Abstract
Background: There are no reliable biomarkers for diagnosis, prediction of outcome or treatment effect monitoring for upper tract urothelial carcinoma (UTUC), which is a uniquely prevalent cancer in Taiwan. In the present study, 128 primary UTUC specimens of various grades and primary tumor status were examined for the expression of cyclooxygenase-2 (COX-2) in tumor and stroma tissues aiming to clarify the association of COX-2 expression with clinical outcomes in Taiwanese patients with UTUC. Materials and Methods: Immunohistochemistry was implemented to investigate COX-2 expression levels in 128 paired tumor and stroma specimens. The association of COX-2 expression with tumor progression and prognosis was analyzed. Results: Our data demonstrated that positive COX-2 expression was more frequent in stromal cells (57.0%), than in tumor sites (53.1%), and the up-regulation of COX-2 was strongly associated with higher cancer-specific death and cancer recurrence rates. In COX-2-negative cases, no similar correlation was found. Conclusion: COX-2 expression was up-regulated in both stromal and tumor cells of more than half of the studied UTUC patients and the positive expression of COX-2 in stromal cells may be a potential predictive and prognostic biomarker for UTUC, especially for cancer-specific death and recurrence.
Urothelial carcinoma is the most common malignancy of the urinary tract. The incidence of renal pelvic and ureteral cancer is relatively rare, accounting for only 4% of all urothelial tumors. The ratio of urothelial carcinoma in renal pelvis, ureter and bladder is reported to be 3:1:51 (1). In the United States, renal pelvic urothelial carcinoma accounted for about only 8% of all renal tumors, and ureteral cancer for about 5% of all urothelial tumors in 2010 (2). In Taiwan, the ratio of renal pelvic, ureteral, and bladder cancer is shifted to 1.1:0.9:8.0 (3), showing that the Taiwanese population may have specific genetic and environmental lifestyle factors responsible for upper tract urothelial carcinomas (UTUC) (4) and clinical outcomes (5). The clinical characteristics and prognosis are quite different in bladder cancer and UTUC. The upper urinary tract has anatomical characteristics such as a thin muscle layer, proximity to the kidney and rich lymphatic drainage (6). Tumor invasion may significantly influence distant metastasis and progression in patients with UTUC.
Our previous reports, implicating on immunochemistry experiments have revealed the roles of osteopontin (7), hypoxia-induced factor-1α (8) and nuclear factor-κB (9) in UTUC, and examined their potential as predictors of poor outcome for patients with UTUC in Taiwan. Patients with UTUC almost always present with hydronephrosis or hematuria. Clinical stage and pathological grade are the only factors to predict disease prognosis. However, the exact molecular mechanisms of tumor invasion and recurrence, and prognosis of UTUC are not clear. There are no reliable biomarkers for diagnosis, outcome prediction or treatment effect monitoring for UTUC. However, different tumor behaviors are still observed even in patients with the same stage or grade of disease.
Several epidemiological studies provided the first evidence that non-steroidal anti-inflammatory drugs reduce the risk of a wide range of tumor types, and that genotypic and phenotypic patterns of cyclooxygenase (COX) expression may be associated with the development of several types of cancer (10-14). COX, also known as prostaglandin endoperoxidase H synthase, is a modifier gene encoding a key enzyme in the conversion of arachidonic acid to prostaglandins (15, 16). Two isoforms of COX have been characterized, COX-1 and COX-2. The former is constitutively expressed in various normal organs and tissues, and is involved in the homeostasis of various physiological functions. In contrast, expression of COX-2 is low or not detectable in most normal tissues, but can be highly-induced in response to cell activation by growth factors, pro-inflammatory cytokines, carcinogens, endotoxins and tumor promoters (17). Increased expression of COX-2 is observed in various tumor types, as well as in bladder urothelial carcinomas (18, 19). COX-2 is also associated with many stages of cancer development, e.g. tumor development and susceptibility, invasion, metastasis, hyperproliferation and transformation (10-14). However, studies on the role on COX-2 in UTUC are still very few.
The purpose of this study was to characterize the relationship between the expression of COX-2 in tumor and stromal tissues and to analyze its association with clinical outcome in Taiwanese patients with UTUC.
Materials and Methods
Surgical specimens and clinicopathological data. Formalin-fixed UTUC samples were obtained from the Department of Urology of Kaohsiung Medical University Hospital from 1997-2006. The pathological grade was classified according to World Health Organzation (WHO) histological criteria (20), and tumor staging was determined according to the International Union Against Cancer tumor-node-metastasis classification (21). The study protocol was reviewed and approved by the Institutional Review Board of Kaohsiung Medical University (KMUH-IRB-20120031 and KMUH-98-8G33).
