERD), is the strongest risk factor for esophageal adenocarcinoma (EAC). Despite similar rates of GERD and other risk factors across racial groups, EAC progression disproportionately impacts Caucasians. We recently reported that elevated tissue levels of the detoxification enzyme GSTT2 in the esophagi of Blacks compared to Caucasians may contribute protection. Herein, we extend our research to investigate whether cranberry proanthocyanidins (C-PAC) mitigate bile acid-induced damage and GSTT2 levels utilizing a racially diverse panel of patient-derived primary esophageal cultures. We have shown that C-PACs mitigate reflux-induced DNA damage through GSTT2 upregulation in a rat esophageal reflux model, but whether effects are recapitulated in humans or differentially based on race remains unknown. We isolated normal primary esophageal cells from Black and Caucasian patients and assessed GSTT2 protein levels and cellular viability following exposure to a bile acid cocktail with and without C-PAC treatment. Constitutive GSTT2 levels were significantly elevated in Black (2.9-fold) compared to Caucasian patients, as were GSTT2 levels in Black patients with GERD. C-PAC treatment induced GSTT2 levels 1.6-fold in primary normal esophageal cells. GSTT2 induction by C-PAC was greatest in cells with constitutively low GSTT2 expression. Overall, C-PAC mitigated bile-induced reductions of GSTT2 and subsequent loss of cell viability regardless of basal GSTT2 expression or race. These data support that C-PAC may be a safe efficacious agent to promote epithelial fitness through GSTT2 induction and in turn protect against bile acid-induced esophageal injury.

Improved methods for early cancer detection arising from Barrett’s oesophagus (BE) are still needed. Imaging molecular expression patterns in BE patients can target neoplasia. We demonstrate a multiplexed fluorescence imaging approach to detect premalignant lesions with two fluorescently labeled heptapeptides specific for EGFR and ErbB2. Twenty-two BE patients underwent endoscopic imaging with a multimodal scanning fiber endoscope (mmSFE). In this pilot study, 92% of neoplastic lesions could be imaged by comparison with pathology, with only 11% false positives. This first-in-human study demonstrates feasibility to concurrently detect multiple targets in vivo and potential for early detection of cancers that are molecularly heterogeneous. ### Background Oesophageal adenocarcinoma (EAC) is a deadly disease that has increased dramatically in incidence over the past several decades.1 2 Endoscopic screening with white light illumination and random biopsy is limited by sampling error.3 Dysplasia often presents with flat architecture and patchy distribution.4 EGFR and ErbB2 are transmembrane tyrosine kinase receptors that stimulate epithelial cell growth, proliferation and differentiation.5 Overexpression of these targets reflects a higher risk for cancer progression.6–8 Multiplexed imaging methods take advantage of the broad spectrum of light over the visible and near-infrared (NIR) regimen. We aim to demonstrate clinical feasibility to visualise EGFR and ErbB2 expression simultaneously in vivo to detect Barrett’s neoplasia. ### Methods Consecutive patients referred for either evaluation or therapy of Barrett’s neoplasia were recruited for the study (NCT03589443). An mmSFE was designed to collect multiplexed fluorescence images concurrently. Target/background (T/B) ratios were calculated for each fluorescence image. More details on the methods and the multiplexed imaging technology can be found in the online supplemental file. ### Supplementary data [gutjnl-2020-322945supp001.pdf] ### Results The peptide QRHKPRE specific for EGFR was labelled with Cy5 via a GGGSK linker, (figure 1A).9 KSPNPRF, specific for ErbB2, was labelled with IRDye800 via a GGGSC linker (figure 1B). …

SummaryEuropean Americans (EA) are more susceptible to esophageal tissue damage and inflammation when exposed to gastric acid and bile acid reflux and have a higher incidence of esophageal adenocarcinoma when compared to African Americans (AA). Population studies have implicated specific genes for these differences; however, the underlying cause for these differences is not well understood. We describe a robust long-term culture system to grow primary human esophagus in vitro, use single cell RNA sequencing to compare primary human biopsies to their in vitro counterparts, identify known and new molecular markers of basal cell types, and demonstrate that in vivo cellular heterogeneity is maintained in vitro. We further developed an ancestrally diverse biobank and a high-content, image based, screening assay to interrogate bile-acid injury response. These results demonstrated that AA esophageal cells responded significantly differently than EA-derived cells, mirroring clinical findings, having important implications for addressing disparities in early drug development pipelines.

Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease (GERD), is the strongest risk factor for EAC development. GERD and esophagitis occur at similar rates among Blacks and Caucasians; yet, progression to EAC is significantly elevated among Caucasians. Unique protective factors in the epithelium of Blacks may contribute to this disparity. Our research team recently reported that the detoxification enzyme GSTT2 is higher in the esophageal squamous epithelium of Blacks compared to Caucasians with potential linkages to previously identified genomic variants in the GSTT2 locus (a 37 kb deletion and a 17 bp promoter duplication among Caucasians). Thus, the current study seeks to evaluate whether primary esophageal cell cultures isolated from Black or Caucasian cohorts can serve as discerning and relevant model systems to investigate risk factors linked to EAC progression, assess efficacy of mitigating agents and differential responses linked to race. We have shown that cranberry proanthocyanidins (C-PAC) mitigate DNA damage associated with reflux through upregulation of GSTT2 in a rat surgical model of reflux-induced EAC, but whether effects are recapitulated in humans or differentially based on race remains unknown. Herein we isolated normal primary esophageal epithelial cells from Black and Caucasian patients and assessed GSTT2 genotype, GSTT2 protein levels and cellular viability following exposure of the cultures to a bile acid cocktail (BAC) [0.2mM] with and without C-PAC [50µg/ml] treatment. Constitutive levels of GSTT2 were 1.7-fold higher in Blacks than Caucasians, with 71% of Blacks identified as high expressors compared to 33% of Caucasians. Pretreatment (48h) of primary cultures with C-PAC induced GSTT2 levels in all but one Black-derived culture which already expressed high basal levels. GSTT2 induction in normal epithelial cultures by C-PAC was greatest in cells with constitutively low GSTT2 expression; however, upon BAC challenge C-PAC effectively mitigated BAC-induced reductions in GSTT2 levels and subsequent loss of normal cell viability regardless of basal GSTT2 expression or race. C-PAC treatment pre- plus post-BAC imparted no additional benefit over pretreatment alone in preserving viability but did further increase GSTT2 levels. Next steps include expanding our panel of primary cultures and conducting nano LC-MS/MS proteomic profiling of Black and Caucasian-derived cultures treated with vehicle, BAC, C-PAC and BAC + C-PAC, with stratification based on GSTT2 basal expression. Taken together these data support that C-PAC may be used as an efficacious non-toxic agent serving to promote epithelial fitness and resiliency against the biologic and molecular sequelae linked bile acid-induced esophageal injury and progression to EAC. Citation Format: Katherine M. Weh, Danielle K. Turgeon, Amy Howell, Laura A. Kresty. Racially diverse primary esophageal cell cultures for evaluating mitigation of bile-induced injury [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2593.

