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This study represents a multiplex cytokine analysis of serum from a 10-month randomized, controlled trial of 238 subjects that investigated the effects of selenomethionine and/or celecoxib in subjects with mild or moderate esophageal squamous dysplasia. The original chemoprevention study found that among those with mild dysplasia, selenomethionine treatment favorably altered dysplasia grade. The current analysis found that selenomethionine down-regulated IL-2 by 9% (p=0.04), while celecoxib down-regulated IL-7 by 11% (p=0.006) and up-regulated IL-13 by 17% (p=0.008). In addition, an increase in IL-7 tertile from baseline to t10 was significantly associated with an increase in dysplasia grade, both overall (OR=1.47, p=0.03) and among those with mild dysplasia at t0 (OR=2.53 p=0.001). An increase in IL-2 tertile from baseline to t10 was also non-significantly associated with worsening dysplasia for all participants (OR=1.32 p=0.098), and significantly associated with worsening dysplasia among those with mild dysplasia at baseline (OR=2.0 p=0.01). The association of increased IL-2 with worsening dysplasia remained significant in those on selenomethionine treatment who began the trial with mild dysplasia (OR=2.52 p=0.03). The current study shows that selenomethionine supplementation decreased serum IL-2 levels, while celecoxib treatment decreased IL-7 levels and increased IL-13 levels during a 10 month randomized chemoprevention trial. An increase in IL-2 or IL-7 was associated with increased severity of dysplasia over the course of the trial, especially in those who began the trial with mild dysplasia. The favorable effect of selenomethionine on esophageal dysplasia in the original trial may have been mediated in part by its effect on reducing levels of IL-2.
The relationship between immunologic competence and cancer is becoming increasingly apparent. Impaired immunologic responses have been identified in many types of cancer patients, including patients with pancreatic, breast (1), lung, ovarian (2), gastric (3), and esophageal (4;5) malignancies. The potential etiologic significance of these altered responses, especially as they may relate to aerodigestive tract cancers, is illustrated by the significantly increased risk of cancer among those with autoimmune diseases and allergic conditions (6;7).
Poor nutrition is a frequent finding in areas with high risk of cancer, and poor nutrition can be associated with impaired immunologic function. Poor nutrition may result in an inadequate intake of immunologically essential nutrients, such as selenium, zinc, vitamin C and vitamin E (8), that influence Th1 and Th2 responses (9) and that are particularly important during critical periods of gestation and neonatal maturation (10).
Our group has conducted multiple studies of the association of nutrition and esophageal and gastric cancers in Linxian, China, a region with some of the highest rates of esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma in the world (11). These include the Linxian General Population Nutrition Intervention Trial (12;13). This study supplemented 29,584 residents for 5.25 years with different combinations of nutrient supplements or placebo. It found that subjects supplemented with selenium, beta-carotene, and alpha-tocopherol had a statistically significant reduction in all-cause mortality (9%) and in total cancer mortality (13%) (13). A prospective study of this cohort during the 5.25 yr trial period found an inverse relation between baseline serum selenium concentrations and risk of ESCC; the relative risk (95% CI) of the highest to lowest quartiles was 0.56 (0.44-0.71) (14). Continued follow-up of this cohort for 10 years after the trial period confirmed the persistence of this inverse association (15). We subsequently conducted a randomized, controlled 10 month intervention of selenomethionine 200ug QD and/or celecoxib 200mg BID (2 × 2 factorial design) that included 238 residents of Linxian with biopsy-proven mild or moderate squamous dysplasia (16). This Linxian Chemoregression Study (CRS) was designed to determine whether supplementation with the antioxidant micronutrient selenium and/or the COX-2 inhibitor celecoxib would affect the progression of esophageal squamous dysplasia, the precursor lesion of invasive ESCC. Among the 115 subjects who had mild dysplasia at baseline, selenomethionine treatment favorably altered dysplasia grade (p=0.02). No effect on dysplasia was seen with celecoxib treatment (16). Further molecular evaluation of the CRS suggested that the beneficial treatment effect may have been related to modulation of immune function (17;18). Individuals whose lesions regressed had higher RNA expression in genes associated with immune stimulation, including antigen presentation, T-cell activation and T-cell survival (17). However, selenomethionine supplementation did not have a measurable effect on the gene expression profiles of normal squamous esophageal mucosa (18).
