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Supplementary Figures 1-4 from A Randomized Clinical Trial of the Effects of Supplemental Calcium and Vitamin D3 on Markers of Their Metabolism in Normal Mucosa of Colorectal Adenoma Patients

Supplementary References from A Randomized Clinical Trial of the Effects of Supplemental Calcium and Vitamin D3 on Markers of Their Metabolism in Normal Mucosa of Colorectal Adenoma Patients

In cancer cell lines and rodent models, calcium and vitamin D favorably modulate cell proliferation, differentiation, and apoptosis in colonic epithelia. These effects may be modulated by local expression of the calcium receptor (CaR), the vitamin D receptor (VDR), and the P450 cytochromes, CYP27B1 and CYP24A1; however, they have yet to be investigated in humans. To address this gap, we conducted a randomized, double-blinded, placebo-controlled 2 × 2 factorial clinical trial. Patients with at least one pathology-confirmed colorectal adenoma were treated with 2 g/d elemental calcium and/or 800 IU/d vitamin D3 versus placebo over 6 months (n = 92; 23 per group). CaR, VDR, CYP27B1, and CYP24A1 expression and distribution in biopsies of normal appearing rectal mucosa were detected by standardized, automated immunohistochemistry and quantified by image analysis. In the calcium-supplemented group, CaR expression increased 27% (P = 0.03) and CYP24A1 expression decreased 21% (P = 0.79). In the vitamin D3-supplemented group, CaR expression increased 39% (P = 0.01) and CYP27B1 expression increased 159% (P = 0.06). In patients supplemented with both calcium and vitamin D3, VDR expression increased 19% (P = 0.13) and CaR expression increased 24% (P = 0.05). These results provide mechanistic support for further investigation of calcium and vitamin D3 as chemopreventive agents against colorectal neoplasms, and CaR, VDR, CYP27B1, and CYP24A1 as modifiable, preneoplastic risk biomarkers for colorectal neoplasms. Cancer Res; 71(2); 413–23. ©2010 AACR.

Supplementary Figure Legends 1-4 from A Randomized Clinical Trial of the Effects of Supplemental Calcium and Vitamin D3 on Markers of Their Metabolism in Normal Mucosa of Colorectal Adenoma Patients

Contains fulltext : 291332.pdf (Publisher’s version ) (Open Access) INTRODUCTION: The 2019 European Society of Cardiology and European Atherosclerosis Society (2019 ESC/EAS) guidelines stress the importance of managing low-density lipoprotein cholesterol (LDL-C) after myocardial infarction (MI) to reduce the risk of cardiovascular events. Information on guideline implementation is limited. The aim of this survey was to describe current clinical practice regarding LDL-C management in the first year post-MI across Europe, improving understanding of the role of ESC/EAS guidelines on clinical practice. METHODS: A qualitative web-based cross-sectional physician survey about the patient pathway and LDL-C management post-MI was conducted in 360 physicians from France, Italy, Germany, The Netherlands, Spain, and the UK (n = 60/country) between December 2019 and June 2020. Secondary and primary care physicians (SCPs/PCPs) described their experiences treating patients post-MI over the preceding 2 months. RESULTS: Physicians reported that on average 90.7% of patients not prescribed lipid-lowering therapy (LLT) before an MI initiated LLT as inpatients; for patients already taking LLT, treatment was intensified for 64.7% of inpatients post-MI. SCPs reported prescribing higher-intensity statins and/or ezetimibe for between 72.3% (Italy) and 88.6% (UK) of patients post-MI. More than 80.0% of SCPs and 51.2% of PCPs stated that they would initiate a change in LLT immediately if patients did not achieve their LDL-C treatment goal by 12 weeks post-MI; 82.0% of SCPs and 55.1% of PCPs reported referring to 2019 ESC/EAS guidelines for management of patients post-MI. Barriers to initiating PCSK9 inhibitors (PCSK9is) included prior prescription of a maximally tolerated dose of statin (49.4%) and/or ezetimibe (38.9%), requirement to reach threshold LDL-C levels (44.9%), and pre-authorization requirements (30.4%). CONCLUSION: Differences in clinical practice post-MI were reported across the countries surveyed, including divergence between 2019 ESC/EAS and local guidelines. Increased use of innovative medicines to achieve LDL-C goals should reduce risk of subsequent cardiovascular events in very high-risk patients post-MI. 01 januari 2023

