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From the Fred Hutchinson Cancer Research Center, Seattle, WA (KFA, PDL, DM, EW, JDP, and JWL); the University of Washington (KFA, PDL, DM, SSE, EW, JDP, and JWL), Seattle, WA; the Center for Health Studies, Group Health Cooperative, Seattle, WA (KMN); and the Gastroenterology, Group Health Cooperative, Seattle, WA (JTY and AF)
2 Supported by NIH grants U01 CA72035, R03 CA92772, and T32 ES07262 (to KFA) and FHCRC Core grant CA15704. 3 Reprints not available. Address correspondence to JW Lampe, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, MP-900, Seattle, WA. E-mail: jlampe{at}fhcrc.org.
ABSTRACT
Background: Soy isoflavones have numerous biological properties that suggest that they may protect against colorectal cancer. Colorectal epithelial cell proliferation has been used extensively as an intermediate endpoint biomarker for colorectal neoplasia.
Objective: We tested the hypothesis that supplementation with soy protein containing isoflavones decreases colorectal epithelial cell proliferation.
Design: A 12-mo randomized intervention was conducted in men and women aged 50–80 y with recently diagnosed adenomatous polyps. One hundred fifty participants were enrolled and randomly assigned to an active treatment group (58 g protein powder/d containing 83 mg isoflavones/d; +ISO) or a control group (ethanol-extracted soy-protein powder containing 3 mg isoflavones; –ISO). Biopsy specimens from the cecum, sigmoid colon, and rectum were collected at baseline and at the 12-mo follow-up. Ki-67 antibody immunohistostaining was used to detect cell proliferation. One hundred twenty-five participants completed the study, and proliferation was measured in the first 91 who completed the study.
Results: In the sigmoid colon, cell proliferation increased by 0.9 (95% CI: 0.09, 1.9) labeled nuclei per crypt more (11%) in the +ISO group than in the –ISO group over the 12-mo intervention, which was opposite the direction predicted. The number of labeled nuclei per 100 µm crypt height also increased more in the +ISO than in the –ISO group. In the cecum and sigmoid colon, but not in the rectum, the proliferation count increased as the serum genistein concentration increased. Proliferation distribution and crypt height were not changed by treatment at any site.
Conclusions: Supplementation with soy protein containing isoflavones does not reduce colorectal epithelial cell proliferation or the average height of proliferating cells in the cecum, sigmoid colon, and rectum and increases cell proliferation measures in the sigmoid colon.
Key Words: Soy isoflavone • genestein • randomized controlled trial • colon • epithelial cell proliferation • Ki-67 antibody
INTRODUCTION
Dietary intake of soy is substantially higher and the incidence of colorectal cancer, with the recent exception of Japan, is much lower (1) in Asia than in the United States (2). Soy contains many biologically active compounds, including isoflavones, and it has been proposed that a high intake of these soy phytochemicals contributes in part to the lower incidence of colorectal cancer in Asia (3). In men and postmenopausal women, who have low endogenous estrogen concentrations, isoflavones are hypothesized to have estrogenic effects (4). Given that exogenous estrogen use has been associated with a decreased risk of colon cancer (5, 6), isoflavones may similarly influence colon cancer risk through their estrogenic action. Soy isoflavones have several anticarcinogenic properties in vitro (7) and genistein, one of the predominant isoflavones in soy, inhibits proliferation of cultured cells, including normal and transformed intestinal epithelial cells (8). However, epidemiologic (7, 9–11) and animal studies (7, 12) of effects of soy or isoflavones on colon cancer or its precursors have yielded mixed results. One human intervention trial providing either a soy or casein supplement for 12 mo reported reductions in colon epithelial cell proliferation in the soy group (13).
Colorectal epithelial cell proliferation has been used as an intermediate endpoint in several dietary intervention studies (14). It is controversial whether proliferation in itself is sufficient to cause colorectal cancer (15); however, for stem cells already having a colon cancer–related mutation, an increased rate of cell division results in a larger accumulation of mutations over time, which increases the likelihood of other colon cancer–related mutations occurring (16–18). Studies in humans show increased cell proliferation in the normal mucosa of patients with adenomas or colorectal cancer and increased proliferation in persons at greater risk of colorectal cancer because of age, family history, previous polyps, or genetic predispositions (reviewed in 19), although many of the findings have been inconsistent (14, 20, 21). Recently, Ki-67 antibody immunohistostaining has become available for labeling proliferation in preserved tissues. Ki-67 is an endogenous nuclear protein associated with the cell cycle. The Ki-67 antibody identifies cells in the cycling phases but not in the resting phase (G0) or early phase (G1) (22, 23). Ki-67 labeling is considered to have several advantages over other commonly used assays for proliferation (24).
