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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
JAMA. Author manuscript; available in PMC Apr 27, 2012.
Published in final edited form as:
PMCID: PMC3087286
NIHMSID: NIHMS287071

Association of CTNNB1 (β-Catenin) Alterations, Body Mass Index, and Physical Activity with Survival in Patients with Colorectal Cancer

Teppei Morikawa, MD, PhD, Aya Kuchiba, PhD, Mai Yamauchi, PhD, Jeffrey A. Meyerhardt, MD, MPH, Kaori Shima, DDS, PhD, Katsuhiko Nosho, MD, PhD, Andrew T. Chan, MD, MPH, Edward Giovannucci, MD, ScD, Charles S. Fuchs, MD, MPH, and Shuji Ogino, MD, PhD, MS

Abstract

Context

Alterations of WNT-CTNNB1 (β-catenin) signaling play roles in colorectal carcinogenesis and metabolic diseases.

Objective

To test the hypothesis that CTNNB1 activation in colorectal cancer might modify prognostic associations of body mass index (BMI) and post-diagnosis physical activity.

Design

We utilized molecular pathological epidemiology (MPE, or molecular pathologic epidemiology) design, investigating interactive effects of tumor and lifestyle factors on tumor behavior. We evaluated CTNNB1 (HGNC ID: 2514) localization by immunohistochemistry in 955 colon and rectal cancers. Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for covariates, including microsatellite instability (MSI), CpG island methylator phenotype (CIMP), LINE-1 methylation, KRAS, BRAF, PIK3CA, and TP53 (p53).

Setting

Two U.S. nationwide prospective cohort studies, the Nurses’ Health Study and the Health Professional Follow-up Study.

Results

In obese patients (BMI ≥30 kg/m2), nuclear CTNNB1 positivity was associated with significantly better cancer-specific survival [adjusted HR=0.24; 95% confidence interval (CI), 0.12-0.49; 5-year survival, 0.85 vs. 0.78 for nuclear CTNNB1-positive vs. negative patients] and overall survival (adjusted HR=0.56; 95% CI, 0.35-0.90; 5-year survival, 0.77 vs. 0.74 for nuclear CTNNB1-positive vs. negative patients), while CTNNB1 status was not associated with prognosis among nonobese patients (Pinteraction=0.0003 and 0.034 for cancer-specific and overall survival, respectively). Among nuclear CTNNB1-negative stage I-III patients, post-diagnosis physical activity was associated with better cancer-specific survival [adjusted HR=0.33; 95% CI, 0.13-0.81; ≥18 vs. <18 MET (metabolic equivalent task) hours/week; 5-year survival, 0.97 vs. 0.89] while physical activity was not associated with survival among nuclear CTNNB1-positive stage I-III patients (Pinteraction=0.046).

Conclusions

This MPE study showed that CTNNB1 activation was associated with better cancer-specific and overall survival only among obese patients. Post-diagnosis physical activity was associated with better cancer-specific survival only among nuclear CTNNB1-negative patients. Our novel MPE data support a role of interactions between tumor and host factors influencing tumor aggressiveness.

Keywords: molecular pathological epidemiology, personalized medicine, colon cancer, transcription factor, exercise, metabolism

INTRODUCTION

Activation of the WNT signaling pathway and its major mediator CTNNB1 (the HUGO-approved official symbol for β-catenin) most commonly by APC loss plays a critical role in colorectal carcinogenesis.1 WNT signaling is a potential therapeutic target.2,3 Inactivation of kinases in the APC complex leads to accumulation of cytoplasmic CTNNB1 and its translocation to the nucleus, where it acts as a coactivator of TCF family transcription factors (Figure 1).4 Proliferative WNT-CTNNB1-regulated genes such as the oncogene MYC and cell cycle regulator CCND1 (cyclin D1) contribute to tumor progression.1

Figure 1
(A-B) The WNT-CTNNB1 pathway. (A) In the absence of WNT, cytoplasmic CTNNB1 binds to the protein complex formed by AXIN, APC, GSK3B, and CSNK1A1 and is phosphorylated by GSK3B and CSNK1A1, leading to proteasomal degradation of CTNNB1. (B) When the WNT ...