Introduction Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of mortality in Italy, accounting for 22% of total deaths. Lowering low-density lipoprotein cholesterol (LDL-C) levels reduces the risk of cardiovascular (CV) events; thus, lipid-lowering therapy (LLT) is the first-line treatment for patients with ASCVD and hypercholesterolaemia. However, many patients with ASCVD fail to reach LDL-C treatment thresholds, leaving them at greater risk of CV events. Inpatient care accounts for 51% of total expenditure on cardiovascular disease in the European Union, but healthcare resource utilization (HCRU) data for ASCVD in Italy is limited. Methods The study analysed healthcare claims data for 17,881 patients with acute coronary syndrome, ischemic stroke or peripheral artery disease from the Umbria 2 and Marche regions of Italy. LLT treatment patterns and CV event rates were collected and HCRU estimated in the year before and after the index event. Results High-intensity LLTs were prescribed to 44.3% of patients and 49.6% received moderate-/low-intensity LLTs during the 6 months after the index event. The first year CV event rate was 18.0/100 patient-years for patients receiving high-intensity LLTs and 17.2/100 patient-years for those on moderate-/low-intensity LLTs. Higher costs were associated with patients untreated with LLT 6 months post-index event (€8323) than patients prescribed high-intensity (€6278) or moderate-/low-intensity LLTs (€6270). Hospitalization accounted for most of the total costs. Conclusions This study found that CV events in secondary prevention Italian patients are associated with substantial HCRU and costs. More intensive LDL-C lowering can prevent CV events, easing the financial burden on the healthcare system. Supplementary Information The online version contains supplementary material available at 10.1007/s12325-021-01960-y.

Purpose Many patients at very-high risk of cardiovascular (CV) events would benefit from lipid lowering therapies (LLT) intensification to decrease their risk. This study aimed to identify the real-world secondary prevention patients potentially eligible for proprotein convertase subtilisin–kexin type 9 inhibitors (PCSK9i) in Spain. Methods Retrospective cohort study included adult patients registered in the IQVIA Spanish Electronic Medical Records outpatient database (2014–2020), diagnosed with myocardial infarction (MI), unstable angina (UA), ischaemic stroke (IS), transient ischaemic attack (TIA), or peripheral artery disease (PAD) and with ≥ 1 low-density lipoprotein cholesterol (LDL-C) or total cholesterol measurements. Longitudinal data were collected from the initial diagnosis to the end of the study period or follow-up loss. Results The study included 9,516 patients, 63.9% male, mean (SD) age 67.7 (12.5) years and mean LDL-C 117.3 (38.8) mg/dL. MI, IS, and PAD were the most severe events reported, during the study period (28.5%,18.7%, and 29.3% of patients, respectively). At the time of last available LDL-C assessment (≥ 3 months post-event), 64.4% patients were on LLT. Of those, 45.4% were on high-, 46.9% on moderate-, and 7.7% on low-intensity LLT. Overall, 9.6% patients achieved LDL-C 100 mg/dL (LDL-C reimbursement threshold for PCSK9i in Spain). Conclusion Up to 82% of atherosclerotic CV disease patients do not achieve LDL-C levels recommended by the 2019 ESC/EAS guidelines despite being on optimized LLT therapy. In 17.9% of these patients LDL-C levels exceed 100 mg/dL, being eligible for PCSK9i in Spain.

Introduction Patients with a history of myocardial infarction (MI) are at very high risk of subsequent cardiovascular events. This study evaluated the association of treatment intensity and adherence to lipid-lowering therapies (LLT) with major adverse cardiovascular events (MACE) among post-MI patients in Germany. Methods We carried out a retrospective cohort study using German health claims data (2010–2015). We included patients ≥ 18 years, with a history of MI and who started an LLT (statin and/or ezetimibe), between 2011 and 2013. The follow-up period started 1 year after the second LLT prescription and continued until MACE, all-cause death or December 31, 2015, whichever occurred first. Treatment intensity was classified based on expected low-density lipoprotein cholesterol reduction; adherence was measured by the proportion of days covered using prescription data. A combined adherence-adjusted intensity variable was created by multiplying intensity and adherence. We used Cox proportional hazards models to control for age, sex, Charlson Comorbidity Index and other cardiovascular risk factors at baseline. Results A total of 14,944 patients were included. Mean age was 66.7 (SD = 13.0) years; 68.7% of patients were men. Each 10% increase in treatment intensity, adherence, or adherence-adjusted intensity was associated with a decrease in the risk of MACE of 17% (HR = 0.83, 95% CI 0.79–0.87), 5% (HR = 0.95, 95% CI 0.94–0.97), and 14% (HR = 0.86, 95% CI 0.83–0.90), respectively. Conclusions Higher treatment intensity and/or adherence of LLT was associated with significantly lower risk of MACE in post-MI patients. Strategies to tailor intensity to patient profiles and improve adherence could reduce the risk of cardiovascular events. Supplementary Information The online version contains supplementary material available at 10.1007/s12325-021-01697-8.

