We thank Dr. Tomomi Hamada of the Iwaki Kusakidai General Clinic for cross-reviewing the pathological diagnoses.
Metformin, an antidiabetic drug, decreases the incidence of various cancers in diabetic patients. Metformin-induced inhibition of cancer cell proliferation has been confirmed in vitro but not in humans. Because endometrial cancer is associated with insulin resistance, the authors investigated whether a diabetes-therapeutic metformin dose inhibits cancer cell growth in patients with endometrial cancer.
A dose of metaformin was administered (1500-2250 mg/day) to 31 patients with endometrial cancer preoperatively for 4 to 6 weeks. Cell proliferation was assessed in patient tissues using immunohistochemical and Western blot analyses and DNA synthesis was measured in serum using a thymidine uptake assay. All statistical tests were 2-sided. P values of < .05 were considered statistically significant.
Preoperative metformin treatment decreased DNA synthesis in sera and significantly reduced the Ki-67 (mean proportional decrease, 44.2%; 95% confidence interval [95% CI], 35.4-53.0 [P < .001]) and topoisomerase IIα (mean proportional decrease, 36.4%; 95% CI, 26.7-46.0 [P < .001]) labeling indices. Levels of phospho-ribosomal protein S6 and phospho-extracellular signal-regulated kinase 1/2 (ERK1/2) were found to be significantly decreased and phospho-adenosine monophosphate-activated protein kinase and p27 levels were significantly increased. Preoperative metformin use caused significant decreases in circulating factors, including insulin, glucose, insulin-like growth factor 1, and leptin. DNA synthesis-stimulating activity in patient sera was significantly decreased during metformin administration.
Metformin, an oral biguanide antihyperglycemic drug, is widely prescribed as first-line therapy against type 2 diabetes mellitus. Metformin suppresses hepatic gluconeogenesis and increases glucose uptake by peripheral tissues, thereby decreasing plasma glucose levels and eventually decreasing plasma insulin levels.
Widespread metformin use over the past decade has inspired several population studies that have identified additional benefits of metformin, including a metformin-induced decrease in cancer incidence and cancer-related mortality in patients with diabetes.[2, 3] This applies to cancers of the breast, colon, lung, prostate, pancreas, ovary, and liver. In addition to cancer prevention, more recent studies have indicated that metformin also possesses antineoplastic properties,[4-9] such as improving response rates to neoadjuvant chemotherapy in patients with breast cancer. These epidemiologic findings suggest that metformin is a promising drug for both cancer prevention and treatment and have facilitated numerous preclinical studies regarding its mechanism of action and therapeutic effects. However, quite a few questions remain to be answered before clinical trials may be initiated. One such question is which type of cancer should be included in introductory clinical studies to establish the benefit of metformin use. Several in vitro studies have demonstrated that metformin causes growth inhibition in various types of cancer cell lines. However, the metformin concentration used for these studies was much higher than the established in vivo concentration of orally administered metformin; thus, these in vitro data do not validate all types of cancer cells as clinical targets. Therefore, additional studies are required to establish relevant study models for the investigation of metformin and its anticancer activity.
In the current study, we investigated the action of metformin in patients with endometrial cancer because endometrial cancer is often associated with obesity, insulin resistance, and diabetes,[12, 13] and a recent retrospective cohort study on metformin use revealed the highest risk reduction was in patients with ovarian/endometrial cancer. We validated the use of endometrial cancer as a study model with a short-duration preclinical study using surrogate markers of therapeutic effects. To the best of our knowledge, the current study is the first report of a prospective study demonstrating the clinical benefit of a diabetes-therapeutic dose of metformin on endometrial cancer cell proliferation and downstream targets of the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways.
MATERIALS AND METHODS
Between January 2011 and May 2013, we recruited 40 consecutive patients with endometrioid adenocarcinoma who were scheduled to undergo surgery at Chiba University Hospital (Chiba, Japan). Eligibility criteria included an Eastern Cooperative Oncology Group performance status of 0 to 1 and normal renal, liver, and cardiac function. Exclusion criteria were as follows: 1) type 2 diabetes requiring medication; 2) history of metformin use; 3) an abnormal blood coagulation profile and/or a history of thromboembolism; and 4) the presence of mental or life-threatening illnesses. Five patients declined study participation, and 35 patients agreed to participate in the study, 4 of whom experienced intolerable nausea at a metformin dose of 750 mg and withdrew consent; this resulted in a final total of 31 patients.