Accumulating evidence indicates a role of WNT-CTNNB1 signaling in adipogenesis, obesity and metabolic diseases.5-7 WNT signaling has been shown to be activated by PRKA (AMP-activated protein kinase),8 which is a key regulator of energy metabolism.9 Considering the dual roles of CTNNB1 in carcinogenesis and energy metabolism, we hypothesized that activation of WNT-CTNNB1 signaling (i.e. accumulation of nuclear CTNNB1) might confer proliferative ability to cancer cells even not under excess energy balance status. In addition, epidemiologic evidence suggests causal effects of obesity or excess energy balance on colon cancer incidence10,11 as well as mortality.12,13 Notably, physical activity (exercise) has emerged as a modifiable lifestyle factor which may improve cancer survival.14,15

We therefore tested the hypothesis that tumor CTNNB1 alterations might modify prognostic associations of body mass index (BMI) and post-diagnosis physical activity, utilizing a database of colorectal cancer patients (N=955) in two nationwide prospective cohort studies. Because patient characteristics and lifestyle factors as well as major tumor molecular features have been accumulated in our database, we were able to analyze an interactive effect of tumor CTNNB1 status and patient’s BMI or post-diagnosis physical activity.

MATERIALS AND METHODS

Study population

We utilized the database of two nationwide prospective cohort studies, the Nurses’ Health Study (N=121,701 women followed since 1976) and the Health Professionals Follow-up Study (N=51,529 men followed since 1986).16 Every two years, cohort participants have been sent follow-up questionnaires to update information on dietary and lifestyle factors (including BMI and physical activity), and to identify newly diagnosed cancers in themselves and their first degree relatives. We collected paraffin-embedded tissue blocks from hospitals throughout the U.S. where patients underwent colorectal cancer resections.16 Hematoxylin and eosin stained tissue sections from all colorectal cancer cases were reviewed by a pathologist (S.O.) unaware of other data. The tumor differentiation was categorized as well-moderate vs. poor (>50% vs. ≤50% gland formation). We excluded cases which were preoperatively treated. Patients were observed until death or June 30 2009, whichever came first. Death of a participant was confirmed by the National Death Index. Returning questionnaire indicated informed consent from all study participants. This study was approved by the Human Subjects Committees at Harvard School of Public Health and Brigham and Women’s Hospital.

Assessment of physical activity

Leisure-time physical activity has been assessed every two years in both cohorts, as previously described and validated against participant diaries.17,18 Participants reported duration of participation (ranging from 0 to 11 or more hours per week) on walking (along with usual pace); jogging; running; bicycling; swimming laps; racket sports; other aerobic exercises; lower intensity exercise (yoga, toning, stretching); or other vigorous activities. Each activity on the questionnaire was assigned a metabolic equivalent task (MET) score.19 One MET is the energy expenditure for sitting quietly. MET scores are defined as the ratio of the metabolic rate associated with specific activities divided by the resting metabolic rate. The values from the individual activities were summed for a total MET-hours per week score. Based on prior studies of physical activity in colorectal cancer survivors,14,15 patients who engaged in at least 18 MET-hours per week had significantly better colorectal cancer-specific mortality. The first physical activity assessment collected at least 1 year but no more than 4 years after diagnosis (median 17 months) was used to avoid assessment during the period of active treatment. To avoid bias due to declining physical activity in a period around recurrence or death, physical activity was not updated, but rather, physical activity was assessed at a single time point after cancer diagnosis.14,15

DNA extraction, Pyrosequencing of KRAS, BRAF and PIK3CA, and microsatellite instability (MSI) analysis

DNA was extracted from paraffin embedded tissue, and we performed PCR and Pyrosequencing targeted for KRAS (HGNC# 6407; codons 12 and 13),20 BRAF (HGNC# 1097; codon 600)21 and PIK3CA (HGNC# 8975; exons 9 and 20).22 MSI analysis was performed using 10 microsatellite markers (D2S123, D5S346, D17S250, BAT25, BAT26, BAT40, D18S55, D18S56, D18S67 and D18S487).23 MSI-high was defined as instability in ≥30% of the markers, and MSI-low/microsatellite stability (MSS) as instability in <30% of the markers.