ObjectivesTo compare treatment patterns, risk factors and cardiovascular disease (CVD) event rates in the UK from 2008 to 2017.DesignRetrospective cohort study using the Clinical Practice Research Datalink.SettingUK primary care.ParticipantsWe selected 10 annual cohorts of patients with documented CVD receiving lipid-lowering therapy and the subsets with myocardial infarction (MI). Each cohort included patients ≥18 years old, with ≥1 year of medical history and ≥2 lipid-lowering therapy prescriptions in the prior year.Primary and secondary outcome measuresFor each annual cohort, we identified cardiovascular risk factors and lipid-lowering therapy and estimated the 1-year composite rate of fatal and non-fatal MI, ischaemic stroke (IS) or revascularisation.ResultsThe documented CVD cohort mean age was 71.6 years in 2008 (N=173 424) and 72.5 (N=94 418) in 2017; in the MI subset, mean age was 70.1 years in 2008 (N=38 999) and 70.4 in 2017 (N=25 900). Both populations had larger proportions of men. In the documented CVD cohort, the proportion receiving high-intensity lipid-lowering therapy from 2008 to 2017 doubled from 16% to 32%; in the MI subset, the increase was 20% to 48%. In the documented CVD cohort, the proportion of patients with low-density lipoprotein cholesterol (LDL-C) ConclusionsDespite an increase in high-intensity therapy use and a decline in revascularisation, more than half of patients did not receive high-intensity lipid-lowering therapy by 2017 and incidence rates of MI and IS remained virtually unchanged.

Background: Cardiovascular (CV) diseases are the leading cause of mortality globally. Reducing low-density lipoprotein cholesterol (LDL-C) is a proven strategy to decrease risk of acute CV events. The study aimed to estimate a PCSK9i-eligible population in Spain. Methods: We conducted a retrospective cohort study based on real-world data from the IQVIA Spanish Electronic Medical Records primary care database. Adult (≥18 y.o.) patients registered in the database between 2014 and 2020, diagnosed with myocardial infarction (MI), unstable angina (UA), ischemic stroke (IS), transient ischemic attack (TIA) or peripheral artery disease (PAD) and with at least 1 measurement of LDL-C or total cholesterol were included in the study and followed up for up to 12 months from the initial diagnosis. Results: The study included 9,516 patients; mean (SD) age was 67.7 (12.5) years, 63.9% were male and mean LDL-C was 117.3 (38.8) mg/dL. In the study population 28.5% of patients suffered an MI as most severe event during the study period, 10.0% had UA, 18.7% were diagnosed with IS, 13.6% with TIA, and 29.3% with PAD. At the time of last available LDL-C assessment (≥3 months after diagnosis), 64.4% of patients were on lipid lowering therapy (LLT). Of those, 45.4% were on high-intensity LLT (high-intensity statins with/without ezetimibe or PCSK9 inhibitors), 46.9% on moderate-intensity statins, and 7.7% on low-Intensity LLT (low-intensity statins, ezetimibe alone or non-statin LLT). Overall, 12.9% of treated patients achieved LDL-C levels 100 mg/dL (LDL-C reimbursement threshold for PCSK9i in Spain). Conclusions: More than 85% of patients at very-high risk of cardiovascular events in our cohort do not achieve LDL-C 100 mg/dL despite being treated with maximally tolerated LLT and, therefore, would be eligible for using PCSK9i within Spanish reimbursement criteria.