The primary objective of the current study was to evaluate the effects of a diabetes-therapeutic dose of metformin on cell proliferation activity and growth-related signaling pathways in endometrial cancer tissues. The secondary objectives were to evaluate the metabolic effect of metformin on endocrine factors and to investigate the effects of metformin on the growth-supporting potential of sera.
Metformin (initial dose, 750 mg/day; increased weekly by 750 mg up to 1500 or 2250 mg/day) was administered for approximately 4 weeks until the day of scheduled surgery. Tissue specimens were obtained via endometrial curettage at the time of initial diagnosis (before treatment) and hysterectomy (after treatment). Changes in cell proliferation were determined by immunohistochemistry and Western blot analysis of paired endometrial tissue specimens. Metformin concentrations were measured in surplus frozen tissue and plasma samples using liquid chromatography-tandem mass spectrometry. Changes in the growth-stimulating potential of serum were assessed in paired sera obtained from 15 patients; thymidine incorporation rates in Ishikawa cells, an endometrial cancer-derived cell line, were measured in the presence of 2% immobilized patient serum. The metabolic effect of metformin treatment was assessed using a 75-g oral glucose tolerance test before and during treatment (usually a few days before surgery and approximately at week 4 of metformin treatment). Pathological diagnoses of endometrial samples were reviewed by 2 independent pathologists.
The Institutional Review Board of Chiba University approved the study protocol, and all patients provided written informed consent before participation. This trial was registered at umin.ac.jp/ctr/index.htm (identifier number UMIN 000004852).
Two endometrial cancer cell lines, HEC-1B and Ishikawa, were cultured in Dulbecco modified Eagle medium (DMEM; Life Technologies Corporation, Carlsbad, Calif) containing 4500 mg/mL of glucose and 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, Mo), 100 U/mL of penicillin, 100 U/mL of streptomycin, and 2 mM glutamine at 37°C and 5% carbon dioxide. The HEC-1B cell line was purchased from the JCRB Cell Bank (Osaka, Japan). The Ishikawa cell line was generously provided by Dr. Nishida (Tsukuba University, Japan).
Cell Proliferation Assay
Cells were seeded in 12-well plates (25,000 cells/well) in DMEM containing 10% FBS for 24 hours. The cells were then treated with increasing doses (0 mM-10 mM) of metformin for 72 hours. The media were replaced every 24 hours. After the addition of AlamarBlue reagent (Invitrogen, Carlsbad, Calif), the plates were incubated at 37°C, and colorimetric changes were measured.
(3H)Thymidine Incorporation Assay
Ishikawa cells (5 × 103 cells/well in 96-well plates) were cultured in DMEM containing 10% FBS for 48 hours until the exponential growth phase was reached, after which they were cultured with increasing doses (0 mM-10 mM) of metformin for 24 hours. Next, the cells were labeled with (3H)thymidine (1 μCi [37 kBq]/well) (PerkinElmer, Waltham, Mass) for 2.5 hours and harvested onto glass fiber filters using the Tomtec Harvester 96 Mach 3 (Wallac Ltd, Turku, Finland). After extensive washing with phosphate-buffered saline, radioactivity was detected on the filter using a Wallac 1450 MicroBeta liquid scintillation counter (Wallac Ltd). Thymidine incorporation was expressed as a percentage of that in untreated (no metformin) controls. Paired samples (premetaformin and postmetformin sera from the same patient) were assayed on the same plate in 5 replicates. A geometrical mean was used for statistical analysis.
Ex Vivo Assay to Assess the Growth-Stimulatory Activity of Serum
Ishikawa cells (96-well plates; 5 × 103 cells/well) were cultured in DMEM containing 10% FBS for 12 hours and cultured for 72 hours in assay media in which 2% immobilized patient serum replaced 10% FBS. At the end of the experiments, the cells were treated as described above, and the number of viable cells and (3H)thymidine incorporation were calculated. A concentration of 2% was determined by preliminary experiments to maximize the difference between premetaformin and postmetformin treatments. Use of 5% serum increased the absolute number of cells and thymidine incorporation but decreased the differences.
