Luz Rodriguez

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.

Randomized phase II trial of sulindac, atorvastatin, and prebiotic dietary fiber for colorectal cancer chemoprevention.

Sulindac, atorvastatin, or prebiotic dietary fiber may reduce colorectal cancer (CRC) risk. However, clinical trial data are currently limited. We conducted a randomized, phase II chemoprevention trial involving subjects 40 years or older, with previously resected colon cancer or multiple/advanced colorectal adenomas. Magnification chromoendoscopy (MCE) was performed to identify and characterize rectal aberrant crypt foci (ACF); eligibility criteria required five or more rectal ACFs at baseline. Intervention assignments were as follows: (a) atorvastatin 20 mg qd; (b) sulindac 150 mg bid; (c) oligofructose-enriched inulin (as ORAFTI®Synergy1) 6 gm bid; or (d) control (maltodextrin) 6 gm bid, for 6 months. Percent change in rectal ACF number (%ΔACF) within arm was the primary endpoint. Secondary endpoints included changes in proliferation (Ki67) and apoptosis (caspase-3), as measured from normal mucosa biopsy samples. Among 85 eligible randomized subjects, 76 (86%) completed the trial per protocol. The median (range) of rectal ACF was 9 (5–34) and 8 (0–37) at baseline and postintervention, respectively. The median (SD) for %ΔACF was 5.6 (−69% to 143%), −18.6 (−83% to 160%), −3.6 (−88% to 83%), and −10.0 (−100% to 117%) in the atorvastatin, sulindac, ORAFTI®Synergy1 and control arms, respectively. Neither within-arm (P = 0.12–0.59) nor between-arm (P = 0.30–0.92) comparisons of %ΔACF were statistically significant. The active and control interventions also seemed to have similar effects on mucosal proliferation and apoptosis (P > 0.05 for each comparison). Data from this multicenter, phase II trial do not provide convincing evidence of CRC risk reduction from 6-month interventions with atorvastatin, sulindac, or ORAFTI®Synergy1, although statistical power was limited by the relatively small sample size. Cancer Prev Res; 4(2); 259–69. ©2011 AACR.

Gene Expression Patterns Distinguish Colonoscopically Isolated Human Aberrant Crypt Foci from Normal Colonic Mucosa

Aberrant crypt foci (ACF) are considered the earliest identifiable preneoplastic colonic lesions; thus, a greater understanding of the nature of genetic changes underlying the transformation of normal colonic mucosa (NM) into ACF may provide insight into the mechanisms of carcinogenesis. ACF were identified by indigo carmine spraying onto colonic mucosa during colonoscopy and isolated as standard pinch biopsies of the mucosal areas containing the ACF. RNAs isolated from ACF and matched NM biopsies from the ascending and descending colons of 13 patients were analyzed on arrays containing 9128 cDNAs. Thirty-four differentially expressed (P < 0.001) genes were found in a paired comparison of the ACF and NM samples, and 25 of 26 matched pairs of ACF and NM could be correctly classified in leave-one-out cross-validation. Differential expression for seven of eight genes was confirmed by real-time reverse transcription-PCR. Furthermore, ACF and NM samples, including six pairs of ACF and NM samples that had not previously been analyzed by array hybridization, can be correctly classified on the basis of the overexpression in ACF of three selected genes (REG4, SRPN-B5, and TRIM29) evaluated by real-time reverse transcription-PCR. In a separate analysis of 13 biopsy pairs from either ascending or descending colon, ACF and NM samples could also be correctly classified by the gene expression patterns. Analysis of gene expression differences in ACF from the ascending and descending colon versus NM samples indicates that ACF from these distinct colonic locations are converging toward similar gene expression profiles and losing differences in gene expression characteristic of NM from the ascending versus descending colon. (Cancer Epidemiol Biomarkers Prev 2006;15(11):2253–62)

Celecoxib treatment alters the gene expression profile of normal colonic mucosa.