Background and aims Statins are the standard of care for patients with dyslipidemia, but some patients develop intolerance to treatment. The experience of statin intolerant (SI) patients is poorly understood. The objective of this study was to identify key aspects of SI that may be associated with treatment discontinuation. Methods Using a previously created questionnaire, we conducted a pilot cross-sectional survey to identify items important for describing patient-centric aspects of SI. The study recruited adult (18+) patients with a history of statin-associated side effects from 9 clinics in 6 countries (France, Italy, Norway, Spain, Sweden, and the United Kingdom), who had received statin treatment within the previous two years. Results We surveyed 104 patients (mean age 61.5 SD=11.2 years, 63.5% men, 50% currently on statins). Patients most frequently reported muscle-related symptoms: pain (90.9%), cramps (63.7%), and stiffness (58.2%). Using a 0–10 point scale, significant differences were found between those continuing versus discontinuing their statins for being bothered by side effects (7.5 vs 9.2, p=0.001), for an inability to tolerate side effects (6.7 vs 9.0, p Conclusions Results of this pilot survey suggest patients who experience greater side effects severity and interference with daily activity, along with lower efforts by clinicians to work with adjusting their statin regimen, are at greater risk for discontinuing treatment. A wider survey and larger study population is needed to confirm the results of this pilot study. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): This study was sponsored by Amgen Inc.

The aim of this study was to estimate patient and caregiver productivity loss and indirect costs following an acute coronary syndrome (ACS) or a stroke in Europe.A cross-sectional study was conducted in seven European countries. A validated questionnaire was used during a cardiologist/neurologist visit 3-12 months post event. We included patients who returned to work ( ≥ 4 weeks prior to recruitment), given specific interest in presenteeism. Patient absenteeism, presenteeism and caregiver loss in the past four weeks were pro-rated to one year and combined with time-off due to initial hospitalisation/sick-leave. Hours lost were valued according to country labour cost (2018 euros).The analysis included 196 ACS (86% myocardial infarction) and 198 stroke (99% ischaemic, 77% modified Rankin Scale 0-1) patients. Mean age in ACS and stroke patients was 53 years, 86% and 78% respectively were men, 28% and 25% had previous cardiovascular event or established cardiovascular disease. Mean (country range) total productivity time loss was 70 (47-91) workdays for ACS and 68 (45-88) workdays for stroke (25% of annual workdays). Particularly, ACS patient lost 59 (37-79) workdays, and caregivers lost 11 (0-16) workdays, with total mean indirect cost per case €13,953 (€6641-23,160). After stroke, 56 (42-70) workdays were lost by patient plus 12 (3-20) days by caregiver, amounting to €13,773 (€10,469-20,215). Patients with previous events or established cardiovascular disease lost 80 (ACS) and 73 (stroke) workdays, costing €16,061 and €14,942 respectively.Our results suggest that lost productive time and indirect costs following ACS/stroke are substantial, with indirect costs comparable to direct costs.

Acute coronary syndrome (ACS) is highly prevalent in Switzerland and a leading cause of death. Associated productivity loss and indirect costs have rarely been studied. We investigated these factors in the first year after ACS in 24 Swiss patients (mean (SD) age 56 (8) years, 79% male). Data on patient productivity loss, absenteeism, presenteeism and caregiver assistance, were collected with the Productivity Cost Questionnaire during a routine cardiologist visit 3 to 12 months after hospitalisation for ACS and at least 4 weeks after patients returned to work. To estimate costs, lost hours were converted into 8-hour workdays, pro-rated to 1 year, combined with time off work due to initial hospitalisation and sick leave, and valued at Swiss labour costs. Additional data came from medical records. ACS patients lost on average (SD, range) 79 (81, 0.3–294) workdays; 38 (36, 0.3–153) days due to the initial hospitalisation and sick leave, 37 (75, 0–243) due to absenteeism after patients returned to work, and 4 (11, 0–41) due to presenteeism. Caregivers lost 10 (23, 0–90) additional workdays. The total indirect costs amounted to CHF 43,205 (44,026, 122–148,648); including CHF 18,514 (17,507, 122-74,619) for initial hospitalisation and sick leave and CHF 17,988 (36,394, 122–143,277) and CHF 1,849 (5181, 0–20,158) for absenteeism and presenteeism after patients returned to work, respectively. Costs of caregiver assistance amounted to CHF 4,855 (11,015, 0–43,843). This study showed that ACS patients lost on average 36% of their annual productive time. Caregivers lost an additional 5%. Lost work time was associated with substantial indirect costs that exceeded estimates of direct costs for ACS during 1 year. This suggests that costs and burden could be reduced through better risk reduction management.