Our objective was to evaluate whether isoflavones in soy protein modulate colorectal epithelial cell proliferation, measured by Ki-67. We hypothesized that a 12-mo dietary intervention providing 83 mg isoflavones/d in a soy-protein matrix would reduce cell proliferation measures in the cecum, sigmoid colon, and rectum.
SUBJECTS AND METHODS
Study design
The study design was described previously (25). The Soy Isoflavone Prevention Study was a 12-month randomized, double-blinded, placebo-controlled dietary intervention evaluating the effects of soy isoflavones on colonic epithelial cell proliferation and other potential markers of colon cancer risk. Participants were recruited among patients who were undergoing colonoscopy at 2 gastroenterology clinics at Group Health Cooperative (GHC) a large managed-care organization. Recruitment began January 1998 and was completed March 2001. Men and women were eligible if they were between the ages of 50 to 80 y and had adenomatous colorectal polyps found on colonoscopy. Persons with previous adenomas were eligible. Exclusions included recent hormone use (testosterone in men, estrogen in women), certain medical conditions (medical history of chronic bleeding disorders, liver or digestive diseases, cancer, laxative or enema dependence, familial polyposis, alcohol or drug dependence, cancer, inflammatory bowel disease, and ulcerative colitis), and soy intake 4 servings/wk. Initially, participants were excluded if they used non-steroidal antiinflammatory drugs (NSAIDs) regularly, but this exclusion was lifted after the first year to increase enrollment. Similarly, the restriction on hormone therapy was revised to allow women to participate who had not used hormone therapy in the past 6 mo. The study activities were approved by the Institutional Review Boards of the Fred Hutchinson Cancer Research Center, Seattle, WA and GHC, Seattle, WA, and informed, written consent was obtained from all study participants.
Subjects
Participants were recruited in a two-phase process. In the first phase, a GHC survey team screened patients scheduled for colonoscopy at 2 GHC gastroenterology clinics and requested interviews at the clinic with age-eligible patients. Age-eligible health plan enrollees scheduled for colonoscopy were contacted by telephone, and permission was requested to discuss the study with the patient at the clinic before the procedure. Of 4085 patients screened, 1513 were eligible based on screening criteria (eg, hormone use, diseases or GI surgeries, NSAID use before criteria change), and 749 consented to have biopsies collected in the event that polyps were found during colonoscopy. The study staff of the Soy Isoflavone Prevention Study attended 648 colonoscopies. GHC pathologists analyzed polyp histology. Two hundred twenty-eight patients were eligible for the dietary intervention on the basis of having adenomatous polyps and not having cancer or other gastrointestinal conditions. Of this group, 150 consented to the dietary intervention. A 1-wk run-in was conducted with the use of an ethanol-extracted soy-protein drink that was low in isoflavones. All patients successfully completed the run-in by consuming 80% of the soy-protein packets and tolerating the soy.
One hundred fifty participants were randomly allocated to 1 of 2 treatment groups and stratified by sex and clinic. From January 1998 onward, participants were also randomly allocated according to NSAID use. One group was provided 58 g/d of soy-drink powder (2 packets/d, 29 g/packet; The Solae Co, formerly Dupont Protein Technologies, St Louis, MO) to consume for 12 mo (+ISO group). A daily serving of soy-drink powder contained 40 g protein, 200 kcal energy, and 1400 mg Ca and provided 45.6 mg genistein, 31.7 mg daidzein, and 5.5 mg glycitein (aglycone units). The other group was provided an ethanol extract of the +ISO powder that provided only small quantities (3 mg total isoflavones; 4% of the isoflavones provided by the +ISO powder) of these isoflavones (–ISO group). The participants were given extensive instruction on how to incorporate the soy drink into their typical diet. Participants who returned <20% of the packets over the 12-mo intervention were considered to be compliant. The participants, investigators, and staff were blinded to the participants' isoflavone treatment.