Introduction: As part of our effort to understand the role of trace elements in colon polyp chemoprevention, we have conducted both colonoid culture (ex vivo) and human subject (in vivo) studies using Aquamin®— a calcium-, magnesium-, trace elements-rich, multi-mineral product derived from red marine algae as an intervention. Calcium alone was used as a comparator. Methods: Thirty subjects at-risk for colorectal cancer were enrolled and randomized to receive Aquamin® or calcium alone (each providing 800-mg calcium per day) or placebo. Colon biopsies were obtained before and after 90 days of intervention. We employed histologically normal colonic tissue from the same subjects (at baseline), to propagate in colonoid culture. Once established and expanded, we used the same interventions for a period of two weeks and compared the response (at 1.5mM) to the outcome of the studies conducted in human colonic biopsies after 90 days of intervention. Specifically, colonoid tissue and biopsies were subjected to analysis for markers of growth and differentiation by quantitative immunohistochemistry and tandem mass tag mass spectrometry-based proteomics. Results: The normal tissue colonoids demonstrated a high degree of differentiation as indicated by gross and microscopic appearance, and cytokeratin 20 (CK20) expression without either intervention. Only modest increases were seen in the CK20 expression with either calcium alone or Aquamin®. Similarly, CK20 expression was higher in baseline biopsies and there was no increase in the expression with calcium but a 5% increase seen with Aquamin® after 90 days of intervention. On proteomic screen, CK20 expression was increased by 40% in colonoid tissue in response to both interventions at 1.5mM, while CK20 expression increased by 10% and 21% with Calcium and Aquamin® in colon biopsies. When the colonoids were assessed for Ki67 (growth marker) expression, it was decreased by 17.2% and 18.4% with calcium alone and Aquamin® respectively as compared to the control. Analyzing colon biopsies after 90 days of intervention revealed that Aquamin® reduced the level of Ki67 expression by 20%, while no change was seen with calcium alone. Differential proteomic expression of proliferating cell nuclear antigen was decreased by 32% and 39% by calcium and Aquamin® at 1.5mM in colonoid tissue, whereas Ki67 protein was decreased by 4% and 22% with calcium and Aquamin®, respectively in colon biopsies. Conclusion: Colon tissue maintained in colonoid culture, thus, provides a good surrogate for human tissue collected in a clinical trial. It can be used to assess personalized responses or can provide a quick snapshot of the response in a relatively short time. Differential proteomic expression appears to be more sensitive than quantitative immunohistochemistry. A combination of calcium and additional trace elements proved to be more effective than calcium alone in altering differentiation and growth markers in either setting. Citation Format: Muhammad N. Aslam, Shannon McClintock, Mohamed Ali H Jawad-Makki, Karsten Knuver, Daniyal M. Nadeem, Haris Ahmad, Durga Attili, Danielle (Kim) Turgeon, James Varani. Cellular Differentiation and Growth Control in the Human Colonic epithelium: Comparing Clinical Trial Outcomes to Responses in Human Colonoid Culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB220.

Introduction: Accurate identification of neoplastic or pre-neoplastic lesions is traditionally dependent on microscopic identification of nuclear abnormalities. It is both time-consuming and challenging in early-stage lesions and difficult to detect. Laser-emission microscopy (LEM) that uses laser emission (not fluorescence) is an emerging technology that takes advantage of the finding that highly-proliferating cells like those in pre-neoplastic lesions can be distinguished from less proliferative cells by a lower laser emission dot density (LDD) when stained with a nuclear dye. We have successfully used this technique to detect (early) nuclear changes in a high-fat induced colon cancer model. LEM has also been used successfully to differentiate healthy tissue from cancerous tissue in human patients. Methods: This LEM system is capable of rapidly scanning tissue as large as 2 cm×2 cm in ~10 min. Both laser emission and regular fluorescence are imaged simultaneously, thus providing information regarding the lasing spot density and location within the human colon tissues. In this study, we employed colon biopsies from 12 human subjects (at risk for colorectal cancer). These subjects participated in a 90-day intervention, placebo-controlled, randomized trial. 8 subjects ingested known chemopreventives and 4 subjects took a placebo. Colon biopsies were taken from the sigmoid colon at baseline and after 90 days. The formalin-fixed, paraffin-embedded tissues were subjected to LEM microscopic assessment. For this, the tissues were sandwiched between two mirrors to form a laser cavity and laser excitation was applied. By comparing the laser emission spectra, highly proliferative tissue could be differentiated from less proliferative tissue. Post-treatment biopsies were compared to their baseline status. These biopsies were also stained with Ki67 to obtain their proliferation index to compare with the LEM results. Results: On average, there was a 20% increase in the LDD among the 8 subjects on chemopreventives while no change was seen in Ki67 positivity. In placebo group, Ki67 was decreased by 14% and LDD was 12% increased. On individual comparison, LDD and Ki67 data were correlated positively in 6 subjects. Conclusion: The preliminary data suggest that in histologically-normal but “at-risk” human colon LEM may pick early nuclear changes that may not be detected by a proliferation marker. Table.A comparison of lasing dot density (LDD) data to Ki67 positivity (Whole slide analysis*)Mean LDDPercent Positive NucleiChange RatioSubject IDPrePostPrePostLDDKi67Data correlated?Placebo 1491336504.75.40.741.15NoPlacebo 2851078159.28.70.920.95YesPlacebo 3573183558.96.01.460.67NoPlacebo 4659288469.76.71.340.69NoChemopreventive#1 1296733436.513.61.132.10YesChemopreventive#1 2528261406.57.71.161.20YesChemopreventive#1 3468453429.67.01.140.73NoChemopreventive#1 4758173358.25.60.970.68YesChemopreventive#2 1323046255.34.91.430.91NoChemopreventive#2 2508078408.29.11.541.11YesChemopreventive#2 35587400110.46.80.720.66YesChemopreventive#2 4491373226.94.91.490.71NoPlacebo combined1.12±0.340.86±0.23Chemopreventive combined1.20±0.281.01±0.49*Both mucosa and submucosa were involved in the analysis Citation Format: Muhammad N. Aslam, Yun-Lu Sun, Ingrid Bergin, Mohamed Ali H. Jawad-Makki, Karsten Knuver, Danielle (Kim) Turgeon, James Varani, Xudong Fan. A novel laser emission microscopy for early detection of colon cancer: Analysis of at-risk human colon from an interventional trial (research in progress) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT113.