Background Over time, guidelines for dyslipidemia management in patients at high risk of atherosclerotic cardiovascular disease (CVD) changed with the goal of improving patient outcomes. Guidelines have been released by the European Joint Task Force in 2007, 2012 and 2016, European Society of Cardiology in 2011, 2016 and 2019, Joint British Societies in 2014, and National Institute for Health and Care Excellence in 2014. Purpose Evaluate cardiovascular risk factors, treatment patterns, and cardiovascular outcomes over time related to dyslipidemia management. Methods Ten prevalent cohorts of patients with documented CVD receiving lipid-lowering therapy (LLT) were created using Clinical Research Practice Datalink (CPRD) records as of January 1, each year from 2008 through 2017. For each cohort, we identified CVD risk factors and LLT, and estimated the 1-year composite rate of fatal and nonfatal myocardial infarction (MI), ischemic stroke (IS), or revascularization. Patient follow-up was censored at the earliest of one year, end of data, or the outcome of interest. Patients in each cohort were required to be ≥18 years old, have ≥1 years of available medical history, and have received ≥2 LLT prescriptions in the prior year. Documented CVD was defined as MI, IS, angina, revascularization, transient ischemic attack, carotid stenosis, abdominal aortic aneurysm, or peripheral arterial disease. Patients could be in multiple cohorts. Results Annual patient counts ranged from 170,501 to 179,137 through 2013 and declined to 94,418 by 2017 (due to fewer patients in the overall CPRD data). Comparing 2008, 2011 (when ESC guidelines were revised) and 2017 showed the following for CVD risk factors: mean age was 71.6, 72.3, and 72.5 years; males were 59.9%, 61.1%, and 63.1%; current smoking was 15.1%, 15.2%, and 13.9%; type 2 diabetes was 18.4%, 20.2%, and 22.4%; stage 3–5 chronic kidney disease was 22.4%, 25.1%, and 22.8%; history of MI was 22.5%, 23.9%, and 27.4%; history of IS was 5.5%, 6.6%, and 7.9%; LDL Conclusions After 2011 in the UK, there was an increased use of high intensity statins, a greater proportion of patients with LDL levels Figure 1 Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Amgen

Introduction Both intensity and adherence to lipid lowering therapies (LLT) play an important role in effectiveness of the therapies in patients at risk for cardiovascular events. Purpose To evaluate the association of adherence and treatment intensity with cardiovascular outcomes and all-cause mortality in very high-risk patients (as defined by the current ESC guidelines) treated with statin and/or ezetimibe. Methods Retrospective cohort study was based on German health claims data (2010–2015) obtained from German Institute for Health Research (InGef) database and included patients ≥18 years with an initial LLT treatment (statin and/or ezetimibe) in 2011–2013, and diagnoses of cardiovascular disease (CVD), stage 4 or 5 chronic kidney disease (CKD) or type 2 diabetes mellitus (DM). Patients must have had at least 2 LLT prescriptions in the first year to ensure intention of treatment. Follow-up period started 1 year after the second LLT prescription and continued until one of the events of the composite study endpoint (hospitalisation for myocardial infarction, unstable angina, ischemic stroke, heart failure, revascularization, or all-cause death) or 31.12.2015, whichever occurred earlier. Adherence was measured annually by the proportion of days covered (PDC) using prescription data. Treatment intensity was quantified based on expected LDL-C reduction as described in the American College of Cardiology and American Heart Association (ACC/AHA) 2018 guidelines. Adherence and treatment intensity were multiplied to create a combined measure of intensity after accounting for adherence. Results 73,257 patients of the CVD cohort were 68 (SD=12) years old, 59% men; the DM cohort (no CVD) had 13,584 patients, age 64 (10), 47% men; 472 patients in the CKD cohort (no CVD) were 65 (15) years old, 46% men. In a Cox proportional hazards model, each 10% increase in treatment intensity (LDL-C lowering) was associated with 18% lower risk of CV event in the CVD (HR 0.82, 95% CI 0.82–0.83), 21% - in the DM (HR 0.79, 95% CI 0.76–0.83), and 15% - in the CKD (HR 0.85, 95% CI 0.75–0.97) cohorts. Similarly, each 10% increase in adherence (PDC) was associated with 6% lower risk of CV event in the CVD (HR 0.94, 95% CI 0.93–0.94), 7% - in the DM (HR 0.93, 95% CI 0.91–0.94), and 7% - in the CKD (HR 0.93, 95% CI 0.89–0.97) cohorts. Each 10% increase in adherence-adjusted intensity was associated with 16% lower risk of CV event in the CVD (HR 0.84, 95% CI 0.83 - 0.85), 19% - in the DM (HR 0.81, 95% CI 0.78–0.85), and 17% - in the CKD (HR 0.83, 95% CI 0.72–0.96) cohorts. The models controlled for age, sex, Charlson comorbidity index and other cardiovascular risk factors at baseline. Conclusions A higher adherence and/or treatment intensity of LLT was associated with significantly lower risk of CV outcomes or all-cause death in German very high-risk patients. Strategies to tailor intensity to patient profile and improve adherence could further lower risk of CV events. Acknowledgement/Funding Amgen Europe GmbH