Both the seleniomethionine and celecoxib supplements in the CRS have possible associations with immune function. Selenium is incorporated as selenocysteine into a variety of selenoproteins and has been shown to enhance cell-mediated immune responsiveness and to inhibit NF-kappa B activation, which leads to reduced expression of NF-kappa B target genes, including inflammatory cytokines (19;20). Celecoxib's immunologic role relates to its effect on COX-2 and is well illustrated by its role in colon carcinogenesis and treatment (21). This includes the association between NSAID use and regression of colorectal polyps in patients with familial adenomatous polyposis and, of particular relevance to the current study, COX-2's role in prostaglandin synthesis and related inflammatory mediators, downstream effectors on gene expression and apoptosis.
To explore the possibility that the selenomethionine effect on mild dysplasia identified in the CRS was mediated by changes in immune function, we measured serum levels of 22 cytokines before and after the trial intervention and evaluated the association of these cytokine levels with selenomethionine or celecoxib treatment, with dysplasia level, and with the CRS intervention effect.
The current study was a multiplex cytokine analysis of serum collected at baseline (at time t0) and at the end of the 10-month CRS intervention (at time t10). Details regarding the CRS subject selection and intervention can be found elsewhere (16). Briefly, a randomized, controlled trial of selenomethionine 200 micrograms daily and/or celecoxib 200 mg twice daily was conducted in a 2 × 2 factorial design among residents of Linxian County, Henan Province, People's Republic of China. All subjects gave written informed consent, and this study was approved by the institutional review boards of the Cancer Institute of the Chinese Academy of Medical Sciences and the U.S. National Cancer Institute. Subjects had histologically confirmed mild or moderate esophageal squamous dysplasia at baseline. Esophagastroduodenoscopy was performed before and after the 10-month intervention. Per-subject change (regression, stable disease, or progression) in the worst dysplasia grade was defined as the primary endpoint. Results were compared by intervention group (selenomethionine versus no selenomethionine; celecoxib versus no celecoxib).
Subjects completed a baseline questionnaire and underwent venipuncture at t0 and t10. The t0 blood samples were all collected within 2 weeks of each other, and the t10 blood samples were all collected during a similar, narrow time frame and immediately placed on ice prior to centrifugation. Seven cc of whole blood was collected in EDTA for hemoglobin, WBC and platelet counts; and an additional 7 cc was collected in a separate tube without anticoagulant which was left to clot and then centrifuged for 10 minutes. An aliquot of the separated serum was sent for chemistry analysis, and the remainder of the serum was stored at −80C until cytokine analysis.
Serum cytokine analysis utilized the Human Cytokine-22-Plex Antibody Bead Kit (‘Beadlyte’, Upstate, Charlottesville, VA). This approach combines the principle of a sandwich immunoassay with Luminex's fluorescent-bead technology. More specifically, beads with defined spectral properties were conjugated to analyte-specific capture antibodies. Samples were pipetted into a 96-well filter-bottom microplate and incubated with the capture antibodies for 2 hours. During a second incubation, the analyte-specific biotinylated detector antibodies were added and incubated with the beads for 1 hour. The analyte-specific biotinylated detector antibodies recognize their epitopes and bind to the appropriate immobilized analytes. Then streptavidin conjugated to the fluorescent protein, R-Phycoerythrin (Streptavidin-RPE), was added and incubated for 30 minutes. The Streptavidin-RPE binds to the biotinylated detector antibodies associated with the immune complexes on the beads, forming a four-member solid phase sandwich. A Luminex 100 instrument (Luminex Corp, Austin, TX) evaluated the spectral properties of the beads and the amount of associated RPE fluorescence. Sixteen control wells per plate were used for a standard serial dilution curve (8 point curve in duplicate).
We utilized a quartet-based design for the sample wells and included four samples from a QC pool on each plate. Each quartet contained a participant from each of the 4 treatment groups. The subjects from each treatment group were randomly permuted and the first person from each of the 4 permuted lists was used to complete the first quartet, then the second person from each list was used to complete the second quartet, and so forth. The t0 and t10 samples of each quartet were included together on the same plate.
Results are presented as the Mean Fluorescent Intensity (MFI) for 100 beads with an individual attached analyte.
The Linxian Chemoregression Study (CRS) had a 2 × 2 factorial design. Both the original trial and the current analysis compared those who received celecoxib (50% of the total study participants) with those who did not receive celecoxib (the other 50% of the study participants) and those who received selenium (50% of the study participants) with those who did not receive selenium (the other 50% of the study participants).