Immunohistochemistry. Tissue cross-sections (5 μm in thickness) were cut from paraffin-embedded tissues and mounted onto poly-lysine-coated slides, de-paraffinized through serial baths in xylene, and rehydrated in a graded series of alcohol and distilled water. Antigen retrieval was enhanced by autoclaving slides in sodium citrate buffer (pH 6.0) for 15 min. Endogenous peroxidase activity was quenched by 10-min incubation in 3% H2O2. The slides were then incubated with a mouse monoclonal primary antibody against COX-2 (diluted 1:500; Dako, Carpenteria, CA, USA) for 60 min. For the detection system, LSAB2 (K0675; Dako) was used with a secondary antibody, as the biotinylated link, for 30 min followed by streptavidin-peroxidase for a further 30 min. The slides were then washed in phosphate-buffered saline (pH 7.4). The reaction was detected by incubating with diaminobenzidine for 5 min. The slides were finally counterstained in aqueous hematoxylin for 30 s, followed by sequential dehydration using graded alcohols and xylene, before mounting and coverslipping. Known strong COX-2-staining colon cancer specimens served as positive controls. Sham-stained sections were used as negative controls. In each set of experiments, positive and negative controls were included for batch comparison.
The results of COX-2 immunostaining were reviewed independently by two pathologists double-blinded to the clinical and pathological status. The cytoplasmic staining of COX-2 in tumor and stroma cells was categorized as positive or negative. More than 10% cells with positive staining, defined samples as positivity or positive expression.
Statistical analysis. Chi-square analysis or Fisher's exact test were used to evaluate the COX-2 expression in patients with different age (dichotomized by medium), gender, cancer location (renal pelvis, ureter or both) and tumor grade (low or high). The nonparametric Spearman's correlation coefficient method was used to evaluate the association between COX-2 expression and tumor stages. Survival analysis was estimated according to the Kaplan–Meier method and the Cox proportional hazard model from the date of primary tumor surgery to the time of recurrence or from the date of diagnosis to the date of death from cancer. The difference in survival curves was examined by means of the log-rank test. Results were considered statistically significant if the p-value was less than 0.05. The data were analyzed using the SPSS package (version 15.0, SPSS, Inc., Chicago, IL, USA); all p-values were two-sided.
Results
The demographic distribution of characteristics such as age, gender, stage and grade of the 128 patients with UTUC recruited in this study are summarized in Table I. As shown in Table I, 73 (57.0%) cases exhibited positive COX-2 staining in stromal cells, while 68 (53.1%) of the patients in the tumor cells. In subgroup analysis of stromal and tumor groups according to their COX-2 expression status, there was no significant correlation between positive COX-2 expression and age, gender, American Joint Committee on Cancer (AJCC) cancer stage, cancer grade or stage of chronic kidney disease (CKD). Compared with tissues from patients with pelvic cancer, those from patients with ureteral cancer had a higher percentage of positive stroma and tumor cell COX-2 staining (p=0.0229 and 0.0021, respectively).
In Table II, the investigated patients are divided according to their death and cancer recurrence status. In cases of COX-2-positive expression, the AJCC cancer stage was positively correlated with cancer-specific survival (p=0.0109) and cancer recurrence (p=0.0235). But in cases with COX-2 negative expression cases, no similar correlation was found.
The clinical and pathological characteristics of patients with upper tract urothelial carcinoma and controls.
Analysis of death and recurrence of disease in patients with upper tract urothelial carcinoma according to the individual COX-2 expression levels.
Analyses for the hazard ratios (HR) of death and recurrence of disease in patients with upper tract urothelial carcinoma according to the (COX-2) expression levels in stromal and tumor cells.
To determine the effects of tissue COX-2 expression on patients' survival status, we examined the effects of stromal and tumor COX-2 expression on both cancer-specific and recurrence-free survival by Cox proportional hazard modeling (Table III). In regard to cancer-specific survival, positive stromal COX-2 expression was associated with a 6.38-fold higher hazard than the negative expression, after adjusted for AJCC cancer stage, age and gender (p=0.0223). Otherwise, tumor COX-2 expression had no effect on cancer-specific survival. In addition, neither stromal nor tumor COX-2 expression had any significant effect on recurrence-free survival.