Objective: To assess the prevalence of patients at very high risk of cardiovascular (CV) events in the United Kingdom (UK) and evaluate low-density lipoprotein cholesterol (LDL-C) values and treatment patterns in these patients. Methods: This cross-sectional study used primary care data from UK electronic medical records in the Clinical Practice Research Datalink (CPRD) in 2013. Very high-risk patients were defined per European Society of Cardiology guidelines as those with hyperlipidemia (assessed by co-medication) and documented cardiovascular disease (CVD) or hyperlipidemia and type 2 diabetes (DM2) without CVD (DM2w/oCVD). All analyses were descriptive. Results: Data from 4,940,226 patients were captured in the CPRD in 2013. Of these, 5% of patients had received ≥2 lipid-modifying therapy prescriptions and were at very high risk of CVD (3% [n = 138,536] had documented CVD, 2% [n = 98,743] had DM2w/oCVD). In documented CVD patients, coronary artery disease (73%) was the most frequent type of event (25% had myocardial infarction [MI]), followed by cerebrovascular disease (18%), and peripheral arterial disease (9%); 21% had experienced multiple CV events, 25% had DM2, and 3% had MI within 1 year. In documented CVD and DM2w/oCVD patients, >95% received statin treatment; 24% received high-intensity statin, and 1.5% statin plus ezetimibe. Across both populations, 64–66% had LDL-C levels ≥1.8 mmol/L, 27–28% ≥2.5 mmol/L, 6–7% ≥3.5 mmol/L, and 3% had levels ≥4.0 mmol/L, respectively. Conclusion: A well-defined proportion of patients remain at very high-risk of CVD. Statin therapy needs optimization, but, for some patients with high LDL-C levels, multiple CV events, MI within 1 year, or CVD and DM2, additional more intensive therapy may be needed.

In cancer cell lines and rodent models, calcium and vitamin D favorably modulate cell proliferation, differentiation, and apoptosis in colonic epithelia. These effects may be modulated by local expression of the calcium receptor (CaR), the vitamin D receptor (VDR), and the P450 cytochromes, CYP27B1 and CYP24A1; however, they have yet to be investigated in humans. To address this gap, we conducted a randomized, double-blinded, placebo-controlled 2 × 2 factorial clinical trial. Patients with at least one pathology-confirmed colorectal adenoma were treated with 2 g/d elemental calcium and/or 800 IU/d vitamin D3 versus placebo over 6 months (n = 92; 23 per group). CaR, VDR, CYP27B1, and CYP24A1 expression and distribution in biopsies of normal appearing rectal mucosa were detected by standardized, automated immunohistochemistry and quantified by image analysis. In the calcium-supplemented group, CaR expression increased 27% (P = 0.03) and CYP24A1 expression decreased 21% (P = 0.79). In the vitamin D3-supplemented group, CaR expression increased 39% (P = 0.01) and CYP27B1 expression increased 159% (P = 0.06). In patients supplemented with both calcium and vitamin D3, VDR expression increased 19% (P = 0.13) and CaR expression increased 24% (P = 0.05). These results provide mechanistic support for further investigation of calcium and vitamin D3 as chemopreventive agents against colorectal neoplasms, and CaR, VDR, CYP27B1, and CYP24A1 as modifiable, preneoplastic risk biomarkers for colorectal neoplasms. Cancer Res; 71(2); 413–23. ©2010 AACR.