The antibodies for phospho-AMPKα (Thr172; #2535), AMPKα (#2603), phospho-ribosomal protein S6 (phospho-rpS6) (Thr389; #2215), rpS6 (#2217), phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204; #4370), p44/42 MAPK (ERK1/2; #9102), cyclin D1 (#2922), p27 (#2552), and β-actin (#4967) were all purchased from Cell Signaling Technology Inc (Danvers, Mass). Antibodies against topoisomerase IIα (sc-56805) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Antibodies against Ki-67 (M7240) were purchased from Dako Denmark A/S (Glostrup, Denmark).
Cell proliferation in endometrial cancer tissues was evaluated by immunohistochemical staining of Ki-67 (positive during all cell cycle stages except for G0) and topoisomerase IIα (positive during S-/G2/M-phase).[16, 17]
Three-μm thick sections were briefly microwaved in 10 mM of citrate buffer (pH 6.0) and immunostained for Ki-67 and topoisomerase IIα. The Envision FLEX system (K8000; Dako Denmark A/S) was used for each antibody to observe the immunostaining using an Autostainer S3400 (Dako Denmark A/S). The primary antibodies were incubated at room temperature for 60 minutes at a dilution of 1:100. The secondary antibody (Envision FLEX/HRP; Dako Denmark A/S) was incubated at room temperature for 60 minutes, and 3,3′-diaminobenzidine tetrahydrochloride (Dako Denmark A/S) was used as a chromogen. The samples were then counterstained with hematoxylin. Labeling indices for Ki-67 and topoisomerase IIα were presented as the percentage of immunoreactive nuclei of 500 tumor cells.
Endometrial cancer specimens embedded in paraffin blocks were retrospectively collected from 10 patients with endometrioid adenocarcinoma who underwent surgery at Chiba University Hospital. Paired specimens obtained at the time of preoperative biopsy and at surgery were stained for Ki-67 and topoisomerase IIα. These patients did not receive metformin, and these specimens were used only to assess the natural change of proliferation markers between 2 intervals.
Western Blot Analysis
Frozen tissue samples were thawed on ice, homogenized using a TissueRuptor (Qiagen, Hilden, Germany), and lysed in Complete-M lysis buffer (Roche Applied Science, Tokyo, Japan) containing Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc, Wayne, Mich). Lysates (10 μg of protein) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (GE Healthcare Japan, Tokyo, Japan). Antibodies were the same as those used for immunohistochemical analyses. The primary antibodies were diluted (1:2000 for phospho-ERK1/2 and ERK1/2; 1:1000 for phospho-rpS6, rpS6, cyclin D, and p27; and 1:5000 for β-actin) and incubated overnight at 4°C. The secondary antibody (enhanced chemiluminescence [ECL] horseradish peroxidase-conjugated antirabbit immunoglobulin G and antimouse immunoglobulin G; GE Healthcare) was incubated at room temperature for 60 minutes. Signals were detected using the ECL Plus or ECL Advance Western Blotting Detection Kit (GE Healthcare). Signal intensity was quantified using a densitometer (CS Analyzer version 3.0 software; ATTO, Tokyo, Japan) and normalized to β-actin levels.
Metformin Concentration in Patient Tissues
Tissue homogenates and plasma samples were deproteinized using methanol and subjected to liquid chromatography-mass spectrometry analysis. Liquid chromatography was performed at ambient temperature using the Shimazu Prominence LC system (Shimadzu Scientific Instruments, Kyoto, Japan) with Capcell Pak columns (5 μm, 50 × 2 mm; Shiseido Co Ltd, Tokyo, Japan) and 10 mM of ammonium acetate with methanol at a ratio of 65:35 (volume/volume) as the mobile phase. Mass spectrometry was performed using the Applied Biosystems Sciex API 4000 mass spectrometer (Applied Biosystems Sciex, Foster City, Calif) with electrospray ionization for ion production. Detection was performed using a multiple reaction monitoring mode, with the transition of the protonated molecular ions of metformin at m/z 130→71.
