D.K. Turgeon

Phase IIA Clinical Trial of Curcumin for the Prevention of Colorectal Neoplasia

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.

Plasma glycoprotein profiling for colorectal cancer biomarker identification by lectin glycoarray and lectin blot

Colorectal cancer (CRC) remains a major worldwide cause of cancer-related morbidity and mortality largely due to the insidious onset of the disease. The current clinical procedures utilized for disease diagnosis are invasive, unpleasant, and inconvenient; hence, the need for simple blood tests that could be used for the early detection of CRC. In this work, we have developed methods for glycoproteomics analysis to identify plasma markers with utility to assist in the detection of colorectal cancer (CRC). Following immunodepletion of the most abundant plasma proteins, the plasma N -linked glycoproteins were enriched using lectin affinity chromatography and subsequently further separated by nonporous silica reversed-phase (NPS-RP)-HPLC. Individual RP-HPLC fractions were printed on nitrocellulose coated slides which were then probed with lectins to determine glycan patterns in plasma samples from 9 normal, 5 adenoma, and 6 colorectal cancer patients. Statistical tools, including principal component analysis, hierarchical clustering, and Z-statistics analysis, were employed to identify distinctive glycosylation patterns. Patients diagnosed with colorectal cancer or adenomas were shown to have dramatically higher levels of sialylation and fucosylation as compared to normal controls. Plasma glycoproteins with aberrant glycosylation were identified by nano-LC-MS/MS, while a lectin blotting methodology was used to validate proteins with significantly altered glycosylation as a function of cancer progression. The potential markers identified in this study for diagnosis to distinguish colorectal cancer from adenoma and normal include elevated sialylation and fucosylation in complement C3, histidine-rich glycoprotein, and kininogen-1. These potential markers of colorectal cancer were subsequently validated by lectin blotting in an independent set of plasma samples obtained from 10 CRC patients, 10 patients with adenomas, and 10 normal subjects. These results demonstrate the utility of this strategy for the identification of N -linked glycan patterns as potential markers of CRC in human plasma, and may have the utility to distinguish different disease states.

The erythromycin breath test predicts the clearance of midazolam

Midazolam, a commonly used sedative and amnestic medication, has recently been shown to be largely metabolized in the liver by a cytochrome P450, termed CYP3A4. There is at least a tenfold intersubject variability in the liver content and catalytic activity of CYP3A4, which may in part account for the known interpatient differences in the kinetics of midazolam. To test this hypothesis, we determined the intravenous midazolam kinetics of 20 medically stable, hospitalized patients, whose hepatic CYP3A4 activities were determined with use of the [14C‐N‐methyl]erythromycin breath test. During the kinetic study, we also performed psychometric testing designed to quantitate the level of sedation and amnesia. We found a significant positive correlation between the erythromycin breath test results and weight adjusted clearance (in milliliters per minute per kilogram) of both total midazolam (r = 0.52; p = 0.03) and unbound midazolam (r = 0.61; p < 0.01). The relatively low dose of midazolam used (0.0145 mg/kg) produced significant but transient sedation and memory impairment in some of the patients. We conclude that interpatient differences in liver CYP3A4 activity in part account for the variations in midazolam kinetics. Our observations account for reported drug interactions involving midazolam and suggest that patients with low CYP3A4 activity may be most susceptible to prolonged amnestic effects occasionally produced by this short‐acting benzodiazepine.

Targeted Imaging of Esophageal Neoplasia with a Fluorescently Labeled Peptide: First-in-Human Results