To characterize the expression of the mismatch repair gene MutL-homolog 1 (MLH1) in normal colorectal crypts in humans, and assess parameters of its expression as a potential biomarker of risk for colorectal neoplasms, we conducted a pilot, colonoscopy-based case-control study (51 cases, 154 controls) of incident, sporadic colorectal adenoma. Biopsies of normal-appearing rectal, sigmoid, and ascending colon mucosa were procured, immunohistochemically processed for MLH1 protein, and analyzed using custom quantitative image analysis procedures. MLH1 expression in the ascending colon was, on average, 49% proportionally lower in cases than controls (P = 0.03), but there was little evidence for case-control differences in the rectum and sigmoid colon. In cases and controls, average MLH1 expression in the ascending colon tended to be lower with increased age [by 56% (P = 0.02) and 25% (P = 0.16), respectively, for those ≥55 years], and with a history of colorectal cancer in a first-degree relative (by 22% [P = 0.56] and 34% [P = 0.16], respectively). Among cases, but not controls, average MLH1 expression tended to be higher with current alcohol consumption, regular aspirin use, and higher total intakes of calcium, vitamin D, and folate. There was little indication of similar differences in the rectum. These preliminary data suggest that lower MLH1 expression in the normal colonic mucosa, at least in the ascending colon, may be associated with increased risk of incident, sporadic colorectal adenoma, as well as with modifiable risk factors for colorectal neoplasms, thus supporting further investigation of MLH1 expression as a potential “treatable” biomarker of risk for colorectal neoplasms. (Cancer Epidemiol Biomarkers Prev 2009;18(5):1599–609)

To further clarify and develop calcium and vitamin D as chemopreventive agents against colorectal cancer in humans and develop modifiable biomarkers of risk for colorectal cancer, we conducted a pilot, randomized, double-blind, placebo-controlled, 2 × 2 factorial clinical trial to test the effects of calcium and vitamin D3, alone and in combination, on key DNA mismatch repair proteins in the normal colorectal mucosa. Ninety-two men and women with at least one pathology-confirmed colorectal adenoma were treated with 2.0 g/d calcium or 800 IU/d vitamin D3, alone or in combination, versus placebo over 6 months. Colorectal crypt overall expression and distribution of MSH2 and MLH1 proteins in biopsies of normal-appearing rectal mucosa were detected by automated immunohistochemistry and quantified by image analysis. After 6 months of treatment, MSH2 expression along the full lengths of crypts increased by 61% (P = 0.11) and 30% (P = 0.36) in the vitamin D and calcium groups, respectively, relative to the placebo group. The estimated calcium and vitamin D treatment effects were more pronounced in the upper 40% of crypts (differentiation zone) in which MSH2 expression increased by 169% (P = 0.04) and 107% (P = 0.13) in the vitamin D and calcium groups, respectively. These findings suggest that higher calcium and vitamin D intakes may result in increased DNA MMR system activity in the normal colorectal mucosa of sporadic adenoma patients and that the strongest effects may be vitamin D related and in the differentiation zone of the colorectal crypt. Cancer Epidemiol Biomarkers Prev; 19(4); 1022–32. ©2010 AACR.

