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To further clarify and/or develop calcium and vitamin D as chemopreventive agents against colorectal cancer in humans, understand the mechanisms by which these agents reduce risk for disease, and develop ‘treatable’ biomarkers of risk for colorectal cancer, a pilot, randomized, double-blind, placebo-controlled, 2x2 factorial clinical trial is being conducted. This clinical trial will test the effects of calcium and vitamin D3, alone and in combination, on the expression of transforming growth factor- (TGF-α) in the normal colorectal mucosa. A stimulatory growth factor and mediator of oncogenesis and malignant progression in colorectal carcinogenesis, TGF-α is a potential growth-related biomarker of risk for colorectal cancer. In this study, 112 men and women with at least one pathology-confirmed colorectal adenoma were treated with calcium 1,200 mg/day or vitamin D3 1,000 IU/day, alone or in combination versus placebo over twelve months. The overall expression and colorectal crypt distributions of TGF-α in biopsies of normal-appearing rectal mucosa will be detected by automated immunohistochemistry and quantified by image analysis.
The Big Picture
Despite advances in screening and treatment, mortality due to colorectal cancer, the second leading cause of cancer deaths in the U.S., has declined only modestly over the past 50 years (1). A similar situation existed with ischemic heart disease nearly three decades ago. The establishment of biological measurements as markers of risk for the disease, including lipid profiles and blood pressures, allowed for individual and population control of the “biomarkers”, and mortality rates from the disease began a dramatic decline which continues today (2). Using biological measurements of risk should likewise result in a decline in colorectal cancer incidence and mortality.
Transforming growth factor-α (TGF-α): A plausible biomarker of risk for colon cancer
Autocrine-paracrine stimulatory growth factor that plays role in proliferation, cell survival, cellular integrity, and injury/inflammation response pathways in gut mucosa. Mediator of oncogenesis and malignant progression in colorectal cancer
The Treatment: Calcium & Vitamin D
Paleolithic calcium intake for Homo Sapiens was twice as much as average American intake (1500-2000 mg/day vs. 740 mg/day). Since only 30% of consumed calcium is absorbed, humans evolved with much higher level of calcium in the colon than modern levels (4)
More than half of all Americans and Europeans have 25-OH-vitamin D levels below optimal range, unlike the Paleolithic population
Both calcium and vitamin D influence bile acid metabolism, affect genes/proteins in colon carcinogenic pathways, and modulate cell proliferation and differentiation
Of 42 observational studies of colorectal cancer and calcium, 30 (71%) found inverse associations (16 statistically significant)
Of 20 cohort, 18 (90%) found inverse associations (8 statistically significant)
Of 25 reported analytic observational studies of dietary vitamin D and colorectal cancer, 15 (60%) found inverse associations
All 5 reported analytic observational studies of 25-OH-vitamin D blood levels and colorectal cancer found inverse associations
Protocol for Quantifying Biomarkers in Normal Colon Crypts (“Scoring”)
Immunohistochemical processing of biopsies by a labeled streptavidin-biotin method was accomplished using a DAKO Automated Stainer (DAKO).
Stained biopsy slides are converted into high quality digitized slides using a Scanscope by Aperio Technologies, Inc.
Once digitized, a customized software program, Cellular Eyes by Diveyes LLC, is used to quantify the staining density of immunohistochemically detected TGF-α. The unit of analysis is the “hemicrypt,” defined as one-half of a crypt bisected from the crypt base to colon lumen. A “scorable” hemicrypt is defined as an intact hemicrypt that extends from the muscularis mucosa to the colon lumen.
Data Analysis
Treatment effects will be evaluated by assessing the differences in the transformed densities from baseline to the 12-month follow-up visit between patients in each active treatment group
Treatment groups will be assessed for comparability at baseline and follow-up by the Fisher’s exact test for categorical variables and analysis of variance (ANOVA) for continuous variables.
Mean density of TGF-α staining in normal colon crypts will be calculated for each patient at baseline and 12-month follow-up
Ratio of TGF-α expression in the upper 40% (differentiation zone) to the lower 60% (proliferation zone), and the ratio of expression in the upper 20% (closest to colon lumen contents) to the lower 20% (furthest from colon lumen contents) will be calculated because they represent the ratios of well recognized functional or exposure zones
There are currently no generally accepted pre-neoplastic biomarkers of risk for colorectal cancer. Colorectal cancer is a complex, multifactorial disease, which, like IHD, will require a multifactorial preventive approach and a panel of biomarkers to describe phenotypes from which to categorize and quantify risk
Based on previous studies, TGF-α expression in the normal-appearing rectal mucosa shows promise as an early, potentially modifiable biomarker of risk for colorectal cancer
There is strong biological plausibility and animal experimental and human evidence for protection against colorectal neoplasms by calcium and vitamin D
We anticipate calcium and vitamin D having beneficial effects on TGF-α expression in the normal-appearing colorectal mucosa
This material is based upon work supported by a fellowship award by the Atlanta Clinical and Translational Science Institute (ACTSI), the Howard Hughes Medical Institute under Grant No.52005873 and by the National Cancer Institute Grant # R01 CA114456
1. Potter JD, Slattery ML, Bostick RM, Gapstur SM. Colon Cancer: A Review of the Epidemiology. Epidemiol Rev
1993;15 (2):499-545
2. Fraser G. Preventive Cardiology. New York, NY: Oxford University Press; 1986.
3. Daniel, C et al. TGF- expression as a potential biomarker of risk within the normal-appearing colorectal mucosa of
patients with and without Incident sporadic adenoma. Cancer Epidemiology Biomarkers & Prevention Jan. 2009;18, 65
4. Eaton, S. B., Shostak, M., and Konner, M. The Paleolithic Prescription. New York: Harper and Row;1988.
5. Potter J. and Lindor N. The Genetics of Colorectal Cancer. Springer New York. 2009; 277-298.
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