Introduction: Based on pre-clinical studies, it was hypothesized that a calcium- and multiple trace element-rich product- Aquamin will promote differentiation/barrier function and reduce proliferation in the human colonic epithelium more effectively than calcium alone. The goal of the study is to begin testing this hypothesis. Methods: A 90-day interventional trial was conducted with human subjects (at risk for colorectal cancer). Thirty subjects were enrolled and randomized to receive Aquamin or calcium alone (each providing 800 mg/day) or placebo. Prior to ingesting the study-agents and following the course of treatment, colon biopsies were obtained by sigmoidoscopy and examined for markers of growth, and differentiation by quantitative immunohistochemistry. Tandem mass tag mass spectrometry-based proteomic screen was used to obtain a broad comparison of protein alterations induced by Aquamin as compared to calcium and placebo. Specimens were pooled by the group and for each group, a pre-, post-treatment “ordinate” was obtained to determine proteins that were up-regulated or down-regulated in the post-treatment specimens as compared to pre-treatment. Results: Quantitation of proliferation marker Ki67 showed a 21% decrease in post-intervention expression with Aquamin to its pre-intervention expression. In contrast, there was no change in differentiation marker (CK20) expression. Calcium did not reduce Ki67 expression (Table). When crypt length was measured, there was no change in the post-intervention biopsies (in all groups), indicating no adverse effect of any intervention on the colonic tissue. On proteomic screen, there was minimal up- and down-regulation of proteins in placebo-treated subjects. In contrast, a large number of proteins demonstrated up- or down-regulation with calcium and Aquamin (at 1.8-fold and Table.Quantitation of Proliferation and Differentiation Markers.PlaceboCalciumAquaminPrePostPrePostPrePostKi67Positive percent nuclei13.1±5.212.7±4.112.1±3.913.9±5.816.6±8.113.1±4.7Positive nuclei / mm2264±119229±89234±79262±117309±191222±105CK20Positive pixel count0.44±0.260.46±0.240.47±0.170.51±0.170.54±0.210.54±0.18Crypt Length (µm)351±71354±72349±67348±86350±81372±79 Conclusion: These results suggest that trace elements in conjunction with calcium can reduce proliferation and improve barrier in the colon epithelium more effectively than calcium alone. This study provides biomarker data needed to plan a more definitive clinical interventional (polyp-prevention) trial. Citation Format: Muhammad N. Aslam, Shannon McClintock, Karsten Knuver, Mohamed Ali H. Jawad-Makki, Haris Ahmad, Danielle (Kim) Turgeon, James Varani. A multi-mineral approach to alter biomarkers of growth and differentiation in the human colon: Results from an interventional trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT112.