The exact antineoplastic effects of calcium and vitamin D3 in the human colon are unclear. Animal and in vitro studies show that these two agents reduce oxidative stress; however, these findings have never been investigated in humans. To address this, we conducted a pilot, randomized, double-blind, placebo-controlled, 2 × 2 factorial clinical trial to test the effects of calcium and vitamin D3 on a marker of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine (8-OH-dG), in the normal colorectal mucosa. Patients (N = 92) with at least one pathology-confirmed colorectal adenoma were treated with 2 g/d calcium and/or 800 IU/d vitamin D3 versus placebo over 6 months. Overall labeling and colorectal crypt distribution of 8-OH-dG in biopsies of normal-appearing rectal mucosa were detected by standardized automated immunohistochemistry and quantified by image analysis. After 6 months of treatment, 8-OH-dG labeling along the full lengths of colorectal crypts decreased by 22% (P = 0.15) and 25% (P = 0.10) in the calcium and vitamin D3 groups, respectively, but not in the calcium plus vitamin D3 group. The estimated treatment effects were strongest among participants with higher baseline colon crypt vitamin D receptor expression (P = 0.05). Overall, these preliminary results indicate that calcium and vitamin D3 may decrease oxidative DNA damage in the normal human colorectal mucosa, support the hypothesis that 8-OH-dG labeling in colorectal crypts is a treatable oxidative DNA damage biomarker of risk for colorectal neoplasms, and provide support for further investigation of calcium and vitamin D3 as chemopreventive agents against colorectal neoplasms. Cancer Epidemiol Biomarkers Prev; 19(1); 280–91

To investigate the potential efficacy of calcium and vitamin D in reducing risk for colorectal neoplasms and to develop “treatable” phenotypic biomarkers of risk for colorectal neoplasms, we conducted a pilot, randomized, double-blind, placebo-controlled, 2 × 2 factorial clinical trial to test the effects of these agents on cell cycle markers in the normal colorectal mucosa. Ninety-two men and women with at least one pathology-confirmed colorectal adenoma were treated with 2 g/day calcium and/or 800 IU/day vitamin D3 versus placebo over 6 months. Overall expression and distributions of p21waf1/cip1 (marker of differentiation), MIB-1 (marker of short-term proliferation), and hTERT (marker of long-term proliferation) in colorectal crypts in the normal-appearing rectal mucosa were detected by automated immunohistochemistry and quantified by image analysis. In the calcium, vitamin D, and calcium plus vitamin D groups relative to the placebo, p21 expression increased by 201% (P = 0.03), 242% (P = 0.005), and 25% (P = 0.47), respectively, along the full lengths of colorectal crypts after 6 months of treatment. There were no statistically significant changes in the expression of either MIB-1 or hTERT in the crypts overall; however, the proportion of hTERT, but not MIB-1, expression that extended into the upper 40% of the crypts was reduced by 15% (P = 0.02) in the vitamin D plus calcium group relative to the placebo. These results indicate that calcium and vitamin D promote colorectal epithelial cell differentiation and may “normalize” the colorectal crypt proliferative zone in sporadic adenoma patients, and support further investigation of calcium and vitamin D as chemopreventive agents against colorectal neoplasms. (Cancer Epidemiol Biomarkers Prev 2009;18(11):2933–41)

Background: Transforming growth factor-α (TGF-α), a stimulatory growth factor and member of the epidermal growth factor family, is a mediator of oncogenesis and malignant progression in colorectal carcinogenesis. Limited evidence suggests its utility as a growth-related biomarker of risk for colorectal cancer. Methods: We measured expression of TGF-α in biopsies of normal-appearing colorectal mucosa using automated immunohistochemistry and quantitative image analysis in a subsample of 29 cases and 31 controls from a colonoscopy-based case-control study (n = 203) of biomarkers of risk for incident sporadic colorectal adenoma. Diet, lifestyle, and medical history were assessed with validated questionnaires. Results: TGF-α expression in the rectum was 51% higher in cases compared with controls (P = 0.05) and statistically significantly associated with accepted risk factors for colorectal neoplasms (36% lower among nonsteroidal anti-inflammatory drug users, 49% lower among women using hormone replacement therapy, 79% higher among persons with a family history of colorectal cancer). Conclusions: TGF-α expression in the normal-appearing rectal mucosa shows promise as an early, potentially modifiable biomarker of risk for colorectal cancer. (Cancer Epidemiol Biomarkers Prev 2009;18(1):65–73)

Of the various agents tested in clinical trials against colorectal neoplasms, only two, calcium and nonsteroidal anti-inflammatory drugs (NSAIDs), have been found to have preventive efficacy, and calcium is the only one for which significant longterm adverse consequences have not been demonstrated. Vitamin D alone, and in combination with calcium, is now being tested in a large, long-term, randomized, placebo-controlled chemoprevention trial of sporadic colorectal adenoma recurrence, and is of current intense interest. This chapter summarizes calcium and vitamin D physiology and metabolism and the mechanistic, genetic, and epidemiologic evidence for these agents in preventing colorectal cancer.