Methylation analyses for CpG islands and LINE-1

Using validated bisulfite DNA treatment and real-time PCR (MethyLight),24,25 we quantified DNA methylation in eight CIMP-specific promoters [CACNA1G (HGNC# 1394), CDKN2A (p16; HGNC# 1787), CRABP1 (HGNC# 2338), IGF2 (HGNC# 5466), MLH1 (HGNC# 7127), NEUROG1 (HGNC# 7764), RUNX3 (HGNC# 10473) and SOCS1 (HGNC# 19383)].23,26 CIMP-high was defined as the presence of ≥6/8 methylated promoters, and CIMP-low/0 as 0/8-5/8 methylated promoters, according to the previously established criteria.27 In order to accurately quantify relatively high LINE-1 methylation levels, we used Pyrosequencing as previously described.28

Immunohistochemical analyses

Method of immunohistochemistry was previously described for TP53 (p53; HGNC# 11998)29 and CTNNB1 (β-catenin; HGNC# 2514).30 For CTNNB1, antigen retrieval was performed; deparaffinized tissue sections in citrate buffer (BioGenex, San Ramon, CA) were treated with microwave in a pressure cooker for 15 minutes. Tissue sections were incubated with 3% H2O2 (15 minutes) to block endogenous peroxidase, with 10% normal goat serum (Vector Laboratories, Burlingame, CA) in phosphate-buffered saline (10 minutes), and with serum-free protein block (10 minutes; DAKO, Carpinteria, CA). Primary antibody against CTNNB1 (β-catenin; clone 14; 1:400 dilution; BD Transduction Laboratories, Franklin Lakes, NJ) was applied for 1 hour at room temperature. Secondary antibody (20 minutes; BioGenex) and then streptavidin peroxidase conjugate (20 minutes; BioGenex) were applied. Sections were visualized by diaminobenzidine (2 minutes) and methyl green counterstain. Appropriate positive and negative controls were included in each run of immunohistochemistry. For CTNNB1, normal colonic epithelial cells served as internal positive controls with membrane staining (Figure 1). Methods of interpretations and validations of cutoffs are described in detail in eAppendix.

Statistical analysis

All statistical analyses were performed by SAS program (Version 9.1, SAS Institute, Cary, NC). All p values were two-sided and statistical significance was set at p=0.05. Nonetheless, when multiple hypothesis testing was performed, a p value for significance was adjusted by Bonferroni correction to p=0.0033 (=0.05/15). For categorical data, the chi-square test was performed. Unpaired t test under the assumption of equal variances was done to compare mean age and mean LINE-1 methylation level. Kaplan-Meier method and log-rank test were used for survival analyses. For analyses of colorectal cancer-specific mortality, deaths as a result of other causes were censored. Based on the significance level at p=0.05 and the total sample size of 955 with 266 events, there was 80% power to detect cancer-specific mortality HR of 1.57, and with 440 events, there was 80% power to detect overall mortality HR of 1.50. We calculated age-adjusted incidence rates of death from colorectal cancer or from all causes in a specific subgroup by dividing the number of deaths by the sum of person-years of follow-up, adjusted for age. To control for confounding, we used multivariate Cox proportional hazards regression models. A multivariate model initially included sex, age at diagnosis (continuous), BMI (<30 vs. ≥30 kg/m2), family history of colorectal cancer in first-degree relative(s) (present vs. absent), tumor location (proximal vs. distal), tumor differentiation (well-moderate vs. poor), MSI (high vs. low/MSS), CIMP (high vs. low/0), LINE-1 methylation (continuous), TP53, KRAS, BRAF, and PIK3CA. Disease stage (I, IIA, IIB, IIIA, IIIB, IIIC, IV, unknown) was used as a stratifying variable using the “strata” option in the SAS “proc phreg” command, to avoid overfitting and residual confounding. A backward stepwise elimination was performed with p=0.20 as a threshold to avoid overfitting. We confirmed that excluding cases with missing information in any of the covariates did not substantially alter results (see eAppendix). Missing data handling is described in detail in eAppendix. The proportionality of hazards assumption was satisfied by evaluating time-dependent variables, which were the cross-product of the CTNNB1 variable and survival time (p>0.10). We tested models removing individual variables and did not detect problems with collinearity.

