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Cyclooxygenase-2 (COX-2, PTGS2) is considered to play an important role in colorectal carcinogenesis, and is often upregulated in colon cancers. However, previous data on the influence of COX-2 expression on patient outcome have been conflicting.
Utilizing 662 colon cancers (stage I-IV) in two independent prospective cohorts (the Nurses' Health Study and the Health Professionals Follow-up Study), we detected COX-2 overexpression in 548 tumors (83%) by immunohistochemistry. Cox proportional hazard models were used to compute hazard ratios (HRs) of colon cancer-specific and overall mortalities, adjusted for patient characteristics and related molecular events, including the CpG island methylation phenotype (CIMP), microsatellite instability (MSI) and p53, KRAS and BRAF mutations.
During follow-up of the 662 cases, there were 283 deaths, including 163 colon cancer-specific deaths. Patients with COX-2-positive tumors showed a trend towards an inferior colon cancer-specific mortality [HR 1.37; 95% confidence interval (CI), 0.87-2.14], which became significant after adjusting for tumor stage and other predictors of clinical outcome (multivariate HR 1.70; 95% CI, 1.06-2.74, p=0.029). Notably, the effect of COX-2 expression on survival might differ according to p53 status (Pinteraction=0.04). Compared to tumors with both COX-2 and p53 negative, COX-2-positive tumors were significantly associated with an increased cancer-specific mortality (multivariate HR 2.12; 95% CI, 1.23-3.65) regardless of p53 status. A similar trend was observed when overall mortality was used as an outcome.
COX-2 overexpression is associated with worse survival among colon cancer patients. The effect of COX-2 on clinical outcome may be modified by p53 status.
Cyclooxygenase-2 (COX-2, PTGS2) converts arachidonic acid to prostaglandins and related eicosanoids, and promotes inflammation and cell proliferation (1, 2). COX-2 is overexpressed in the majority of human colon cancers (2-4). Supporting the importance of COX-2 in colorectal carcinogenesis, randomized trials have demonstrated that aspirin and COX-2 selective inhibitors reduce risk of recurrent adenoma among high-risk patients (5-7).
Despite the well-accepted role of COX-2 in tumor development (2), studies are conflicting regarding prognostic significance of COX-2 in colorectal cancer with some (3, 8, 9) supporting and others (4, 10-16) refuting an independent adverse effect of COX-2 overexpression. COX-2 overexpression has been positively associated with p53 alteration (17, 18), and inversely associated with microsatellite instability (MSI) (18-20), which generally predicts longer survival of colon cancer patients (21). Moreover, COX-2 and p53 appear to regulate each other in a complex manner (17, 22, 23). Thus, effect of COX-2 on patient survival can possibly be confounded by p53 alteration, MSI and other related molecular events.
In this study using a large number (N=662) of colon cancer patients in two independent cohort studies, we have examined the effect of tumoral COX-2 expression on patient outcome, adjusted for tumor stage and other potential predictors of clinical outcome. Since we concurrently assessed tumoral molecular alterations including p53, KRAS and BRAF mutations, MSI, and the CpG island methylator phenotype (CIMP), we could evaluate the independent effect of COX-2 expression after controlling for these related molecular events.
We utilized the databases of two large prospective cohort studies; the Nurses' Health Study (N = 121,700 women followed since 1976) (24, 25), and the Health Professional Follow-up Study (N = 51,500 men followed since 1986) (25). On each biennial follow-up questionnaire, participants were asked whether they had a diagnosis of colon cancer during the previous 2 years. When a participant (or next of kin for decedents) reported colon cancer, we sought permission to obtain medical records. Study physicians, while blinded to exposure data, reviewed all records related to colon cancer, and recorded AJCC (American Joint Committee on Cancer) tumor stage and tumor location. For nonresponders, we searched the National Death Index to discover deaths and ascertain any diagnosis of colon cancer that contributed to death or was a secondary diagnosis. Approximately 96% of all incident colon cancer cases were identified through these methods. We collected paraffin-embedded tissue blocks from hospitals where colon cancer patients underwent resections of primary tumors (25). Tissue sections from all colon cancer cases were reviewed and confirmed by a pathologist (S.O.). Tumor grade was categorized as high (≤50% glandular area) or low (>50% glandular area). Based on availability of tissue samples, we included a total of 662 colon cancer cases (287 from the men's cohort and 375 from the women's cohort) diagnosed up to 2002. Written informed consent was obtained from all subjects. This study was approved by the Human Subjects Committees at Brigham and Women's Hospital and the Harvard School of Public Health.