Ki-67 labeling of colonic epithelial cell proliferation
Colonic epithelial cell biopsy specimens were collected during the initial colonoscopy, before potential participants were enrolled in the dietary intervention, and at the end of the 12-mo intervention. Before the colonoscopy was performed, 136 participants received a standard preparation of Fleet Phospho-Soda (CB Fleet Co, Inc, Lynchburg, VA), whereas 14 received Colyte (Scharz Pharma, Milwaukee, WI). The participants received the same bowel preparation during the follow-up procedure as they received during the initial colonoscopy. During the colonoscopy, 1-mm thick biopsy specimens were collected by physicians trained in the procedure. Fifteen biopsy specimens were collected from each participant with the use of jumbo biopsy forceps (Olympus FB-50U1-1; Olympus America Inc, Melville, NY). Nine specimens were processed for the analysis of proliferation; 3 each from the cecum, the sigmoid colon (10–25 cm from the level of the external anal aperture), and the rectum (up to 10 cm from the level of the external anal aperture).
The biopsy specimens were oriented and placed in 10% neutral buffered formalin for 2–3 h and then transferred to 70% ethanol. Eight to 12 sections, 50 microns apart, were cut from each specimen and mounted with positive control (human tonsil) and negative control (colon biopsy tissue from the same participant with all reagents except primary antibody) tissues. Sections were deparaffinized with the use of xylene, ethanol, and water. All slides from a single participant (baseline and follow-up, for all 3 sites) were stained in the same run to minimize the effect of run-to-run variability in staining intensity inherent in the procedure. Slides were placed in either boiling Antigen Retrieval Buffer (DAKO Corp, Carpenteria, CA) or in boiling citrate buffer (10 mmol/L; pH 6.0) and steamed for 30 min. After being cooled for 20 min, the slides were stained on a Techmate 1000 Staining System automatic immunostainer (Ventana Medical Systems Inc, Tucson, AZ) with the use of detection reagents from the Vectastain Elite Standard ABC Kit (Vector Laboratories, Inc, Burlingame, CA). Slides were blocked for 5 min with 4% normal horse serum in phosphate-buffered saline (PBS) with Tween-20 and then buffer-washed. The biopsy specimens were stained with the Ki-67 (MIB-1) monoclonal antibody (Immunotech Inc, Miami, FL) at a 1:500 dilution for 1 h in PBS plus 1% bovine serum albumin (BSA) and Tween-20 and then buffer-washed. They were immersed in the secondary antibody, biotinylated rabbit anti-mouse immunoglobulin G (Jackson ImmunoResearch, West Grove, PA), at a dilution of 1:2000 or biotinylated horse anti-mouse immunoglobulin G (Vector Laboratories Inc) at a dilution of 1:200 for 30 min in PBS with 1% BSA with Tween-20 and then buffer-washed. Endogenous peroxidases were inactivated by treatment with hydrogen peroxide for 5 min. Slides were then washed and immersed in Avidin-Biotin Complex reagent for 30 min. After being washed, the slides were developed by using diaminobenzidine tetrahydrochloride solution at a concentration of 0.5 mg/mL in PBS with 0.01% hydrogen peroxide. The stained slides were then dehydrated, and cover slips were applied.
The biopsy specimens were scanned under a Nikon E400 microscope (Nikon Inc, Melville, NY) with a 4x objective to identify acceptable colon crypts. Acceptable crypts were defined as being midaxial, with U-shaped sections extending from the muscularis to the lumen with an intact structure. Images of the crypts were captured by using a digital camera (Hamamatsu Photonics KK, Hamamatsu City, Japan) attached to the microscope with 20x objective and set at constant illumination and exposure settings. Nuclei were counted if they were positioned on the edge of the crypt cell columns and fully darkened. Partially darkened nuclei were not counted to exclude nuclei lying below the plane of the cell column. Macros developed for the NIH Image software program (available on the Internet: http://rsb.info.nih.gov/nih-image) allowed us to collect and analyze images. Biopsy tissue was stained generally in the order that follow-up biopsy specimens were collected from the participants, and the slides were scored generally in the order that they became available. We aimed to capture 3 crypts for each combination of person, time, and site. A sequential sampling plan was used that could potentially allow scoring fewer than the total number of samples if the results allowed hypotheses to be addressed conclusively.