Discussion
Currently, there are few reliable biomarkers for UTUC, and clinical stage and pathological grade are the only indices for the prediction of UTUC prognosis. The studies, which aimed at determining common identified prognostic markers for outcomes following radical nephroureterectomy among multi-institutions, have shown that it may be a better strategy to identify specific markers for individual populations, due to the rare cases among populations with different genetic backgrounds (22-25). Taiwan has a remarkably high incidence of UTUC, and is famous for the widespread use of Aristolochia herbal remedies, which may be closely related to cause an increased risk of developing end-stage renal disease or urothelial carcinoma (26). Interestingly, about 43% of the carcinomas studied here were located in the upper urinary tract. Taiwanese may have a unique etiology of UTUC, together with several unique markers for different stages of carcinogenesis.
COX-2, the inducible enzyme involved in the conversion of arachidonic acid to prostaglandin and other eicosanoids, has been revealed to be positively stained in many types of cancer, thus making it an attractive therapeutic target for the prevention and treatment of a number of malignancies, such as breast (27), gastric (28), colorectal (29), lung (30), and bladder cancer (31). Elevated tumor COX-2 is associated with increased angiogenesis, tumor invasion and promotion of tumor cell resistance to apoptosis (32). Although great attention is being attributed to COX-2 as a beneficial target for cancer chemotherapy, the detailed mechanism and role of COX-2 in carcinogenesis of UTUC, together with its inhibitors, remains unclear.
To the best of our knowledge, there are few studies evaluating the predictive accuracy of tumor markers with outcomes of patient with UTUC. In 2008, Remzi and colleagues performed a multi-institutional study, with 1,363 patients with UTUC, showing that tumor architecture is an independent predictor for both cancer recurrence and cancer-specific death after nephroureterectomy (25). In this study, we firstly showed that COX-2 is expressed both in tumor and stromal cells of UTUC and used the expression level as a convenient outcome predictor of outcome. It is interesting and surprising that COX-2 is expressed not only in tumor cells but also in stromal cells (Table I). In addition, the AJCC cancer stage was positively correlated with cancer-specific survival (p=0.0109) and cancer recurrence (p=0.0235) only in cases with positive COX-2 expression, and not in the negative ones (Table II). Furthermore, COX-2 positive expression in stromal cells was found to be a prognostic factor of cancer-specific survival for patients with UTUC (Table III). Kaplan–Meier survival curves were drawn, showing that stromal COX-2-positivity predicted poor cancer-specific survival (data not shown). Stromal COX-2-positive staining may predict poor cancer-specific survival. However, tumor COX-2 staining was not correlated with either cancer-specific survival or recurrence-free survival (data not shown).
Human solid tumors are histologically complex mixtures composed of carcinoma and stromal cells such as fibroblasts, endothelial, smooth muscle, and specialized immune cells (33, 34). The interaction between carcinoma cells and surrounding stromal cells has a critical role in tumor growth, progression, and metastasis (35). Cancer cells can crosstalk with stromal cells, such as macrophages and fibroblasts, through several paracrine pathways, resulting in an increased expression of factors involved in cancer cell invasiveness, together with an increase in inflammatory effects (36, 37). The significance of COX-2 up-regulation was firstly found in colonic cancer, and increased COX-2 expression was correlated with reduced patient survival (38).
There are several limitations of the present study. Firstly, there is limited information in the retrospective analyses, such as smoking habit. Several of the patients could not provide their individual habits for smoking and drinking, which made the analysis of the association of these habits and UTUC incomplete in the current study. In addition, the present patients underwent surgery under different surgeons at various sites, and had their specimens evaluated by different pathologists. Fortunately, all specimens were re-examined by dedicated pathologists according to strict criteria, as agreed by all the participating pathologists. Furthermore, the sample size was limited, which made the significant findings not so obvious. The enlargement of sample size is urgently needed, but at the same time, the background information of the patient population should be well-recorded together with individual behaviors. A multi-institutional study of patient populations in Taiwan may add more information to our understanding of UTUC.
There is increasing evidence for the important role of COX-2 in several types of tumors, and experimental and epidemiological studies show that tumor development can be reduced by inhibitors of COX-2. The finding that inhibition of COX-2 reduced the risk of cancer development, indicates that the expression of COX-2 in tumor samples might be used as a prognostic factor (39). In conclusion, we found that COX-2 is up-regulated in not only tumor cells but also stromal cells, and COX-2 overexpression in stromal cells could be a prognostic factor for poor clinical outcomes, such as lower cancer-specific survival in patients with UTUC after surgery.
- Received May 17, 2012.
- Revision received July 23, 2012.
- Accepted July 23, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