The statistical analysis explored 3 objectives: (1) whether selenium or celecoxib treatments altered cytokine levels; (2) whether baseline cytokine levels were associated with baseline dysplasia, dysplasia at t10, or change in dysplasia, after controlling for treatment effects; and (3) whether the effect of treatment on dysplasia (regression, stable disease, progression) may have been mediated by cytokine levels.
For (1), we compared the t10-t0 differences in median MFI values between treated and untreated subjects for each intervention agent. We used Wilcoxon rank tests with plate-specific ranks of cytokine levels (PROC TWOSAMPL, StatXact 7, Cytel Inc.) because the cytokine distributions had many outliers and were strongly non-normally distributed even under log or square root transformations. We repeated all Wilcoxon tests without plate-specific ranks, but the results were always similar to the results of the Wilcoxon tests with plate-specific ranks (data not shown). We did not formally adjust for multiple hypothesis testing because this study was hypothesis-generating in nature.
Treatment effects were quantified as the difference of the median t10 cytokine level minus the median t0 cytokine level, between participants on versus not on the individual treatments. For example, the selenium treatment effect on t10-t0 cytokine changes was calculated as (median(ct10,sel) − median(ct0,sel)) − (median(ct10,no sel) − median(ct0,no sel)) where c denotes a cytokine level, and the subscript “sel” denotes selenium treatment and “no sel” denotes no selenium treatment. These treatment effects are expressed both as actual change in median MFI values and as percent change in median MFI values.
For (2), we conducted trend tests for each cytokine, using linear models with dysplasia outcomes coded as consecutive integers reflecting ordinality (for dysplasia at one time point: 0=no dysplasia, 1=mild dysplasia, 2=moderate dysplasia, 3=severe dysplasia; for change in dysplasia: −1=regression (mild to no dysplasia, or moderate to mild or no dysplasia), 0=no change, 1=progression (mild to moderate or severe dysplasia, or moderate to severe dysplasia)). Separate trend test models were run for each cytokine at t0, with the cytokine levels categorized into tertiles. The MFI tertiles were coded 1, 2, and 3 for the first, second, and third tertiles, respectively. For the t10-t0 cytokine differences, we subtracted each cytokine's tertile coding at t0 from its tertile coding at t10. Adjusting covariates included plate number and treatment. We fit these models including everyone, and also including only those with mild dysplasia at t0 or those with moderate dysplasia at t0, to look for different effects by initial dysplasia.
For (3), we conducted trend tests using linear models (as in objective (2)) to see if changes in cytokine levels (t10-t0) were associated with changes in dysplasia. If an individual cytokine's t10-t0 change was associated with a change in dysplasia and that cytokine's level was also associated with a treatment that changed dysplasia in the same direction (in objective 1), then we considered this to be evidence that this cytokine may have mediated the treatment effect. For cytokines with significant trend tests, we fit proportional odds logistic regression models (22) to estimate odds ratios for a worse change in dysplasia as the cytokine increased by one tertile from t0 to t10.
To assess the reproducibility of the MFI measurements, we randomly placed 23 pooled-serum QC aliquots across the 6 plates. The within-plate and overall coefficients of variation (CV) for each analyte's MFI were estimated using a variance components model (SAS code available upon request).
The current analysis utilized the CRS cohort (Table 1) that included a selenomethionine plus celecoxib treatment group (N=64), a celecoxib only group (N=57), a selenomethionine only group (N=59) and a placebo group (N=58) (from Limburg et al. 2005 (16)). These groups were similar with regards to age, sex, tobacco use, alcohol consumption and histologic composition. The original intervention study found that among the 115 subjects who had mild dysplasia at baseline, selenomethionine treatment favorably altered dysplasia grade (p=0.02, Table 2). No effect was seen with celecoxib treatment. In the current study of serum cytokine levels, the sample size for the MFI analyses was 108 for those with and 114 for those without selenomethionine supplementation, and it was 111 for those with and 111 for those without celecoxib supplementation.
Exploratory data analysis showed that the cytokine distributions were greatly skewed, with many large outliers. Consequently, we present the summary data using medians, rather than means, with interquartile range (IQR) as a measure of spread, rather than variance.
The within-plate CVs for MFI readings for each cytokine averaged 23% (range: 7%-33%) and the overall CVs averaged 32% (range: 7%-58%). Our analyses reduced the effect of between-plate variation by either stratifying on plate or including plate as a covariate for regression adjustment. Thus, the within-plate CVs are more relevant to our analyses.