The microbiome has been implicated in the development of colorectal cancer (CRC) and inflammatory bowel diseases (IBD). The specific traits of these diseases vary along the axis of the digestive tract. Further, variation in the structure of the gut microbiota has been associated with both diseases. Here we profiled the microbiota of the healthy proximal and distal mucosa and lumen to better understand how bacterial populations vary along the colon. We used a two-colonoscope approach to sample proximal and distal mucosal and luminal contents from the colons of 20 healthy subjects that had not undergone any bowel preparation procedure. The biopsies and home-collected stool were subjected to 16S rRNA gene sequencing and Random Forest classification models were built using taxa abundance and location to identify microbiota specific to each site. The right mucosa and lumen had the most similar community structures of the five sites we considered from each subject. The distal mucosa had higher relative abundance of Finegoldia, Murdochiella, Peptoniphilus, Porphyromonas and Anaerococcus. The proximal mucosa had more of the genera Enterobacteriaceae, Bacteroides and Pseudomonas. The classification model performed well when classifying mucosal samples into proximal or distal sides (AUC = 0.808). Separating proximal and distal luminal samples proved more challenging (AUC = 0.599) and specific microbiota that differentiated the two were hard to identify. By sampling the unprepped colon, we identified distinct bacterial populations native to the proximal and distal sides. Further investigation of these bacteria may elucidate if and how these groups contribute to different disease processes on their respective sides of the colon.

Introduction: The bacteria making up the microbial community of the gastrointestinal tract, by virtue of their ability to produce pro-carcinogenic bile acids and anti-carcinogenic short chain fatty acids (SCFA), are thought to influence the colonic health. Methods: Thirty healthy human subjects (at risk for colorectal cancer) were enrolled in a three-arm, 90-day double-blinded, randomized interventional trial in which Aquamin, a calcium- and magnesium-rich, multi-mineral product was compared to calcium alone and placebo for effects on the colonic microbial community and metabolomic profile. Aquamin and calcium (carbonate) alone groups provided 800 mg of calcium per day from the respective sources. Colonic tissue and stool samples were obtained endoscopically from unpreped sigmoid colon with a flexible sigmoidoscope before and after the intervention and were subjected to microbial and metabolomic analysis. Illumina® MiSeq sequencing of the V4 region of 16S rRNA genes was used to assess the bacterial populations represented in both colon mucosal biopsies and fecal material and qPCR was used to obtain an estimate of total bacterial DNA in each tissue and stool specimen. In parallel, a 2-step extraction procedure followed by liquid chromatography-mass spectrometry was used to assess bile acids and SCFA. Results: As far as safety and tolerability is concerned, there were no serious adverse events to report during the trial with Aquamin or calcium. Compared to pretreatment values from both colonic biopsies and stool specimens, intervention with Aquamin led to a reduction in total bacterial load (based on qPCR). Treatment with calcium alone produced intermediary changes in total bacterial DNA while no reduction was observed with placebo. Based on sequencing of the V4 region of 16S rRNA genes, there were significant pre-post differences, but intra-subject variability was less than the variability between individuals. In spite of the high variability, greater pre-post interventional differences were seen with Aquamin relative to calcium alone and placebo. Differences between Aquamin and calcium alone reached statistical significance. Several operational taxonomic units representing major microbial phyla present in the colon (i.e., actinobacteria, bacteroidetes, and firmicutes) were reduced in response to Aquamin compared to placebo. Reductions were also seen with calcium but these were intermediary between Aquamin and placebo. In parallel with microbial changes, we observed a reduction in total bile acid levels and an increase in the level of acetate (an SCFA) in stool specimens from Aquamin-treated subjects. No or minimal change in bile acids or SCFAs was observed with calcium alone or placebo. Some of the primary bile acids (cholate and Chenodeoxycholate/deoxycholate) and the secondary bile acids (alpha-Murocholate, hyocholate, hyodeoxycholate, and omega-Muricholate) were significantly reduced with Aquamin. Additionally, on eicosanoids assessment in colon biopsies, 13S-HODE was reduced with Aquamin. Conclusion: The combination of calcium, magnesium and additional trace elements (as present in Aquamin) can produce alterations in the colonic microbial community and the attendant metabolomic profile that could be beneficial to colonic health in subjects at risk for colorectal cancer. Citation Format: Muhammad N. Aslam, Christine Bassis, Ingrid Bergin, Karsten Knuver, Ananda Sen, Danielle Kim Turgeon, James Varani. The alteration of the colonic microbial community and metabolomic profile in humans: Results from a 90-day double-blinded, randomized interventional trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-250.