The statistical analysis for the cell proliferation assay was performed using the Mann-Whitney U test. Means, 95% confidence intervals (95% CIs), and standard deviations were calculated for continuous variables. Comparisons between paired values were made using the Wilcoxon signed rank test. All comparisons were planned, and the tests were 2-sided. A P value of < .05 was considered statistically significant. All statistical analyses were performed using SAS software (version 9.3; SAS Institute Inc, Cary, NC) and SPSS software (version 20; IBM-SPSS Inc, Chicago, Ill).
A total of 31 patients were included in the current study, and patient clinical characteristics are listed in Table 1. The median patient age was 51 years (range, 27 years-72 years). Nineteen patients (61%) had a body mass index of ≥ 25 (mean, 28; range, 18-42), and 20 patients (65%) were insulin-resistant, as indicated by a homeostasis model assessment of insulin resistance (HOMA-R) score of 2.5 (mean score, 3.2; range, 0.6-9.8). Nineteen patients (61%) had abnormal glucose tolerance, including 5 patients with a BMI < 25 and 6 patients with a HOMA-R < 2.5.
Table 1. Patient Characteristics
Abbreviations: BMI, body mass index; DM, diabetes mellitus; HOMA-R, homeostasis model assessment of insulin resistance; OGTT, oral glucose tolerance test.
The International Federation of Gynecology and Obstetrics (FIGO) stage and histological grade.
Preoperative metformin treatment resulted in significantly reduced Ki-67 expression in 28 patients (90%) (labeling index reduced from 51.0 [95% CI, 42.9-59.1] to 30.3 [95% CI, 21.4-39.3]; P < .001) and reduced topoisomerase IIα expression in 25 patients (81%) (labeling index reduced from 49.9 [95% CI, 41.4-58.4] to 28.5 [95% CI, 23.0-34.0]; P < .001) (Fig. 1A-F). This corresponded to a mean proportional decrease of 44.2% in the Ki-67 labeling index (95% CI, 35.4-53.0) and 36.4% in the topoisomerase IIα labeling index (95% CI, 26.7-46.0). To confirm this finding was not the result of a natural change between 2 sampling intervals, we conducted a supplemental retrospective analysis on 10 additional patients who were not administered metformin. No significant changes in either Ki-67 or topoisomerase IIα expression were found (Table 2).
Table 2. Effect of Preoperative Metformin on Ki-67 and Topoisomerase IIα Expressiona
Using samples from 15 patients, we then examined changes in 2 cell signaling pathways through which metformin reportedly inhibits cell proliferation: AMPK/mTOR/rpS6 and MAPK pathways.[4, 5, 18] Metformin administration resulted in significantly increased phospho-AMPK levels (mean proportional increase of 113.2%; 95% CI, 13.6-212.8 [P = .03]) and significantly decreased phospho-rpS6 levels (mean proportional decrease of 53.2%; 95% CI, 36.8-69.5 [P = .002]) (Fig. 2A-B). In addition, metformin significantly decreased phosphorylated extracellular signal-regulated kinase (ERK1/2) levels (mean proportional decrease of 65.8%; 95% CI, 45.6-95.9 [P = .002]), thereby activating p27 (mean proportional increase of 59.0%; 95% CI, 12.5-105.5 [P = .02]) and inhibiting cyclin D1 (mean proportional decrease of 20.9%; 95% CI, 17.1-58.9 [P = .67]) (Fig. 2C-E).
Tissue Metformin Concentrations In Vivo
After preoperative oral administration of 3 doses of 750 mg each of metformin daily for 4 to 9 weeks, plasma concentrations on the day of the second oral glucose tolerance test were 1.7 μM and 6.8 to 18.1 μM before and 2 hours after the patient ingested the last dose, respectively (Fig. 3A). Concentrations in endometrial cancer tissues were 1.2 to 5.1 μmol/kg wet weight, which equated to approximately 20% of the plasma concentration, assuming that 1 g of wet tissue is equivalent to 1 mL.
Using the endometrial cancer-derived cell lines, we then found that at least 1 mM of metformin was necessary to inhibit cell proliferation in vitro (Fig. 3B-E), a dose < 1/400 of the minimum concentration (approximately 1 mM) required to suppress growth in vitro.