First, we tested if selenomethionine or celecoxib treatments were associated with changes in cytokine levels from t0 to t10. Selenomethionine treatment was associated with a lower t10-t0 difference in IL-2 (a median effect (IQR) of −1.0 (7.4) MFI, a −9.0% change, p=0.04) (Table 3). Celecoxib treatment was associated with decreased IL-7 (a median effect (IQR) of −2.5 (18) MFI, a −11% change, p=0.006) and increased IL-13 (1.5 (4.0) MFI, a 17% change, p=0.008) (Table 3).
Second, we tested for an association between a trend in baseline cytokine tertiles with baseline dysplasia, t10 dysplasia, and change in dysplasia between t0 and t10. None of these trend tests was significant (Table 4), and there were no significant Treatment by Cytokine interactions for any trend-test model for any of the outcomes on either scale (MFI or concentration), including the most complex model of t0 or t10 cytokine level, and dysplasia, stratified by plate, treatment intervention, and baseline dysplasia level (data not shown).
Third, we tested whether an increase in cytokine MFI tertile from t0 to t10 was associated with a change in dysplasia during that time. All 22 cytokines were evaluated (data not shown); the results for IL-2, IL-7 and IL-13, the three cytokines that were significantly associated with intervention treatments (Table 3), are shown in Table 5. An increase in cytokine IL-2 tertile was non-significantly associated with an increase in dysplasia grade for all participants (OR=1.32, p=0.098), and was significantly associated with worsening dysplasia among those with mild (OR=2.0, p=0.01), but not moderate (OR=1.04, p=0.8), dysplasia at baseline. An increase in IL-7 tertile was also significantly associated with an increase in dysplasia grade both overall and among those with mild dysplasia at t0 (all participants OR=1.47 (p=0.03); mild dysplasia at t0 OR=2.53 (p=0.001), moderate dysplasia at t0 OR=0.84 (p=0.5)) (Table 5).
We also studied the association between change in cytokine MFI tertiles with change in dysplasia for those in strata defined by active treatment arm and baseline dysplasia levels (Table 6). The association of increased IL-2 with increased dysplasia remained statistically significant in those on selenomethionine treatment who began the trial with mild baseline dysplasia (OR=2.52 p=0.03), and the association of increased IL-7 with increased dysplasia also remained significant in these same subjects (OR=2.51, p=0.02). There were no statistically significant associations between change in cytokine tertiles and change in dysplasia status among subjects on selenomethionine who began the trial with moderate dysplasia or among subjects on celecoxib treatment.
We sought to determine whether use of the selenomethionine supplementation or the celecoxib treatment given in the Linxian Chemoregression Study altered cytokine levels; whether baseline cytokine levels were associated with baseline dysplasia (t0), dysplasia at the end of the ten month intervention (t10), or a change in dysplasia during this period (t10-t0); and whether changes in cytokine levels, especially those that were associated with active treatment, were associated with changes in dysplasia. Such associations would support the possibility that the effects of treatment on dysplasia that were observed in the parent trial were mediated by their effects on cytokine levels. We measured 22 cytokines simultaneously in each serum sample, and found that selenomethionine supplementation was associated with lower IL-2 levels and celecoxib treatment was associated with lower IL-7 levels and higher IL-13 levels.
None of the baseline cytokine levels were associated with dysplasia status at baseline or after 10 months of intervention, or with the change in dysplasia from baseline to 10 months after intervention. However, an increase in IL-2 or IL-7 between baseline and the end of intervention was associated with an increase in dysplasia grade. These associations were most evident in participants who had mild dysplasia at baseline and were given selenomethionine treatment. Since selenomethionine treatment was associated with a reduction in dysplasia grade in the parent trial and a reduction in IL-2 levels in this study, and changes in IL-2 level paralleled the changes in dysplasia grade, it is plausible that the beneficial effect of selenomethionine treatment on dysplasia observed in the original trial may have been partially mediated by its effects on lowering IL-2. This hypothesis would be consistent with reports of a direct relationship between IL-2 serum levels and disease progression in other tumors (23). The same hypothesis cannot be made for IL-7, because selenomethionine treatment did not affect its levels in this trial.
There are several possible mechanisms for anticarcinogenic effects of selenium compounds, including immunologic modulation (24). The potential therapeutic importance of enhancing immunologic function, as may have occurred with the selenomethionine treatment in this trial, stems from the fact that host-derived cytokines can modulate an anti-tumor response and, consequently, can play a beneficial role in suppressing tumor formation. This response has been associated with a reduced frequency of metastasis and improved patient survival in early-stage cancers of several types (25).