A clinical trial was recently conducted to evaluate the safety and efficacy of a selective inhibitor of cyclooxygenase-2 (celecoxib) in hereditary nonpolyposis colon cancer patients. In a randomized, placebo-controlled phase I/II multicenter trial, hereditary nonpolyposis colon cancer patients and gene carriers received either celecoxib at one of two doses or placebo. The goal was to evaluate the effects of these treatment arms on a number of endoscopic and tissue-based biomarker end points after 12 months of treatment. As part of this trial, we analyzed gene expression by cDNA array technology in normal descending (rectal) colonic mucosa of patients before and after treatment with celecoxib or placebo. We found that treatment of patients with celecoxib at recommended clinical doses (200 and 400 mg p.o. bid), in contrast to treatment with placebo, leads to changes in expression of >1,400 genes in the healthy colon, although in general, the magnitude of changes is <2-fold. Twenty-three of 25 pairs of colon biopsies taken before and after celecoxib treatment can be classified correctly by the pattern of gene expression in a leave-one-out cross-validation. Immune response, particularly T- and B-lymphocyte activation and early steps of inflammatory reaction, cell signaling and cell adhesion, response to stress, transforming growth factor-β signaling, and regulation of apoptosis, are the main biological processes targeted by celecoxib as shown by overrepresentation analysis of the distribution of celecoxib-affected genes across Gene Ontology categories. Analysis of possible cumulative effects of celecoxib-induced changes in gene expression indicates that in healthy colon, celecoxib may suppress the immune response and early steps of inflammation, inhibit formation of focal contacts, and stimulate transforming growth factor-β signaling. (Cancer Epidemiol Biomarkers Prev 2006;15(7):1382–91)

Metformin does not reduce markers of cell proliferation in esophageal tissues of patients with Barrett's esophagus.

BACKGROUND & AIMS Obesity is associated with neoplasia, possibly via insulin-mediated cell pathways that affect cell proliferation. Metformin has been proposed to protect against obesity-associated cancers by decreasing serum insulin. We conducted a randomized, double-blind, placebo-controlled, phase 2 study of patients with Barretts esophagus (BE) to assess the effect of metformin on phosphorylated S6 kinase (pS6K1), a biomarker of insulin pathway activation. METHODS Seventy-four subjects with BE (mean age, 58.7 years; 58 men [78%; 52 with BE >2 cm [70%]) were recruited through 8 participating organizations of the Cancer Prevention Network. Participants were randomly assigned to groups given metformin daily (increasing to 2000 mg/day by week 4, n = 38) or placebo (n = 36) for 12 weeks. Biopsy specimens were collected at baseline and at week 12 via esophagogastroduodenoscopy. We calculated and compared percent changes in median levels of pS6K1 between subjects given metformin vs placebo as the primary end point. RESULTS The percent change in median level of pS6K1 did not differ significantly between groups (1.4% among subjects given metformin vs -14.7% among subjects given placebo; 1-sided P = .80). Metformin was associated with an almost significant reduction in serum levels of insulin (median -4.7% among subjects given metformin vs 23.6% increase among those given placebo, P = .08) as well as in homeostatic model assessments of insulin resistance (median -7.2% among subjects given metformin vs 38% increase among those given placebo, P = .06). Metformin had no effects on cell proliferation (on the basis of assays for KI67) or apoptosis (on the basis of levels of caspase 3). CONCLUSIONS In a chemoprevention trial of patients with BE, daily administration of metformin for 12 weeks, compared with placebo, did not cause major reductions in esophageal levels of pS6K1. Although metformin reduced serum levels of insulin and insulin resistance, it did not discernibly alter epithelial proliferation or apoptosis in esophageal tissues. These findings do not support metformin as a chemopreventive agent for BE-associated carcinogenesis. ClinicalTrials.gov number, NCT01447927.

Spectral biomarkers for chemoprevention of colonic neoplasia: a placebo-controlled double-blinded trial with aspirin

Objective A major impediment to translating chemoprevention to clinical practice has been lack of intermediate biomarkers. We previously reported that rectal interrogation with low-coherence enhanced backscattering spectroscopy (LEBS) detected microarchitectural manifestations of field carcinogenesis. We now wanted to ascertain if reversion of two LEBS markers spectral slope (SPEC) and fractal dimension (FRAC) could serve as a marker for chemopreventive efficacy. Design We conducted a multicentre, prospective, randomised, double-blind placebo-controlled, clinical trial in subjects with a history of colonic neoplasia who manifested altered SPEC/FRAC in histologically normal colonic mucosa. Subjects (n=79) were randomised to 325 mg aspirin or placebo. The primary endpoint changed in FRAC and SPEC spectral markers after 3 months. Mucosal levels of prostaglandin E2 (PGE2) and UDP-glucuronosyltransferase (UGT)1A6 genotypes were planned secondary endpoints. Results At 3 months, the aspirin group manifested alterations in SPEC (48.9%, p=0.055) and FRAC (55.4%, p=0.200) with the direction towards non-neoplastic status. As a measure of aspirins pharmacological efficacy, we assessed changes in rectal PGE2 levels and noted that it correlated with SPEC and FRAC alterations (R=−0.55, p=0.01 and R=0.57, p=0.009, respectively) whereas there was no significant correlation in placebo specimens. While UGT1A6 subgroup analysis did not achieve statistical significance, the changes in SPEC and FRAC to a less neoplastic direction occurred only in the variant consonant with epidemiological evidence of chemoprevention. Conclusions We provide the first proof of concept, albeit somewhat underpowered, that spectral markers reversion mirrors antineoplastic efficacy providing a potential modality for titration of agent type/dose to optimise chemopreventive strategies in clinical practice. Trial Number NCT00468910