A fluorescently labeled peptide enables first-in-human targeted endoscopic imaging of esophageal neoplasia. Fluorescent Peptide Probe for Esophageal Cancer Detecting cancerous tissue isn’t always easy—and it can be particularly difficult for the early stages of esophageal cancer because the new lesions are often flat (versus a bulky tumor, for example) and thus invisible to the naked eye. To confidently detect esophageal adenocarcinoma (EAC), Sturm and colleagues designed a fluorescently labeled synthetic peptide, named ASY*-FITC, that recognizes cancer tissue and allows for in vivo imaging with a clinical endoscope. The cancer-targeting ASY*-FITC peptide was discovered using phage display technology and was found to bind tightly to human EAC cells and tissues ex vivo, but not to normal (squamous) tissue or metaplastic tissue, such as Barrett’s esophagus (BE). The tissues identified as cancerous were confirmed via histology. The authors then took this peptide into 25 patients. The fluorescent peptide was administered as would be expected during clinical exam: sprayed on the suspect area and then imaged with an endoscope. No ASY*-FITC bound to the squamous areas, and only minimal amounts of peptide bound to BE. However, areas of EAC as well as a high-grade dysplasia (HGD) were brightly illuminated and easily detected. The peptide was found to be safe and well tolerated in both humans and animals, and was synthesized according to good manufacturing practices (GMPs), suggesting that translation to a clinical setting will be possible in the near future. Further testing is needed to address optical limitations, such as imaging depth. Nevertheless, this first-in-human study paves the way for detection of HGD and EAC and other neoplasias, potentially without invasive biopsy. Esophageal adenocarcinoma is rising rapidly in incidence and usually develops from Barrett’s esophagus, a precursor condition commonly found in patients with chronic acid reflux. Premalignant lesions are challenging to detect on conventional screening endoscopy because of their flat appearance. Molecular changes can be used to improve detection of early neoplasia. We have developed a peptide that binds specifically to high-grade dysplasia and adenocarcinoma. We first applied the peptide ex vivo to esophageal specimens from 17 patients to validate specific binding. Next, we performed confocal endomicroscopy in vivo in 25 human subjects after topical peptide administration and found 3.8-fold greater fluorescence intensity for esophageal neoplasia compared with Barrett’s esophagus and squamous epithelium with 75% sensitivity and 97% specificity. No toxicity was attributed to the peptide in either animal or patient studies. Therefore, our first-in-human results show that this targeted imaging agent is safe and may be useful for guiding tissue biopsy and for early detection of esophageal neoplasia and potentially other cancers of epithelial origin, such as bladder, colon, lung, pancreas, and stomach.

Microsatellite instability and DNA mismatch repair protein deficiency in Lynch syndrome colorectal polyps.

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.

Effect of ginger root on cyclooxygenase-1 and 15-hydroxyprostaglandin dehydrogenase expression in colonic mucosa of humans at normal and increased risk for colorectal cancer.

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.

Relationships between serum and colon concentrations of carotenoids and fatty acids in randomized dietary intervention trial.

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.

Development of exchange lists for Mediterranean and Healthy Eating Diets: implementation in an intervention trial

BACKGROUND There has been little research published on the adaptation of diabetic exchange list diet approaches for the design of intervention diets in health research despite their clinical utility. The exchange list approach can provide clear and precise guidance on multiple dietary changes simultaneously. The present study aimed to develop exchange list diets for Mediterranean and Healthy Eating, and to evaluate adherence, dietary intakes and markers of health risks with each counselling approach in 120 subjects at increased risk for developing colon cancer. METHODS A randomised clinical trial was implemented in the USA involving telephone counselling. The Mediterranean diet had 10 dietary goals targeting increases in mono-unsaturated fats, n-3 fats, whole grains and the amount and variety of fruits and vegetables. The Healthy Eating diet had five dietary goals that were based on the US Healthy People 2010 recommendations. RESULTS Dietary compliance was similar in both diet arms, with 82-88% of goals being met at 6 months, although subjects took more time to achieve the Mediterranean goals than the Healthy Eating goals. The relatively modest fruit and vegetable goals in the Healthy Eating arm were exceeded, resulting in fruit and vegetable intakes of approximately eight servings per day in each arm after 6 months. A significant (P < 0.05) weight loss and a decrease in serum C-reactive protein concentrations were observed in the overweight/obese subgroup of subjects in the Mediterranean arm in the absence of weight loss goals. CONCLUSIONS Counselling for the Mediterranean diet may be useful for both improving diet quality and for achieving a modest weight loss in overweight or obese individuals.