Little is known about the effect of preventive diets on colonic nutrient concentrations. This study randomized 120 persons at increased risk of colon cancer to a Mediterranean versus a Healthy Eating diet for six months. The former targeted increases in whole grains, fruits, vegetables, monounsaturated, and n3 fats. The Healthy Eating diet was based on Healthy People 2010 recommendations. At baseline, dietary fat and carotenoid intakes were poorly associated (Spearman ρ < 0.4) with serum and colon concentrations. Strong associations were observed between serum and colon measurements of β-cryptoxanthin (ρ = 0.58; P < 0.001), α-carotene (ρ = 0.48; P < 0.001), and β-carotene (ρ = 0.45; P < 0.001). After six months, the Healthy Eating intervention increased serum lutein, β-, and α-carotene significantly (P < 0.05). In the Mediterranean arm, the significant increases were in serum lutein, β-cryptoxanthin, β-carotene, monounsaturated, and n3 fats. A significant group-by-time interaction (P = 0.03) was obtained for monounsaturated fats. Colonic increases in carotenoids and n3 fats were significant only in Healthy Eating arm, whereas the group-by-time interaction was significant for β-carotene (P = 0.02) and α-carotene (P = 0.03). Changes in colon concentrations were not significantly associated with reported dietary changes. Changes in colon and serum concentrations were strongly associated for β-cryptoxanthin (ρ = 0.56; P < 0.001) and α-carotene (ρ = 0.40; P < 0.001). The associations between colonic and serum concentrations suggest the potential use of using serum concentration as a target in dietary interventions aimed at reducing colon cancer risk. Cancer Prev Res; 6(6); 558–65. ©2013 AACR.

Elevated tissue levels of prostaglandin E2, produced by cyclooxygenase (COX), are an early event in colorectal cancer (CRC). Data suggest the efficacy of nonsteroidal anti-inflammatory drugs, such as cancer preventives, in the inhibition of COX activity; however, side effects of nonsteroidal anti-inflammatory pose unacceptable limitations. Ginger has been reported to have anti-inflammatory activities with significant CRC preventive potential. We investigated whether consumption of 2.0 g ginger daily regulated the level of two key enzymes that control prostaglandin E2 production, COX-1 and NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Thirty participants at normal and 20 participants at increased risk for CRC were randomized and given 2.0 g/day ginger or placebo for 28 days. Flexible sigmoidoscopy was used to obtain colon biopsies at baseline and the end of the study. Tissue levels of COX-1 and 15-PGDH were assessed using western blotting. After ginger consumption, participants at increased risk for CRC had a significantly reduced colonic COX-1 protein level (23.8±41%) compared with the placebo group (18.9±52%; P=0.03). Protein levels of 15-PGDH in the colon were unchanged. In participants who were at normal risk for CRC, neither protein levels of COX-1 nor 15-PGDH in the colon were altered by ginger consumption. Ginger significantly lowered COX-1 protein expression in participants at increased risk for CRC but not in those at normal risk for CRC. Ginger did not alter 15-PGDH protein expression in either increased or normal-risk participants. Further investigation, in larger studies with a longer ginger intervention, is needed to examine the ability of ginger to impact tissue levels of prostaglandin.