Five outcome variables were defined, including 2 measures of proliferation count, 2 measures of proliferation distribution, and crypt height. Cell proliferation count (number of proliferating cells per crypt) was measured in absolute and relative scales as the number of labeled (stained) nuclei per crypt and as the number of labeled nuclei per 100 µm crypt height. The distribution of labeled nuclei (height of proliferating cells within a crypt) was measured in absolute and relative (proportional) scales as the average height of a labeled nucleus in the crypt and the average height of a labeled nucleus divided by the height of the crypt. Crypt height, the distance from base to lumen, was also analyzed as an outcome variable.
Serum isoflavones
We measured serum genistein concentrations in fasting morning blood samples collected at baseline and at 4, 8, and 12 mo with the use of a gas chromatography–mass spectrometry method that was described previously (25). Most of the samples were collected between 0700 and 1200.
Statistical analysis
Measurements for each crypt were averaged to obtain single values at the level of participant, site, and time. The statistical analysis involved a comparison of mean changes in Ki-67 count and mean height between the +ISO and –ISO groups after a 12-mo intervention with the use of a linear regression model with robust SE estimates to account for unequal variances (Stata 8.0; Stata Corp, College Park, TX). After proliferation was measured in the first 91 participants for whom biopsy specimens were available, the data were analyzed and a level 0.025 one-sided symmetric stopping rule with O'Brien-Fleming boundaries (26) was applied to assess whether additional biopsy specimens were needed to be scored. During this analysis it was determined that the data were sufficiently precise to be highly confident in the decision to stop scoring. For the primary analysis we adjusted treatment-effect means, CIs, and P values by using group sequential methods (27) to account for the fact that we stopped staining and scoring biopsy specimens after performing such for the first 91 participants with available biopsy specimens (S+SeqTrial; Insightful Corp, Seattle, WA). We used a one-sided of 0.025 to test the null hypothesis that the intervention did not reduce proliferation. A one-sided test was used because our intention was to stop staining and scoring crypts if we did not find a reduction in proliferation. The primary analysis was by intention-to-treat, based on comparisons between groups as defined at randomization. We adjusted for baseline values of the response variable. We did not adjust for multiple comparisons. In secondary analyses, which are exploratory, we did not use group sequential methods because the results had no potential to affect our decision of whether to stain and score the remaining biopsy samples. We used 2-sided tests (overall of 0.05) because we were interested in exploring changes in proliferation in either direction.
RESULTS
We conducted an analysis using measurements obtained from the first 91 participants from whom biopsy specimens were available. Of 150 participants who were randomly assigned to treatment and began the study, 125 completed the dietary intervention and underwent the follow-up colonoscopy. Because women who received hormone therapy were excluded (60% of the women screened), the study population was mostly men (Table 1) . Although randomization was blocked on treatment allocation, sex, clinic, and NSAID use in this interim analysis, the 2 groups were somewhat unbalanced in terms of treatment allocation, sex, and the proportion of NSAID users. By chance, the 91 participants we analyzed included fewer participants from the +ISO group than from the –ISO group (40 compared with 51). The +ISO group contained more women (20.0% compared with 5.9%) and fewer NSAID users (30.0% compared with 39.2%). The proportion of participants who consumed any soy outside the study at baseline was also lower in the +ISO group (7.5% compared with 21.6%). On the basis of packet counts, our primary measure of compliance, 31 (77.5%) of the 40 +ISO participants and 44 (86.3%) of the 51 –ISO group participants (P = 0.3) were compliant over the 12-mo intervention. Participants who adhered to the study tended to be highly compliant, and consumed 94% of their soy over the entire 12 mo. However, the proportion of participants who were compliant decreased as the study progressed: from 88.5% in the +ISO group in the first 4 mo to 72.5% from 5 to 12 mo.
View this table:
TABLE 1. Baseline characteristics of the Soy Isoflavone Prevention Study participants, by intervention group1
Serum genistein concentration, a secondary measure of compliance, was similar in the +ISO and –ISO groups at baseline and increased substantially in the +ISO group at 4, 8, and 12 mo (Figure 1), whereas genistein concentration in the –ISO group changed little over the course of the intervention. The average numbers of crypts scored per person at each time point were 6.1 in the cecum, 7.4 in the sigmoid colon, and 6.2 in the rectum. Median weight gain was not significantly different between groups: 0.3 kg in the +ISO group and 0.5 kg in the –ISO group.