Fluorescence Detection of Colonic Neoplasia Using a Novel Peptide

Background. Overexpression of AnnexinA2 (ANXA2) has been reported in several epithelial cancers, including colorectal cancers (CRC) (Cancer Letters, 2007). Increasing expression of gastrin gene, and hence progastrin (PG), has also been reported in CRCs. We recently discovered that ANXA2 serves as a non-conventional receptor for PG (Oncogene, 2007). We additionally reported that binding of PGwith extracellular, membrane associated, ANXA2 results in endocytosis and co-localization of PG/ANXA2 intracellularly (Gastro, 2010), and that ANXA2 is required for mediating mitogenic and anti-apoptotic effects of PG on colon cancer cells. ANXA2 is normally involved with intracellular trafficking. During the course of our studies we discovered that ANXA2 is also present on outer membranes of CRC cells, bound to a 29KDa transmembrane protein, and also released into the medium. To further understand physiological significance of these unexpected findings, we examined if ANXA2 is present in serum of patients positive for colorectal growth and if ANXA2 co-localizes with PG in tumor sections, in relation to disease status. Methods, Results and Conclusions. Secretion of ANXA2 (10-50ng/106 cells) was confirmed in conditioned medium (CM) of CRC cells using quantitative Western Blot assay using standard curve with rhANXA2; nontransformed cells were negative. Serum from CRC tumor bearing mice were positive for ANXA2, while control mice were negative. Next, serum samples were obtained from consented patients at time of endoscopy or as discarded samples from CRC patients on day of surgery, as per our IRB protocols. Serum from patients, negative for colonic growths (Normal, N) had low levels of ANXA2 (<1ng/ml), while serum from patients with hyperproliferative growths (Hp), Adenomas (Ad) and adenocarcinomas (AdCA) were on an average positive for 42, 80, and 490 ngs/ml, respectively, suggesting elevation of serum ANXA2 in relation to disease status. Paraffin blocks of Hp, Ads, AdCAs and adjoining N mucosa were obtained as discarded specimens from Department of Pathology and stained immunofluorescently for PG/ANXA2. Hp, Ads and AdCAs were increasingly positive for ANXA2 and PG expression compared to N specimens; ~ 10, 25 and 60 % of respective specimens were positive for intracellular co-localization of PG/ANXA2, suggesting presence of functional PG/ANXA2 axis, confirmed by progressive elevation of nuclear β-catenin and stem cell markers (DCAMKL+1, Lgr5) in relation to disease status. It remains to be determined if, 1) presence of co-localized PG/ANXA2 intracellularly represents a prognostic marker for predicting treatment/recurrence, and 2) if serum ANXA2 levels can be used as a non-invasive diagnostic marker for predicting presence of colorectal growths, stage of disease and/or relapse. Supported by NCI grants CA97959 and CA114264 to PS.

654 Quantitative Targeted Wide Area In Vivo Imaging of Neoplasia in Barrett's Esophagus

Introduction and aim: Colonoscopy is considered to be the most sensitive tool to detect colorectal neoplasms, including small and flat lesions. Nevertheless, detection of such lesions is still incomplete, giving rise to interval cancers, particularly in patients with Lynch syndrome and other hereditary disorders. To improve the detection of (pre)malignant lesions nearinfrared endoscopy, guided bymolecular targeted fluorescent tracers, may hold great promise. Therefore, we developed a combined white-light and near-infrared (WLNI) endoscopic system, as well as two clinically approved fluorescent labeled antibodies, targeting vascular endothelial growth factor-A (VEGF-A) and epithelial growth factor receptor (EGFR). In the present study we investigated VEGF-A and EGFR expression in adenomas and CRC of Lynch patients, as potential targets for (pre)malignant detection. Subsequently we validated the newly developed WLNI endoscopic system, together with intravenously injected fluorescent anti-VEGF-A and anti-EGFR antibodies, in human CRC mouse models. Materials and Methods: VEGF-A and EGFR expression was analyzed by immunohistochemical staining of 34 CRC, 64 high-grade dysplastic (HGD) adenomas, and adjacent normal colon crypts of Lynch patients. The endoscopic system consisted of a custom made clinically approved WLNI camera, comprising a color camera and an ultra-sensitive camera for concurrent white-light and NIR fluorescence acquisition, attached to either a clinical fiberscope or a multi-modal fiber-optic bundle. The WLNI system was evaluated in vivo in CRC subcutaneous (sc) and intraperitoneal (ip) mouse models of bioluminescent CRC cell lines (HCT116-luc, HT29luc2), using bevacizumab-800CW (anti-VEGF-A) and cetuximab-800CW (anti-EGFR). Results: VEGF-A and EGFR were significantly overexpressed in 95% and 74% of HGD adenomas and in 100% and 85% of CRC, compared to adjacent normal crypts (resp. P,0.001/P,0.05 and P,0.05/P,0.001). Tumor-to-background ratio was high (3.2±0.9 for bevacizumab-800CW and 5.7±2.9 for cetuximab-800CW in the HCT116-luc sc model). WLNI endoscopy demonstrated excellent instant visualization of the sc and ip tumors (diameter ≥1 mm), with clear tumor boundaries and a low background fluorescence, demonstrating very high sensitivity and specificity of our WLNI endoscopic system. Conclusion: VEGF-A and EGFR are attractive targets for molecular targeted fluorescence endoscopy in Lynch patients based on their expression patterns. The newly developed WLNI endoscopic system using clinically approved molecular targeted fluorescent antibodies, enables instant visualization of very small tumor lesions in CRC mice models, with an excellent sensitivity and specificity. These results support clinical evaluation of WLNI endoscopy, in order to enhance early detection of colorectal (pre)malignancies and improve potentially outcome in patients.