Patients were observed until death or June 2006, whichever came first. Ascertainment of deaths included reporting by the family or postal authorities. In addition, the names of persistent nonresponders were searched in the National Death Index. The cause of death was assigned by physicians blinded to other clinical and lifestyle information. More than 98% of deaths in the cohorts were identified by these methods.
Tissue microarrays (TMAs) construction and immunohistochemical examination for COX-2 and p53 were performed as previously described (18). p53 positivity was defined as 50% or more of tumor cells with unequivocal strong nuclear staining. These criteria were based on the observations that the 50% cutoff appeared to increase specificity of p53 immunohistochemistry to correlate with the presence of TP53 mutation (26-28). Our data also indicated that MSI-high or CIMP-high was uncommon (6.3-8.1%) in tumors with p53 positivity in ≥50% tumor cells, while CIMP-high or MSI-high was more frequent (22-27%) in tumors with p53 positivity in <50% tumor cells as well as tumors without p53 staining.
For COX-2 immunohistochemistry, antigen retrieval was performed by incubating deparaffinized tissue sections in citrate buffer (BioGenex) by a microwave for 15 min, and let the sections cool for at least 40 min. Tissue sections were incubated with 3% H2O2 (20 min) to block endogenous peroxidase, and then incubated with Avidin Block (Vector Laboratories) (15 min), than with Biotin Block (Vector Laboratories) (15 min). Primary anti-COX-2 antibody (Cayman Chemical, Ann Arbor, MI) (dilution 1:300) was applied for overnight at 4°C. Then, secondary anti-mouse antibody (Vector Laboratories) was applied (20 min), avidin biotin complex conjugate (Vector Laboratories) was added and sections were visualized by diaminobenzidine (DAB) (5 min) and methyl-green counterstain. For each assay run, we included a positive control (cancer with COX-2 overexpression) and a negative control (normal colonic tissue). We also treated a positive control specimen with phosphate-buffered saline without anti-COX-2 antibody. A pathologist (S.O.), unaware of other data, interpreted cytoplasmic COX-2 expression in tumor as either absent, weak, moderate, or strong staining compared to adjacent normal colonic epithelium. Inflammatory cells served as internal built-in positive controls (18, 25). Consistent with other investigators (8, 29), if immunostaining intensity was moderate or strong, tumors were classified as cancers with COX-2 overexpression. If immunostaining intensity was weak or absent, tumors were classified as cancers with negative COX-2 overexpression (Figure 1). This classification has been previously shown to associate well with p53 expression and inversely with MSI and CIMP in colorectal cancer (18).
Appropriate positive and negative controls were included in each run of immunohistochemistry. All immunohistochemically-stained slides were interpreted by a pathologist (S.O.) unaware of other data. A random sample of 108 cases was re-examined for COX-2 expression by a second observer (R.D.) unaware of other data, and the concordance between the two observers was 0.92 (κ=0.62, p<0.0001), indicating substantial agreement. Another random sample of 118 tumors were re-examined for p53 by another observer (K.N.) unaware of other data, and the concordance between the two observers was 0.87 (κ=0.75, p<0.0001).
Genomic DNA from paraffin-embedded tissue and whole genome amplification of genomic DNA was performed as previously described (30). PCR and sequencing targeted for KRAS codons 12 and 13, and BRAF codon 600 were performed as previously described (30, 31).
MSI status was determined using a microsatellite marker panel consisting of D2S123, D5S346, D17S250, BAT25, BAT26, BAT40, D18S55, D18S56, D18S67 and D18S487 (i.e., 10-marker panel) (32). A high degree of MSI (MSI-high) was defined as the presence of instability in ≥30% of the markers, MSI-low as the presence of instability in <30% of markers, and microsatellite stability (MSS) as no unstable marker.