FIGURE 1.. Geometric mean fasting serum genistein concentrations (and 95% CIs) over 12 mo in subjects supplemented with either 83 mg soy isoflavones/d (+ISO group) or 3 mg isoflavones/d (control; –ISO group). Concentrations in the +ISO group differed significantly from those in the –ISO group at 4, 8, and 12 mo, P = 0.05 (independent-sample t test).
Overall, we found little change in mean proliferation measures over the course of the intervention, and none of the measures of cell proliferation were reduced (Table 2). In the sigmoid colon, the 2 measures of proliferation count increased with +ISO treatment, which was opposite our hypothesis. The number of labeled nuclei per crypt increased by 0.9 (95% CI: 0.09, 1.8) more (11%) in the +ISO group than in the –ISO group (control group). The high one-sided P value (P = 0.99) strongly argues that intervention does not reduce proliferation. Similarly, the number of labeled nuclei per crypt per 100 µm crypt height increased by 0.2 (95% CI: 0.07, 0.36) more (15%) in the +ISO group than in the –ISO group.
View this table:
TABLE 2. Effect of treatment group on colorectal cell proliferation after a 12-mo intervention1
The increase in proliferation count (number of labeled nuclei per crypt) in the sigmoid colon was reduced slightly after adjustment for baseline NSAID use. After adjustment for NSAID use, the number of labeled nuclei increased by 0.8 per crypt more in the +ISO group than in the –ISO group (95% CI: –0.007, 0.14 labeled nuclei per crypt; NS), which was slightly less than the 0.9 increase observed for this variable in the unadjusted analysis. Adjustment for sex, clinic site, or other covariates did not significantly change the results. The results were similarly null when the analysis was restricted to men. We had too few women to consider them separately. Restricting the analysis to participants who were compliant, based on packet counts, also had little effect on the estimates.
We also analyzed the results observationally, using serum genistein concentrations as an indicator of internal dose or compliance (Table 3). This analysis included the combined intervention and control group participants. We observed increases in proliferation count in the sigmoid colon in participants with higher serum genistein concentrations, consistent with the intention-to-treat analysis. In contrast with our intention-to-treat analysis, we found higher proliferation count in the cecum in association with higher serum genistein concentrations in the observational analysis. In none of our analyses did higher serum genistein concentrations at 12 mo correspond with a decrease in proliferation.
View this table:
TABLE 3. Associations between changes in colorectal epithelial cell proliferation measures from baseline to 12 mo and serum genistein concentrations at 12 mo of the soy isoflavone intervention in both treatment groups combined
DISCUSSION
We found no reduction in colorectal epithelial cell proliferation after a 12-mo dietary soy isoflavone intervention in a study population of predominantly men aged 50- to 80 y with previous adenomatous polyps. Our results did not change significantly when we restricted the analysis to subjects who were compliant or when we analyzed the dependence of proliferation measures on the serum genistein concentration—an internal marker of isoflavone intake.
Bennink et al (13, 28) conducted a randomized intervention study in which participants with a history of colon polyps or cancer consumed 39 g/d of isolated soy protein containing isoflavones (n = 29) or 40 g/d of casein (n = 13) for 12 mo. Although details about the study were limited, the authors reported that isolated soy protein reduced both the labeling index and proliferation distribution in colonic mucosa (site not specified) when measured by proliferating cell nuclear antigen (PCNA) but not when measured by Ki-67. The authors interpreted the difference in results to be due to methodologic differences: PCNA measures the capability for proliferation, whereas Ki-67 measures cells in the S-phase. Our choice of Ki-67 was based on evidence that it is less sensitive to variations in laboratory conditions and more selective than is PCNA for cycling cells (24, 29).
Our measures differ slightly from those of previously reported studies that analyzed colon or rectal cell proliferation, primarily because we measured the length of the crypt rather than counted the number of unlabeled cells in the crypt column; the former method is definitely more time-efficient and possibly more accurate than are counts of unlabeled cells. Our relative measure of proliferation count, the number of labeled nuclei adjusted for crypt length, is similar to the labeling index used in most studies. We used the average height of a labeled cell as our measure of proliferative distribution, rather than the proportion of labeled cells in the upper compartment (30). This measure, also known as phih, is similar to proliferative height, a measure used by others (31, 32), and because it is a continuous variable, the average height of a labeled cell is richer in information than is the proportion of labeled cells in the upper compartment. We considered crypt height as a measure of crypt morphology. Crypt height probably reflects the joint effects of proliferation and cell loss. Certain agents may induce cell loss, which results in decreased crypt length (33). The crypt may then increase proliferation and inhibit apoptosis as a compensatory response. Alternatively, an increase in crypt length suggests proliferation unopposed by apoptosis. A related question is whether the absolute or relative measure of proliferation is more important, ie, whether proliferation should be adjusted for length of crypt. We reported both. In our study, the lack of difference in crypt height between the +ISO and –ISO groups suggests that the small increase in proliferation was counterbalanced by apoptosis or that the increase was not sufficient to alter the measure of crypt height.