Growth-Stimulatory Potential of Sera
We assessed the humoral factor-mediated action of metformin on cell proliferation. Preoperative metformin use caused significant decreases in the profiles of circulating factors, such as insulin, glucose, insulin-like growth factor 1 (IGF-1), and leptin (Table 3). Oral administration of metformin decreased insulin and glucose by approximately 40% and 25%, respectively. In addition, oral metformin decreased the levels of circulating IGF-1 by approximately 15%.
Table 3. Changes in Metabolic Profiles After Preoperative Administration of Metformina
95% CI, 95% confidence interval; BMI, body mass index; HOMA-R, homeostasis model assessment of insulin resistance; IGF-1, insulin-like growth factor 1.
Group comparisons were performed using the Wilcoxon matched pairs test.
Metformin lowered the thymidine uptake activity of sera in all 15 patients (Fig. 4). It is interesting to note that thymidine uptake remained low even 2 hours after loading with 75 g glucose. Moreover, serum insulin and glucose levels increased, whereas IGF-1 and leptin levels remained low.
To the best of our knowledge, the current study is the first prospective evidence that an antidiabetic dose of metformin suppresses endometrial cancer cell growth in vivo.
We found that metformin reduced the expression of cell proliferation markers by approximately 50%. To our knowledge this is the most significant difference among studies that have evaluated cancer tissue growth during metformin administration.[19-21] Only 3 studies to date have assessed the in vivo effect of metformin on cancer tissue, all of which were histological studies on breast cancer tissues obtained by preoperative needle biopsy and mastectomy.
Bonanni et al examined changes in Ki-67 staining in breast cancer tissues harvested from nondiabetic women who received metformin (850 mg/twice a day for 4 weeks). Overall, the effect of metformin on the Ki-67 proliferative index was not statistically significant. However, metformin administration caused an insignificant decrease (10.5%) in the Ki-67 index of insulin-resistant women (≥ 2.8 HOMA-R index) and a significant increase (11%) in insulin-sensitive women (< 2.8 HOMA-R index). Therefore, the association between metformin and the HOMA-R index was statistically significant (P = .045). This indicates that metformin treatment is beneficial for insulin-resistant women only when compared with medication-free insulin-sensitive women. However, the detected benefits were marginal (P = .045), and multiple testing may have resulted in nonsignificant results. Hadad et al evaluated tumor cell growth in patients with breast cancer and reported that preoperative metformin treatment (1000 mg/day) for 2 weeks significantly decreased the Ki-67 index by 3.4% relative to baseline values. However, insulin levels were measured only once during a preoperative nonfasting period, thereby limiting interpretation of the results. Recently, Niraula et al conducted a similar study in patients with breast cancer and reported that preoperative metformin treatment (1500 mg/day) for 2 to 3 weeks significantly decreased the Ki-67 index by approximately 8% relative to baseline values.
Thus, 2 of the 3 studies of metformin use in patients with breast cancer demonstrated a significant reduction in the Ki-67 index; however, the changes were subtle and heterogeneous between individuals. In comparison, the results of the current study demonstrated a more profound (approximately 50%) and consistent decrease in proliferation.
This finding may be explained by the differences in target tissues, clinical demography, and duration of metformin administration. First, the planned dose of metformin in the current study was 2250 mg/day, whereas previous studies used 1000 to 1700 mg/day. Second, we studied the effects of metformin on patients with type I endometrial cancer, a malignancy more commonly associated with insulin resistance and diabetes than breast cancer.[22, 23]
In addition, endometrial cancer has other advantages over breast cancer as an in vivo study model. Preoperative total ablation of the endometrial cavity is obligatory for diagnosis; however, endometrial tissue will regenerate in several weeks, thus providing a sufficient amount of tissue for repetitive analyses with minimal ethical difficulties. Moreover, total ablation eliminates the possibility that the pathological diagnosis is skewed because of limited sampling, whereas in patients with breast cancer, preoperative evaluation is limited to the core diagnostic biopsy samples. Given the histological response and accessibility to tissues samples, we propose that endometrial cancer is a better-suited model for window-of-opportunity clinical trials with metformin.