Sodium bisulfite treatment on DNA and MethyLight assays were validated and performed as previously described (33). We used ABI 7300 (Applied Biosystems, Foster City, CA) for quantitative real-time PCR [MethyLight (34)] on 8 CIMP-specific markers (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) (32, 35). CIMP-high was defined as ≥6/8 methylated markers using the 8-marker CIMP panel, CIMP-low/0 as ≤5/8 methylated markers, and CIMP-0 as 0/8 methylated markers, according to the previously established criteria (32).
We used Cox proportional hazard models to calculate hazard ratios (HRs) of death according to tumoral COX-2 status, unadjusted as well as adjusted for age, sex, year of diagnosis, tumor location, stage, grade, MSI, CIMP, KRAS, BRAF and p53. For analyses of colon cancer-specific mortality, death as a result of colon cancer was the primary end point and deaths as a result of other causes were censored. To adjust for potential confounding, age and year of diagnosis were used as continuous variables, and all of the other covariates were used as categorical variables. We dichotomized tumor location (proximal vs. distal), tumor grade (high vs. low), CIMP (high vs. low/0), MSI (high vs. low/MSS), p53 (positive vs. negative), KRAS (mutated vs. wild-type) and BRAF (mutated vs. wild-type). We assigned a separate indicator variable to each tumor stage (as in Table 1) in order to minimize residual confounding. When there was missing information on tumor location (1.2% missing), stage (7.4%), tumor grade (0.5% missing), MSI (3.0% missing), p53 (0.8% missing), KRAS (2.6% missing) or BRAF (4.8% missing), we assigned a separate (“missing”) indicator variable and included those cases in the multivariate analysis models. We confirmed that excluding cases with a missing variable did not significantly alter results (data not shown). An interaction was assessed by including the cross product of the COX-2 variable and another variable of interest in a multivariate Cox model, and the likelihood ratio test was performed. To assess an interaction of COX-2 and stage, we dichotomized tumor stage (I-II vs. III-IV). The Kaplan-Meier method was used to describe the distribution of colon cancer-specific and overall survival time, and the log-rank test was performed. The chi square test was used to examine an association between categorical variables. The t-test assuming unequal variances was performed to compare mean age. All analyses used SAS version 9.1 (SAS Institute, Cary, NC) and all p values were two-sided.
Among the 662 tumors, 548 (83%) were positive for overexpression of COX-2, while 114 (17%) were negative for COX-2. We also examined p53 status (by immunohistochemistry), microsatellite instability (MSI), the CpG island methylator phenotype (CIMP), KRAS and BRAF mutations, because expressions of COX-2 and p53 were inversely related with MSI and CIMP (18), and MSI, CIMP and BRAF mutation have been related with patient outcome (21, 36-39). Table 1 summarizes clinical and molecular features of colon cancer according to COX-2 status. Notably, compared to COX-2 negative tumors, COX-2 positive tumors are more likely distal, low-grade, and p53-positive, and less likely MSI-high and CIMP-high.
Among the 662 eligible patients with adequate follow-up, there were 283 deaths, including 163 colon cancer-specific deaths. We assessed the influence of COX-2 expression on patient survival. Five-year colon cancer-specific survival was 84% among patients with COX-2-negative tumors and 78% among patients with COX-2-positive tumors (log-rank p=0.17) (Figure 2). Five-year overall survival was 79% among patients with COX-2-negative tumors and 72% among those with COX-2-positive tumors (log-rank p=0.63).
In univariate Cox regression analysis, COX-2 positivity was associated with a non-significant increase in colon cancer-specific mortality [hazard ratio (HR) 1.37; 95% confidence interval (CI), 0.87-2.14] (Table 2). In a multivariate model that adjusted for other clinical, pathologic and molecular predictors of survival, COX-2 positivity was associated with a significant increase in colon cancer-specific mortality (multivariate HR 1.70; 95% CI, 1.06-2.74). The increase in the effect of COX-2 positivity on survival in the multivariate analysis was mainly the result of adjusting for tumor stage; when we simply adjusted for tumor stage, the HR for colon cancer-specific mortality in COX-2 positive tumors was 1.57 (95% CI, 1.00-2.46). When we excluded stage IV cases, multivariate HR for colon cancer-specific mortality in COX-2 positive cases (vs. COX-2 negative cases) was 1.60 (95% CI, 0.81-3.13). Thus, the results did not change substantially.