An interaction was assessed by the Wald test on the cross product of CTNNB1 and another variable of interest (without data-missing cases) in a multivariate Cox model. Based on our a-priori hypothesis on an interaction between CTNNB1 and energy balance status, we examined an interaction between nuclear CTNNB1 and pre-diagnosis BMI (or post-diagnosis physical activity). In exploratory analyses, we examined a potential interaction between nuclear CTNNB1 and any of the other covariates (including age, sex, family history of colorectal cancer, tumor location, stage, MSI, CIMP, LINE-1 methylation, KRAS, BRAF, PIK3CA, and TP53), and found no significant interaction (all Pinteraction>0.01; given multiple hypothesis testing, a statistical significance level was adjusted to Pinteraction=0.0033). Notably, the effect of nuclear CTNNB1 did not significantly differ between the two independent cohort studies (Pinteraction=0.86 for cancer-specific survival and Pinteraction =0.78 for overall survival).

RESULTS

CTNNB1 (β-catenin) status in colorectal cancer

Based on the availability of tumor tissue and data, 955 stage I-IV colorectal cancer cases diagnosed up to 2004 were included in this study (Table 1). Among the 955 cases, 455 (48%) showed cytoplasmic CTNNB1 positivity (defined as moderate-strong expression), 439 (46%) showed nuclear CTNNB1 positivity (defined as moderate-strong expression) and 488 (51%) showed loss of membrane CTNNB1 (defined as no or weak membrane expression). Cytoplasmic and nuclear CTNNB1 positivities were correlated with each other (p<0.0001) and with loss of membrane CTNNB1 (p<0.0001). As in Table 1, proximal tumor location, MSI-high, CIMP-high, and BRAF mutation were associated inversely with cytoplasmic/nuclear CTNNB1 positivity and loss of membrane CTNNB1 (all p<0.0001).

Table 1
CTNNB1 (β-catenin) alterations in colorectal cancers

CTNNB1 alterations and colorectal cancer survival

During follow-up (141 months of median follow-up time for all censored cases with an interquartile range of 105-192 months), there were 440 deaths, including 266 colorectal cancer-specific deaths. For either colorectal cancer-specific or overall mortality, CTNNB1 status (cytoplasmic, nuclear, or membrane) was not significantly associated with patient survival in log-rank test, or univariate or multivariate Cox regression analysis (Table 2).

Table 2
CTNNB1 (β-catenin) alterations in colorectal cancer and patient mortality

Modifying effect of pre-diagnosis BMI on prognostic association of CTNNB1 alterations

Since WNT-CTNNB1 signaling has been implicated in obesity and metabolic diseases,5-7 we examined a potential modifying effect of pre-diagnosis BMI on the relation between CTNNB1 alterations and patient survival (Table 3). Notably, there was a significant modifying effect of BMI. In obese patients (BMI ≥30 kg/m2), nuclear CTNNB1 positivity was associated with significantly better cancer-specific mortality (adjusted HR, 0.24; 95% CI, 0.12-0.49) and overall mortality (adjusted HR, 0.56; 95% CI, 0.35-0.90). In contrast, among nonobese patients (BMI <30 kg/m2), nuclear CTNNB1 positivity was not significantly associated with cancer specific or overall survival (Pinteraction=0.0003 and 0.034 for cancer-specific and overall mortality, respectively). Similar, albeit weaker, modifying effect of BMI was found in survival analyses using cytoplasmic or membrane CTNNB1 (see eTables 1 and 2).

Table 3
Colorectal cancer patient mortality according to nuclear CTNNB1 (β-catenin) status and pre-diagnosis body mass index (BMI)

Effect of post-diagnosis physical activity on survival in strata of nuclear CTNNB1 status