In vitro experiments have demonstrated that metformin inhibits cancer cell growth through the activation of AMPK and the resulting inhibition of the mTOR/S6K pathway.[4-9] Recent reports have identified the MAPK pathway as an additional growth inhibition target; metformin inhibits MAPK and increases phosphorylation of p27, thereby resulting in cell cycle inhibition.[8, 24, 25] The results of the current study confirmed similar effects of metformin in vivo; metformin intake activated AMPK and inhibited MAPK in the endometrium.
There is currently controversy regarding whether metformin inhibits cancer cell growth directly or indirectly. This is based on experimental findings that the concentration needed to inhibit cell growth is much higher (approximately ≥ 1000 times) than that expected in patient sera. Supporting the indirect mechanism, metformin prevented carcinogen-induced lung tumorigenesis in mice through the suppression of mTOR. Rather than directly activating AMPK, metformin administration resulted in downregulation of phosphorylated IGF-1 receptor/insulin receptor, Akt, and ERK in mice lung tissues. Thus, the findings supported the indirect mechanism of metformin, in which metformin reduces IGF-1 production from liver, thereby resulting in decreased mTOR and ERK levels.
To address the mechanism of action, we first compared the metformin concentrations in vitro and in vivo and found that the concentration in tissues was < 1/400 times lower than that of the minimum requirement to inhibit cell growth in vitro. The enhanced responses observed in vivo might be explained by metformin-induced changes in humoral factor(s). To assess this possibility, we performed an ex vivo assay and found that metformin reduced the growth-supporting potential of patient sera. Among the humoral factors examined, IGF-1 and leptin, but not insulin, were factors potentially responsible for this result. In fact, IGF-1 is known to play an important role in tumorigenesis and the promotion of endometrial cancer.[27-29] Leptin has also been shown to stimulate endometrial cell proliferation through the activation of signal transducer and activator of transcription 3 (STAT3), AKT, and MAPK signaling pathways.[30, 31]
Although these findings are compatible with an indirect action of metformin, additional confirmation is necessary because there are several alternative explanations. First, we estimated the concentration in tissues by measuring tissue mass; thus, we did not measure the actual concentration of metformin in cancer cells in vivo. It might be possible that organic anion transporter-1, a powerful transporter of metformin expressed in liver cells, may concentrate metformin in vivo, resulting in a higher concentration than we calculated as a mass average. Another possibility is that the established cancer cell line used in the current study lost sensitivity to metformin, for example by no longer expressing the transporter. Third, it is unknown whether the growth-supporting potential detected by the ex vivo assay is a true reflection of the in vivo potential. Further experiments are necessary to define the action of metformin in vivo.
Recently, 2 retrospective studies revealed that metformin use was associated with improved recurrence-free survival and overall survival.[33, 34] In these studies, 24% to 25% of patients with endometrial cancer were diabetic, and 50% of these diabetic patients with cancer used metformin. Greater than 80% of patients with endometrial cancer were overweight or obese and were insulin-resistant. In this regard, the results of the current study clarified that metformin reduced levels of glucose, insulin, IGF-1, leptin, and other factors, an important mechanism for the management of endometrial cancer.
There were several limitations to the current study, including the lack of a placebo control. In this study, we did not consider natural changes in cell proliferation markers between the 2 sampling times, which were separated by an average 4-week interval. Therefore, we conducted supplemental retrospective analyses on 10 additional patients who were not administered metformin. The results indicated no significant changes in any proliferation markers between the paired samples from the same individuals.
Although further studies to identify the humoral factor(s) that may mediate the actions of metformin are warranted, the findings of the current study provide a reasonable basis for the initiation of clinical trials using antidiabetic doses of metformin for the treatment of patients with endometrial cancer.
Supported in part by a grant from the Japan Society for Promotion of Science (grant numbers 24592504, 23132503, 25670694, and 25253092). The funding source had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.
CONFLICT OF INTEREST DISCLOSURES
Dr. Sato has received a grant from Kowa Souyaku, acted as a paid member of the Independent Data Monitoring Committee of Pfizer Japan Inc, received speaking fees from Elekta, received lecture fees from Siemens, and acted as a paid member of the advisory committee for the Ministry of Health, Labor and Welfare of Japan and the Japan Medical Association Center for Clinical Trial.