Neither NSAID use by the study participants nor calcium supplementation with the soy-protein product likely explain our results. Aspirin was shown to reduce adenomatous polyps in chemoprevention trials (34, 35) and to inhibit proliferation in vitro (36); however, overall, NSAIDS have not been found to inhibit colorectal cell proliferation in chemoprevention trials (reviewed in 14). In our study, adjustment for NSAID use did not appreciably affect the results. Participants in both treatment groups were supplemented with 1400 mg Ca/d. Calcium reduced proliferation in some controlled interventions (19, 37). However, we did not observe a decrease in proliferation over time in either treatment group; thus, an effect of calcium was unlikely.
Contrary to expectations, we found with some measures that soy isoflavones increased proliferation rather than decreased it. Our exploratory analysis showed increases in proliferation in the cecum and sigmoid colon, but not in the rectum, with increasing serum genistein concentrations. This may reflect a luminal dose effect along the length of the colon or a difference in tissue response. It also suggests that rectal biopsy specimens are not sufficient to monitor interventions in other parts of the colon. In vitro, genistein appears to increase proliferation at low concentrations but to decrease it at higher concentrations (7, 38). Animal studies have yielded contradictory results regarding protective effects of soy or its components on tumors (7, 39). Consistent with our findings, one study found that dietary soy protein increased colon cell proliferation in rats (40); compared with milk protein, soy protein, in the context of a high-fat, low-calcium diet, doubled proliferation in rat colon.
Our study had several strengths. It was a randomized intervention and was thus free of the biases that are inherent in observational studies. The participants and study personnel were blinded to the participants' treatment assignment. We measured 3 sites in the colon, including the cecum. In contrast, many studies measured the effects of interventions on the rectum only. Biopsy specimens from the same participant were processed and scored together to minimize intraindividual variability due to batch-to-batch differences in immunostaining. For each combination of person, time, and colon site, we scored 2 biopsy specimens and multiple crypts to increase precision. The study also had limitations. The completion rate was less than desired and was lower in the +ISO than in the –ISO group. This finding raises the question of whether dietary isoflavones are difficult to tolerate. However, the proportion of participants who withdrew because of health-related reasons was similar in the 2 groups (25). Compliance was also less than optimal, although most participants who completed the study consumed at least one-half of the soy packets.
This study measured cell proliferation, a potential intermediate endpoint, rather than adenomatous polyp recurrence or colon cancer. Polyps and incident colon cancer occur too rarely to be measured without an extremely large study population. It is possible that soy isoflavones affect the recurrence of polyps or incident colon cancer without affecting cell proliferation in flat mucosa. The measurement of colorectal epithelial cell proliferation has been shown to be imprecise (32), and our null findings may have been due to measurement error. However, given the small effects we observed, we conclude that soy isoflavones have no effect on proliferation, at least as measured by the methods used.
In conclusion, we analyzed the effect of a soy isoflavone intervention on 3 sites in the colon on the basis of 5 different measurements. We found no evidence that a 12-mo dietary soy isoflavone intervention decreases colorectal epithelial cell proliferation in a study population of mostly men aged 50–80 y with previous adenomatous polyps.
ACKNOWLEDGMENTS
We thank the study staff of the GHC (Kelly Ehrlich, Linda Palmer, and Tina Stroh) and the clinic staff and physicians of the Central (Lanie Belic, Rubeela Malik, Venkatchala Mohan, David Perera, Shie-Pon Tzung, and Herbert Weisberg) and Eastside (Janet Chu, Charles Janeway, and Flavio Manela) GHC gastroenterology clinics, without whom this study would not have been possible. We also thank Elizabeth Levy, JoAnn Prunty, Heidi Warner, Vivian Li, and the staff of the Research Pathology Shared Resource at the Fred Hutchinson Cancer Research Center.
None of the authors had a conflict of interest.
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