Hereditary cancer syndromes as model systems for chemopreventive agent development

Research in chemoprevention has undergone a shift in emphasis for pragmatic reasons from large, phase III randomized studies to earlier phase studies focused on safety, mechanisms, and utilization of surrogate endpoints such as biomarkers instead of cancer incidence. This transition permits trials to be conducted in smaller populations and at substantially reduced costs while still yielding valuable information. This article will summarize some of the current chemoprevention challenges and the justification for the use of animal models to facilitate identification and testing of chemopreventive agents as illustrated though four inherited cancer syndromes. Preclinical models of inherited cancer syndromes serve as prototypical systems in which chemopreventive agents can be developed for ultimate application to both the sporadic and inherited cancer settings.

Abstract A21: A Phase IIa trial of metformin for colorectal cancer risk reduction among patients with a history of colorectal adenomas and elevated body mass index

Background: Despite advances in colorectal cancer (CRC) screening, early detection, and treatment, CRC remains the 2nd most common cancer cause of death in the U.S.. Obesity is increasing in incidence in the U.S., and has been implicated in colorectal adenoma (CRA) risk, risk of CRA recurrence, and risk of CRC. Obese patients with history of CRA are a high-risk group that may benefit from novel CRC prevention strategies. There is early evidence for reduced cancer mortality among metformin users. The signaling pathway activated by metformin (LKB1/AMPK/mTOR) is implicated in tumor suppression in ApcMin/+ mice, as evidenced by metformin-induced reduction in polyp burden, increased ratio of pAMPK/AMPk, decreased ratio of pmTOR/mTOR, and decreased ratio of pS6Ser235/S6Ser235 in polyp specimens. We hypothesized that metformin would affect colorectal tissue S6Ser235 similarly in humans, targeting obese patients with recent history of CRAs as a high-risk group. Methods: A phase IIa clinical biomarker trial was conducted across 3 clinical sites via the NCI-funded Southern California Chemoprevention Program. Eligible participants included non-diabetic, obese patients (BMI >30) with history of colorectal adenoma within the past 3 years, age ≥ 35 years and ≤ 80 years. All patients received an upward titration of metformin over 3 weeks to 1000mg po bid, which was continued until the end-of-study (EOS) at 12 weeks. Rectal mucosa biopsies were obtained at baseline (BL) and at time of EOS endoscopy. Tissue S6Ser235 immunostaining was analyzed in a blinded fashion by the study pathologist using Histo Score (HScore) analysis. A paired t-test was used to examine the effect of metformin on activated S6serine235 (i.e., the ratio of pS6serine235/ S6serine235). Results: 45 patients were consented to achieve 32 eligible subjects. 4 subjects were removed from study due to Adverse Events (1 SAE, unrelated). In order of frequency, the most common AEs were diarrhea, cramping, flatulence, nausea, stomach pain; 80% of participants had Grade 1 AE, 27% had grade 2 AE. Mean (SD) weight and body mass index at BL were 105.2 (17.42) kg and 34.9 (5.57) respectively. Weight did not significantly differ over the course of the study. Glucose levels at EOS did not significantly differ from BL. Vitamin B12 levels were significantly reduced at EOS vs. BL (-46.7 pg/mL, 95% CI -73.2 to -20.2). Comparing EOS to BL tissue S6Ser235 by IHC HScore analysis, no significant differences were observed. Mean (SD) Hscore at BL was 1.1 (0.57) and 1.1 (0.51) at EOS. Median HScore change was 0.032 (p=0.77). Conclusions: Among obese CRA patients, 12 weeks of oral metformin 1000mg twice daily does not reduce pS6 levels in the rectal mucosa. Other potential mechanisms of action have not yet been analyzed. Data from this clinical trial indicate that metformin can be used safely in a non-diabetic population. Further research is needed to determine what effects, if any, metformin has on the target tissue of origin (colorectum) relevant to colorectal carcinogenesis if metformin is to be pursued as a CRC chemopreventive agent. Citation Format: Jason A. Zell, Christine E. McLaren, Timothy R. Morgan, Michael J. Lawson, Sherif Rezk, Gregory C. Albers, Wen-Pin Chen, Joseph C. Carmichael, Luz Rodriguez, Eva Szabo, Leslie Ford, Michael Pollak, Frank L. Meyskens. A Phase IIa trial of metformin for colorectal cancer risk reduction among patients with a history of colorectal adenomas and elevated body mass index. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A21.