COX-2 expression did not significantly influence overall mortality in both univariate and multivariate analyses (Table 2). High tumor grade was associated with an increased colon cancer-specific mortality (multivariate HR 2.07; 95% CI, 1.17-3.66). p53 positivity was not a significant predictor of survival in both univariate and multivariate analyses (multivariate HR for colon cancer-specific mortality, 1.34, 95% CI, 0.92-1.94).
We examined whether the effect of COX-2 expression on survival was modified by any of the clinical and molecular variables (Figure 3). The effect of COX-2 overexpression on colon cancer-specific mortality was not significantly different across most strata of patient and disease characteristics. Notably, the effect of COX-2 overexpression was similar across the two independent cohort studies (p for interaction =0.87). We did observe an apparently significant effect of p53 expression on the association between COX-2 and mortality (p for interaction =0.04). A significant adverse effect of COX-2 overexpression was present in p53-negative tumors, but not among p53-positive tumors.
Since there was evidence for effect modification by p53 status on the association between COX-2 and patient survival, we stratified tumors by combined status of COX-2 and p53 (Table 3). Compared to tumors that were negative for both COX-2 and p53, COX-2-positive tumors (regardless of p53 status) were associated with a significant increase in cancer-specific mortality (multivariate HR 2.12, 95% CI, 1.23-3.65). At the same time, p53 status had little effect on mortality among COX-2-positive tumors.
We conducted this study to examine the influence of cyclooxygenase-2 (COX-2, PTGS2) expression on outcome of colon cancer patients. We have found that COX-2 overexpression appears to predict an inferior cancer-specific survival, independent of various clinical and molecular variables. The adverse effect of COX-2 overexpression was consistent across most strata of patient and tumoral characteristics, in particular across the two independent cohort studies. Our data support an adverse effect of COX-2 overexpression on survival of colon cancer patients.
Considerable experimental evidence supports a role of COX-2 in colorectal carcinogenesis (2). Randomized, placebo-controlled trials have uniformly shown that selective COX-2 inhibitors prevent adenoma recurrence among patients with a prior history of adenoma (5, 6). COX-2, possibly through production of inflammatory prostaglandins, may regulate angiogenesis, apoptosis, or tumor cell invasiveness (2, 40). We have previously shown that aspirin use decreases a risk for colon cancers that are positive for COX-2, but not a risk for COX-2-negative cancers, providing additional evidence for a role of COX-2 in colon carcinogenesis (25).
Studying molecular alterations and clinical outcome is important in cancer research (41-48). Our data support a role of COX-2 in determining biological behavior of colon cancer. COX-2 has been examined as a predictive biomarker in cancer (3, 8, 9). Previous studies are conflicting regarding prognostic significance of COX-2 in colorectal cancer with some (3, 8, 9) supporting and others (4, 10-16) refuting independent adverse effect of COX-2. These discrepant results are likely due to differences in patient cohorts, COX-2 detection methods, criteria for COX-2 overexpression, and multivariate survival analysis models. Our current study has comprehensively examined the effect of COX-2 on patient survival independent of clinical characteristics and other molecular events, including statuses of p53 alterations, mutations in KRAS and BRAF, microsatellite instability (MSI) and the CpG island methylator phenotype (CIMP). All of these molecular events are potential confounders for the association between COX-2 and patient survival.
The relationship between COX-2 overexpression and p53 alteration has been examined previously. In one in vivo study, inhibition of COX-2 by celecoxib led to p53 activation in colon cancer cells (22). In other studies, COX-2 expression was inhibited by wild-type p53 in murine embryo cell lines (17), whereas COX-2 overexpression was induced by p53 and nuclear factor-kappa B (NFKB1) in esophageal and colon cancer cells (23). It may be possible that COX-2 and p53 regulate each other to form a feedback loop. Thus, it may not be surprising to find a significant interactive effect of COX-2 and p53 alterations on patient survival. This possible interaction of COX-2 and p53 alterations needs to be further examined and confirmed by future studies.
Our study has several advantages including a large number of colon cancers in the two prospective cohort studies with adequate follow-up, as well as extensive data on disease characteristics and other important tumoral molecular events. Thus, we have been able to demonstrate an effect of COX-2 on patient survival, independent of clinical and other tumoral predictors of clinical outcome.