Colorectal cancers associated with Lynch syndrome are characterized by deficient DNA mismatch repair (MMR) function. Our aim was to evaluate the prevalence of microsatellite instability (MSI) and loss of MMR protein expression in Lynch syndrome–associated polyps. Sixty-two colorectal polyps—37 adenomatous polyps, 23 hyperplastic polyps, and 2 sessile serrated polyps (SSP)—from 34 subjects with germline MMR gene mutations were tested for MSI using a single pentaplex PCR for five mononucleotide repeat microsatellite markers, and also for expression of MLH1, MSH2, MSH6, and PMS2 proteins by immunohistochemistry. High-level MSI (MSI-H) was seen in 15 of 37 (41%) adenomatous polyps, one of 23 (4%) hyperplastic polyps, and one of two (50%) SSPs. Loss of MMR protein expression was seen in 18 of 36 (50%) adenomatous polyps, zero of 21 hyperplastic polyps, and zero of two SSPs. Adenomatous polyps 8 mm or larger in size were significantly more likely to show MSI-H [OR, 9.98; 95% confidence interval (CI), 1.52–65.65; P = 0.02] and deficient MMR protein expression (OR, 3.17; 95% CI, 1.20–8.37; P = 0.02) compared with those less than 8 mm in size. All (six of six) adenomatous polyps 10 mm or larger in size showed both MSI-H and loss of MMR protein expression by immunohistochemistry. Our finding that the prevalence of MMR deficiency increases with the size of adenomatous polyps suggests that loss of MMR function is a late event in Lynch syndrome–associated colorectal neoplasia. Although testing large adenomatous polyps may be of value in the diagnostic evaluation of patients with suspected Lynch syndrome, the absence of an MMR-deficient phenotype in an adenoma cannot be considered as a strong evidence against Lynch syndrome, as it is with colorectal carcinomas. Cancer Prev Res; 5(4); 574–82. ©2012 AACR.

Curcumin is derived from the spice tumeric and has antiinflammatory and antineoplastic effects in vitro and in animal models, including preventing aberrant crypt foci (ACF) and adenomas in murine models of colorectal carcinogenesis. Inhibiting the production of the procarcinogenic eicosanoids prostaglandin E2 (PGE2) and 5-hydroxyeicosatetraenoic acid (5-HETE) can suppress carcinogenesis in rodents. Curcumin reduces mucosal concentrations of PGE2 (via inhibition of cyclooxygenases 1 and 2) and 5-HETE (via inhibition of 5-lipoxygenase) in rats. Although preclinical data support curcumin activity in many sites, the poor bioavailability reported for this agent supports its use in the colorectum. We assessed the effects of oral curcumin (2 g or 4 g per day for 30 days) on PGE2 within ACF (primary endpoint), 5-HETE, ACF number, and proliferation in a nonrandomized, open-label clinical trial in 44 eligible smokers with eight or more ACF on screening colonoscopy. We assessed pre- and posttreatment concentrations of PGE2 and 5-HETE by liquid chromatography tandem mass spectroscopy in ACF and normal-tissue biopsies; ACF number via rectal endoscopy; proliferation by Ki-67 immunohistochemistry; and curcumin concentrations by high-performance liquid chromatography in serum and rectal mucosal samples. Forty-one subjects completed the study. Neither dose of curcumin reduced PGE2 or 5-HETE within ACF or normal mucosa or reduced Ki-67 in normal mucosa. A significant 40% reduction in ACF number occurred with the 4-g dose (P < 0.005), whereas ACF were not reduced in the 2-g group. The ACF reduction in the 4-g group was associated with a significant, five-fold increase in posttreatment plasma curcumin/conjugate levels (versus pretreatment; P = 0.009). Curcumin was well tolerated at both 2 g and 4 g. Our data suggest that curcumin can decrease ACF number, and this is potentially mediated by curcumin conjugates delivered systemically. Cancer Prev Res; 4(3); 354–64. ©2011 AACR.