Abstract 5402: Synergism of Metformin and DFMO on colon cancer proliferation and migration.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Metformin has been shown to inhibit cancer cell growth via activation of MAP kinase with resultant inhibition of mTOR signaling pathway. ODC, a key enzyme in polyamine biosynthesis, was found to be overexpressed in colon cancer. We determined the effect of metformin in combination with DMFO, an ODC inhibitor, on tumor proliferation and migration in human colon cancer cells. Methods: Human colon cancer cells, HCT 116 and HT 29, were treated with Metformin, DFMO, or combination. Cell proliferation assay was performed using Cell Titer Blue proliferation assay. Cell migration assay was performed by seeding colon cancer cells in 24-well plate. After overnight incubation, an artificial homogenous wound was created onto the monolayer with a plastic micropipette tip. After wounding, the migration of cells into the wound was observed up to 72 hours at various time points. Quantitative RT-PCR for ODC1 detection was performed using Assay-on-Demand Hs00159739-ml. GAPDH served as control. Western blotting was performed to determine the expression of AMP kinase, mTOR, p70S6K and 4E-BP1. Data were presented as means ± SD for the three separate experiments. For comparison between groups, the students t test was used and p< 0.05 was considered to be statically significant. Results: There was dose-dependent (1-10 mM for DFMO and 1-20 mM for metformin) time-dependent (up to 96 hours) inhibition of cell proliferation and migration with either metformin or DFMO in both HCT 116 and HT 29 cells. Synergistic inhibition was observed with the combination treatment (5 mM DFMO and 5 mM Metformin) on both cell migration and proliferation at 24 hours compared with either agent alone. There was synergistic inhibition of ODC expression with the combined treatment compared with DFMO alone at 24 hours (p<0.01). The combination treatment also lead to significant enhancement of AMPK activation and increased phosphorylation of AMPKα at Thr-172 compared with metformin alone (p<0.01). The combination treatment resulted in significant inhibition of mTOR signaling with decreased phosphorylation of mTOR, p70S6K and 4E-BP1 in treated cancer cells, when compared with individual drug treatments. Inhibition of proliferation and migration was significantly greater in the mismatch-repair defective HCT 116 cells than HT 29. Conclusion: We have observed a synergistic effect of metformin and DFMO in suppressing human colon cancer proliferation and migration. The synergism reflects enhanced inhibition of both polyamine synthesis and mTOR signaling pathways by the drug combination. Citation Format: Yanping Zhang, Luz M. Rodriguez, Ronald Lubet, Eddy C. Hsueh. Synergism of Metformin and DFMO on colon cancer proliferation and migration. [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 5402. doi:10.1158/1538-7445.AM2013-5402

Prevention of colonic neoplasia with polyethylene glycol: A short term randomized placebo-controlled double-blinded trial

Chemoprevention represents an attractive modality against colorectal cancer (CRC) although widespread clinical implementation of promising agents (e.g. aspirin/NSAIDS) have been stymied by both suboptimal efficacy and concerns over toxicity. This highlights the need for better agents. Several groups, including our own, have reported that the over-the-counter laxative polyethylene glycol (PEG) has remarkable efficacy in rodent models of colon carcinogenesis. In this study, we undertook the first randomized human trial to address the role of PEG in prevention of human colonic neoplasia. This was a double-blind, placebo-controlled, three-arm trial where eligible subjects were randomized to 8g PEG-3350 (n = 27) or 17g PEG-3350 (n = 24), or placebo (n = 24; maltodextrin) orally for a duration of six months. Our initial primary endpoint was rectal aberrant crypt foci (ACF) but this was changed during protocol period to rectal mucosal epidermal growth factor receptor (EGFR). Of the 87 patients randomized, 48 completed study primary endpoints and rectal EGFR unchanged PEG treatment. Rectal ACF had a trend suggesting potentially reduction with PEG treatment (pre-post change 1.7 in placebo versus -0.3 in PEG 8+ 17g doses, p = 0.108). Other endpoints (proliferation, apoptosis, expression of SNAIL and E-cadherin), previously noted to be modulated in rodent models, appeared unchanged with PEG treatment in this clinical trial. We conclude that PEG was generally well tolerated with the trial failing to meet primary efficacy endpoints. However, rectal ACFs demonstrated a trend (albeit statistically insignificant) for suppression with PEG. Moreover, all molecular assays including EGFR were unaltered with PEG underscoring issues with lack of translatability of biomarkers from preclinical to clinical trials. This data may provide the impetus for future clinical trials on PEG using more robust biomarkers of chemoprevention. Trial registration: ClinicalTrials.gov NCT00828984