We examined the effect of post-diagnosis physical activity on mortality (in stage I-III patients, N=497) in strata of nuclear CTNNB1 status (Figure 2 and Table 4). We excluded stage IV cases as in our previous analysis,31 because of likely influence of disease severity on patient’s ability to exercise. Notably, for nuclear CTNNB1-negative cases, high post-diagnosis physical activity was associated with significantly better colorectal cancer-specific mortality (adjusted HR, 0.33; 95% CI, 0.13-0.81; ≥18 vs. <18 MET-hours/week) and there was a trend towards better overall survival (adjusted HR=0.68; 95% CI, 0.42-1.09; ≥18 vs. <18 MET-hours/week), while physical activity was not associated with survival in CTNNB1-positive cases (adjusted HR for cancer-specific survival, 1.07; 95% CI, 0.50-2.30; adjusted HR for overall survival, 0.86; 95% CI, 0.55-1.34) (Pinteraction=0.046 and 0.47 for cancer-specific and overall survival, respectively). In Kaplan-Meier method, the differential effect of post-diagnosis physical activity on patient survival according to nuclear CTNNB1 status was also evident (Figure 2).

Figure 2
(A-B) Kaplan-Meier curves for colorectal cancer-specific (A) and overall survival (B) in nuclear CTNNB1-negative stage I-III cases according to post-diagnosis physical activity. (C-D) Kaplan-Meier curves for colorectal cancer-specific (C) and overall ...
Table 4
Colorectal cancer (stage I-III) mortality by level of post-diagnosis physical activity in strata of nuclear CTNNB1 (β-catenin) status

DISCUSSION

We performed this study to test the hypothesis that CTNNB1 (β-catenin) status in colorectal cancers interact with patient’s BMI or post-diagnosis physical activity, and modify tumor cell behavior. We found a substantial interactive prognostic associations of tumor CTNNB1 and self-reported BMI or post-diagnosis physical activity. Specifically, nuclear CTNNB1 positivity was associated with significantly better cancer-specific and overall survivalin obese patients, while nuclear CTNNB1 status was not associated with survival among nonobese patients. Furthermore, in stage I-III nuclear CTNNB1-negative cases, high post-diagnosis physical activity (≥18 MET hours/week) was associated with significantly better cancer-specific survival, while physical activity was not associated with survival among stage I-III nuclear CTNNB1-positive cases. These results provide evidence for a possible interactive effect of tumor CTNNB1 and patient’s energy balance status in determining tumor cell behavior. Our data support the hypothesis that progression of WNT-CTNNB1-inactive tumor might be influenced by energy intake and expenditure, whereas WNT-CTNNB1-active tumor might progress independent of energy balance status. Although our data need to be confirmed by independent datasets, tumor CTNNB1 status may serve as a predictive biomarker for response to exercise prescription in clinical practice. Because physical activity is a modifiable lifestyle factor, our data may have considerable clinical implications.

Examining molecular changes or prognostic factors is important in cancer research.32,33 Previous prognostic data on CTNNB1 alterations in colorectal cancer are inconclusive.34-52 Two large studies42,43 (N>540) demonstrated no prognostic role of CTNNB1 alterations. However, none of the previous studies34-52 has examined interactive associations of tumor CTNNB1 and patient’s BMI or physical activity.

A host-tumor interaction that appears to modify tumor cell behavior has been first described between BMI and FASN expression in colon cancer.53 Examining host-tumor interactions has been a novel research paradigm in the evolving interdisciplinary field of “Molecular Pathological Epidemiology (MPE)”.54,55 Investigating whether lifestyle interventions are more or less beneficial based on tumor molecular subtypes provides important information regarding mechanisms of action that can, in turn, inform mechanistically-driven clinical trials to optimize the efficacy of such lifestyle interventions. We have previously examined interactions of several molecular markers with physical activity, and found that the benefit of physical activity may be influenced by tumor CDKN1B (p27) status.31 Specifically, physical activity after colon cancer diagnosis was associated with better cancer-specific survival in CDKN1B-expressing tumors but not in CDKN1B-lost tumors.31 It would be of particular interest to examine interactive effects of CDKN1B, CTNNB1 and physical activity in well-powered trial settings in the future. In addition, further studies are warranted to examine the exact mechanism of how physical activity modulates the CTNNB1 or CDKN1B signaling pathway to influence tumor cell behavior.