As a limitation of this study, data on cancer treatment are limited in our cohorts. Nonetheless, it is unlikely that chemotherapy use differed according to tumoral COX-2 status, especially since such data were not available to patients or treating physicians. In addition, beyond cause of mortality, data on cancer recurrences were not available in these cohorts. Nonetheless, given the median survival for metastatic colon cancer was approximately 10 to 12 months during much of the time period of this study, colon cancer-specific survival should be a reasonable surrogate for cancer-specific outcomes. Despite the apparent effects of COX-2 expression on colon cancer-specific mortality, the influence of COX-2 on all-cause mortality was considerably attenuated. This is likely due to deaths unrelated to colon cancer in our cohort studies.
There is variability in grading COX-2 expression and presently there is no widely accepted standardized classification scheme. False positive and false negative results are well-known problems in immunohistochemistry. Nonetheless, previous studies have demonstrated that Western and Northern blot analysis highly correlate with immunohistochemical expression of COX-2 (49), and our classification of COX-2 overexpression resulted in a similar proportion of COX-2 overexpressing tumors as other investigators (3, 4, 8-16). Moreover, we assessed COX-2 overexpression through central, blinded review of tumor specimens with rigorous comparison to internal controls with the substantial inter-observer agreement (92%, κ=0.62). Our COX-2 expression data in relation to MSI and CIMP are in agreement with studies by other investigators (18, 19, 50). Finally, any random misclassification of COX-2 status would have conservatively biased our results toward finding no significant difference in patient survival according to tumoral COX-2 expression.
In conclusion, this large prospective study of colon cancer patients suggests that COX-2 upregulation is independently associated with a worse colon cancer-specific mortality. In addition, when compared to patients with tumors negative for both COX-2 and p53, patients with tumors positive for COX-2 exhibit longer survival regardless of p53 status. Our finding that COX-2 overexpression is associated with poor patient outcome may have significant clinical implications, considering an emerging role of COX-2 and its pathway as chemotherapeutic and chemopreventive targets.
We thank the Nurses' Health Study and Health Professionals Follow-up Study cohort participants who have generously agreed to provide us with biological specimens and information through responses to questionnaires. We thank hospitals and pathology departments throughout the United States for generously providing us with tissue materials from the participants who underwent resection of their colon cancer. We thank Frank Speizer, Walter Willett, Susan Hankinson, Graham Colditz, Meir Stampfer, and many other staff members who implemented and maintained the cohort studies.
This work was supported by the U.S. National Institute of Health (NIH) grants P01 CA87969, P01 CA55075, P50 CA127003 and K07 CA122826 (to S.O.), and in part by the Bennett Family Fund for Targeted Therapies Research, and by the Entertainment Industry Foundation (EIF) through the National Colorectal Cancer Research Alliance (NCCRA). K.N. was supported by a fellowship grant from the Japanese Society for Promotion of Science. Any of these funding agencies has not had any role in design or conduct of the study; collection, management, analysis, or interpretation of the data; or preparation, review, or approval of the manuscript.
This work was supported by the US National Institute of Health (NIH) grants P01 CA87969, P01 CA55075, P50 CA127003 and K07 CA122826 (to S.O.), and in part by the Bennett Family Fund for Targeted Therapies Research, and by the Entertainment Industry Foundation (EIF) through the EIF National Colorectal Cancer Research Alliance (NCCRA). K.N. was supported by a fellowship grant from the Japanese Society for Promotion of Science.
Statement of Translational Relevance COX-2 (cyclooxygenase-2) has been shown to play an important role in carcinogenesis in various organ systems including colon. COX-2 inhibitors (aspirin, NSAIDs and celecoxib) have been shown to be effective in preventing colorectal adenoma and cancer. However, the relation between COX-2 expression in colon cancer and patient survival has been controversial. We have utilized the database of more than 600 colon cancer in two independent, prospective cohort studies, with available clinical information, adequate follow-up, and other important molecular events in colon cancers. To our knowledge, this is the first study to demonstrate adverse effect of COX-2 overexpression on clinical outcome independent of related molecular events including BRAF mutation, MSI (microsatellite instability) and CIMP (the CpG island methylator phenotype), all of which are associated with both COX-2 expression and clinical outcome in colon cancer. Thus, our findings are relevant to practice in oncology.