Introduction: In order to advance a primary cell culture model of human colonic carcinogenesis we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomatous polyps. Methods: Fresh biopsy samples were transported in culture medium containing 1.5mM Ca2+ and antimicrobials. The mucosa/submucosa was surgically separated from the muscularis propria and incubated in 10mM dithiothreitol followed by 8mM ethylenediaminetetraacetic acid. Crypts were agitated free, washed with slow spins, and immediately embedded in a 3-dimensional environment at a density of 50 crypts/50μl Matrigel. After 3 hours, media was replaced to remove non-viable cellular products. Serum-free medium, containing low Ca2+ (0.15mM), was used to maintain crypt cultures. Cell proliferation and differentiation were assessed with markers Ki67 and E-cadherin by confocal microscopy. The fluorescence signal was quantified for the bottom 1/3 of the crypts (35 cells from base on one crypt edge) using morphometric imaging software. In parallel studies, abnormal crypt-like structures were isolated from adenomatous polyps/adenocarcinoma and maintained in a similar culture system. Results: At day-1 in culture, Ki67 expression was strong in the proliferating cells at the crypt base, with E-cadherin staining increasing towards the crypt apex. By day-3, E-cadherin expression became much more prominent and Ki67 decreased dramatically. This culture system preserved the in vivo phenotype of the crypts and allowed for the colonic epithelial cells to follow the physiological sequence of progression from proliferation to differentiation. Adenomatous tissue in culture at day-1 showed expression for Ki67 and E-cadherin but lacked spacial organization. Conclusion: Intact colonic crypts from normal and abnormal human mucosa can be viably maintained in 3-D culture for 72 hours. This primary human in vitro 3-D model may be used to model efficacy of colonic cancer chemopreventives. Epithelial Cell Proliferation and Differentiation in Cultured Human Crypts (fold changes) Markers Zero-time 24 hours 48 hours 72 hours Ki67 1.00 0.36 0.18 0.00 E-cadherin 1.00 2.00 2.89 3.22 Citation Format: Michael K. Dame, Yan Jiang, Danielle Kim Turgeon, Henry Appelman, Muhammad N. Aslam, Kelly Copley, Durga Attili, Dean E. Brenner, James Varani. Human colon mucosal crypts in culture: a model to assess colon chemoprevention. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3711. doi:10.1158/1538-7445.AM2013-3711

1570 Background: Studies using drugs that inhibit cyclooxygenase enzymes (COX-1,-2) indicate that up-regulation of inflammatory eicasanoids, and in particular prostaglandin E2 (PGE2) are early events in the development of CRC. Ginger root has demonstrated down regulation of COX-1,-2, apoptosis in CRC cell lines and decreased incidence and multiplicity of adenomas in rats. This study was conducted to determine if 2.0 g/day of ginger extract could decrease the levels of PGE2, 5-,12-, & 15-hydroxyeicosatetraenoic acid (5-,12-, & 15-HETE), 13-hydroxyoctadecadienoic acid (13-HODE) and Leukotriene B4 (LTB4), in the gut mucosa of people at increased risk of colorectal cancer when taken for 28 days. Methods: A total of 20 subjects were randomized to 2.0 g/day ginger or placebo for 28 days. Flexible sigmoidoscopy at baseline and day 28 was used to obtain rectal biopsies. A liquid chromatography mass spectrometry (LC/MS/MS) method was used to simultaneously determine PGE2, 5-HETE, 12-HETE, 15-HETE, LTB4 and 13-HODE...

Curcumin, a dietary polyphenol derivative of turmeric, has potent cancer preventive activity in in vivo colon carcinogenesis models. To determine whether curcumin has pharmacodynamic effects on the human colon, we administered curcumin at doses of 2 g or 4 g to separate groups of 20 healthy human smokers who had 8 or more aberrant crypt foci on screening colonoscopy. We removed 3 aberrant crypt foci and 4 flat mucosal biopsies under magnifying endoscopy prior to and after 28 days of daily curcumin dosing. We enrolled 41 subjects (66% African Americans, 27% Caucasian, mean age 55+/−6.5 years). PGE2 and 5-HETE concentrations on tissue biopsies (assayed with liquid chromatography tandem mass spectroscopy) are shown in the Table. Curcumin concentrations assayed by HPLC in the colonic mucosa were undetectable ( (Support: NCI N01-35610, F. Meyskens PI Univ California Irvine) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2906.