Prospective observational data suggest that physically active colorectal cancer survivors have lower rates of cancer recurrence and better survival compared with inactive survivors.14,15,56 Physical activity is a modifiable lifestyle factor, and thus its beneficial effect on cancer survivors has considerable clinical implications. However, as with any other oncological intervention, it is unlikely that all patients will universally gain a benefit from exercise. As evidence grows that nondrug interventions such as physical activity can influence outcome of patients with established cancer, there is a need to better delineate subpopulations of cancers that may or may not be more likely to be impacted by such an intervention. Thus, it is of particular interest to identify patient characteristics or a tumor biomarker which can predict response to exercise intervention. In the current study, we have found CTNNB1 status as such a candidate predictor. Although our data need to be confirmed by additional studies, nuclear CTNNB1 status may improve the identification of patients who will benefit most from physical activity. Future prospective intervention studies may be warranted.

There are limitations in this study. For example, data on cancer treatment were limited. Nonetheless, it is unlikely that chemotherapy use substantially differed according to CTNNB1 alterations in tumor, since such data were not available for treatment decision making. In addition, our multivariate survival analysis finely adjusted for disease stage (I, IIA, IIB, IIIA, IIIB, IIIC, IV, unknown), on which treatment decision making was mostly based. We recognize that we cannot exclude the possibility that dosing, completion, or dose modification rates of adjuvant therapy might vary according to BMI or physical activity. However, approximately 60% of patients included in our analysis had stage I or stage II disease, for whom surgery alone would generally be the standard care, and no interaction between physical activity and disease stage was observed in our previous analyses of this cohort.56 Thus, we consider that the confounding effects, if any, of different dosing, etc, of adjuvant therapy, would not have been substantial. As another limitation, beyond cause of mortality, data on cancer recurrences were not available in these cohorts. Nonetheless, colorectal cancer-specific survival is a reasonable surrogate of colorectal cancer-specific outcome.

There are advantages in utilizing the database of the two prospective cohort studies. Data on anthropometric measurements, physical activity, cancer staging, and other clinical, pathologic, and tumoral molecular variables were prospectively collected, blinded to patient outcome. Cohort participants who developed cancer were treated at hospitals throughout the U.S., and thus more representative colorectal cancer cases in the general U.S. population than patients in one to a few academic hospitals. In addition, we assessed the effect of CTNNB1 alterations independent of other critical molecular events such as BRAF and PIK3CA mutations, LINE-1 hypomethylation, CIMP and MSI, all of which have been associated with colorectal cancer prognosis.23,57,58

In conclusion, our data provide evidence for an interaction between CTNNB1 alterations in colorectal cancer and patient’s energy balance status, which influences tumor cell behavior. Notably, there appear to be substantial modifying effects of tumor CTNNB1 status on the beneficial prognostic role of post-diagnosis physical activity. Physical activity is associated with better cancer-specific survival only in patients with nuclear CTNNB1-negative colorectal cancers, whereas physical activity is not associated with survival in nuclear CTNNB1-positive cases. Our findings may have considerable clinical implications because physical activity is a modifiable lifestyle factor.

Supplementary Material

Supplementary data

Acknowledgments

We deeply thank the Nurses’ Health Study and Health Professionals Follow-up Study cohort participants who have agreed to provide us with information through questionnaires and biological specimens; hospitals and pathology departments throughout the U.S. for generously providing us with tissue specimens. We thank the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY.

Funding/Support: This work was supported by U.S. National Institute of Health (NIH) grants P01CA87969 (to S.E. Hankinson), P01CA55075 (to W.C. Willett), P50CA127003 (to C.S.F.), K07CA122826 (to S.O.), R01CA151993 (to S.O.), and R01CA137178 (to A.T.C.) and by grants from the Bennett Family Fund and the Entertainment Industry Foundation through National Colorectal Cancer Research Alliance. A.T.C. is a Damon Runyon Clinical Investigator. T.M. was supported by a fellowship grant from the Japan Society for Promotion of Science. The content is solely the responsibility of the authors and does not necessarily represent the official views of NCI or NIH. Role of the Sponsor: The sponsors had no role in the design and conduct of the study; in the collection, management, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript.

Abbreviations

BMI
body mass index
CI
confidence interval
CIMP
CpG island methylator phenotype
HR
hazard ratio
MET
metabolic equivalent task
MSI
microsatellite instability
MSS
microsatellite stability
OR
odds ratio

Footnotes

A.T.C is a consultant of Bayer Healthcare. No other conflict of interest exists.

T.M. and S.O. had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

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