Of particular relevance to the current findings are several possible mechanisms by which a selenium-induced reduction in IL-2 might protect against tumor development. Selenium inhibits NF-kappa B expression, which can result in reduced expression of NF-kappa B's target genes, including IL-2 (19;20). A reduction in serum IL-2 might be especially protective against IL-2 receptor alpha (IL-2Ra)-expressing tumors (26;27). These IL-2 promoted tumors include squamous cell carcinoma of the lung and cervical intraepithelial neoplasias, entities with histologic and etiologic similarities to esophageal squamous cell carcinoma (23;27). And in the evaluation of cervical intraepithelial neoplasia, IL-2Ra expression was also associated with the grade of squamous dysplasia (27), suggesting that serum IL-2 may specifically help regulate pre-invasive squamous disease, which was the focus of the Linxian chemoprevention trial.
Selenium-induced reduction in IL-2 might also protect against tumor development by reducing the activity of regulator T-cells (Tregs), which are T-lymphocytes that can impair cancer immunosurveillance. IL-2 activated Tregs protect against autoimmune reactivity but suppress the activation, proliferation, and effector functions of tumor infiltrating lymphocytes (TILs) (28-31), which may be particularly relevant in modulating immune responses during the earliest stages of neoplastic disease. Thus, the reduction in IL-2 seen with selenomethionine treatment could lead to reduced Treg activity and enhanced tumor immune surveillance.
In a separate set of studies, we have previously shown that the people of Linxian are highly exposed to polycyclic aromatic hydrocarbons (PAHs), carcinogenic products of incomplete combustion of organic matter (32-34). It is interesting to note that PAHs can also have harmful effects on immunologic function. The intracellular receptor for PAHs is the aryl hydrocarbon receptor (AhR), and increased exposure to PAHs stimulates the production of AhR target genes (35). AhR in turn can be immunotoxic, because it stimulates Tregs, which impair cancer immunosurveillance. Thus PAH exposure and selenium exposure are probably both impacting cancer immunosurveillance in Linxian (in opposite directions) via their effects on Tregs.
IL-7 decreased with celecoxib supplementation and was positively associated with a worsening of preneoplastic disease. IL-7 is a member of the common gamma-chain family of cytokines which are involved in homeostatic proliferation and survival (36;37). It has a potential regulatory role affecting the interaction between mucosal lymphocytes and intestinal epithelium (38-40), is constitutively expressed by gastrointestinal epithelial cells (39), and is involved in the development and persistence of chronic inflammatory bowel disease (40). Thus, the decrease in IL-7 seen with celecoxib supplementation could serve to lessen a potentially adverse chronic inflammatory response that might potentiate cancer formation (41).
IL-13, which was increased with ten months of celecoxib supplementation, also plays an important role in various inflammatory diseases, including cancer, asthma, and allergy (42;43). In this study, IL-13 was not associated with a change in preneoplastic disease and, thus, the potential clinical significance of its association with celecoxib could not be further assessed.
A significant strength of the current study was the original intervention trial, with its accurate, paired, endoscopic categorization of existing esophageal disease (44) before and after an intervention that showed a significant beneficial effect. Thus, this study provided an opportunity to explore potential mechanisms to explain the observed changes in preneoplastic disease in the parent intervention trial.
A limitation of the study was the relatively high within-plate and plate-to-plate variability, the reasons for which remain unclear. This variation was similar to that recently reported by others but still deemed successful at yielding high intraclass correlation coefficients that were useful for epidemiologic studies (45). Another limitation of all cytokine studies is the fact that serum cytokine levels may change rapidly over time in an individual in response to changes in diet and other factors, so that a single serum measurement may not represent long-term cytokine levels. This issue may be less important in a population with chronic nutrient deficiencies such as our study population. Finally, it is also possible that some of the associations that we observed may have occurred by chance, since we examined 22 cytokines and did not adjust for multiple comparisons. Thus our observations must be regarded as exploratory and need independent confirmation.
In conclusion, we explored 22 cytokines and found that selenomethionine treatment decreased serum IL-2 levels and that celecoxib treatment decreased IL-7 and increased IL-13 levels over a 10-month intervention conducted in Linxian, China. Importantly, we found that increased IL-2 and IL-7 levels during the trial were associated with neoplastic progression to increased grades of esophageal squamous dysplasia at the end of the trial. Thus, the favorable effects of selenomethionine treatment on dysplasia that were observed in this trial may have been mediated in part by the reduction in IL-2 levels associated with this treatment.
This research was supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health.