Int. J. Cancer: 119, 1082–1087 (2006) ' 2006 Wiley-Liss, Inc.
Cyclooxygenase-2 (cox-2) expression is an independent predictor of prostate cancer recurrence Brian L. Cohen1, Pablo Gomez1, Yohei Omori1, Robert C. Duncan2, Francisco Civantos1,3, Mark S. Soloway1, Vinata B. Lokeshwar1,4,5 and Bal L. Lokeshwar1,5,6* 1 Department of Urology, Miller School of Medicine, University of Miami, Miami, FL, USA 2 Epidemiology and Public Health, Miller School of Medicine, University of Miami, Miami, FL, USA 3 Department of Pathology, Miller School of Medicine, University of Miami, Miami, FL, USA 4 Department of Cell Biology and Anatomy, Miller School of Medicine, University of Miami, Miami, FL, USA 5 Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL, USA 6 Department of Radiation Oncology, Miller School of Medicine, University of Miami, Miami, FL, USA Lack of reliable prognostic markers hinders accurate prediction of disease progression in prostate cancer. The inducible proinflammatory enzyme cyclooxygenase-2 (COX-2) is implicated in prostate carcinogenesis, but its role in cancer progression is less clear. We examined whether COX-2 expression evaluated by immunohistochemistry (IHC) in radical prostatectomy (RP) specimens can predict biochemical recurrence. Archival prostate cancer specimens (n 5 60) were obtained from patients who underwent RP, but had not received neoadjuvant hormonal therapy. Twentythree patients had biochemical or clinical recurrence (mean time of recurrence: 38.2 months), and 37 patients were recurrence free (mean follow-up: 95 months). COX-2 expression was determined by IHC, using an anti-COX-2 antibody. Three individuals scored the staining independently, as high- or low-expression. COX-2 was expressed in prostate cancer cells, in adjacent normal glands and in specimens from patients who later progressed. At 62-months follow-up, COX-2 staining predicted progression with 82.4% sensitivity and 81.3% specificity. Sensitivity (86.4%) and specificity (86.7%) improved at 100-months follow-up. In univariate analysis, Gleason score, preoperative PSA, extraprostatic extension, margin, seminal vesicle invasion, and high COX-2 expression were significant predictors of biochemical recurrence (p < 0.05). In multivariate analysis, preoperative PSA (hazard ratio/unit PSA change 1.080; p 5 0.0036) and COX-2 expression (hazard ratio 16.442; p < 0.0001) were independent prognostic indicators. Patients with PSA > 7 ng/ml and high COX-2 expression had the highest probability of recurrence (Kaplan-Meier analysis). COX-2 expression is an independent predictor of prostate cancer progression following RP and underscores the significance of inflammatory factors in this process. ' 2006 Wiley-Liss, Inc. Key words: inflammation; immunohistochemistry; PSA; cancer progression; prognostic indicators; multivariate analysis; tumor markers
Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) are prostaglandin endoperoxide synthases, which are rate limiting enzymes in the conversion of the fatty acid arachidonic acid into physiologically active eicosanoids such as prostaglandin, thromboxane and prostacyclin.1 COX-2 is the inducible form of COX that is frequently elevated in cancer tissues.2–4 Although the mechanism of COX-2 action in carcinogenesis or progression is not well established, COX-2 inhibitors have been shown to be chemopreventive in cancer, specifically in colorectal cancer.5–7 The expression and role of COX-2 in prostate cancer has been a topic of several reports over the decade.8,9 There exists some controversy over the expression, detection and the putative role of COX-2 in prostate carcinogenesis and progression. While early studies reported high-level expression of COX-2 in prostate, findings from subsequent studies have been mixed.10–12 Typically, later studies reported low or no expression in normal, benign prostatic hyperplasia or low-grade cancer tissues but elevated levels in prostatic intraepithelial neoplasia (PIN) and high-grade cancer.13– 26 A prominent study, however, discounted COX-2 specific staining in all epithelial tissues and claimed that COX-2 is expressed Publication of the International Union Against Cancer
only in infiltrating lymphocytes and proliferative inflammatory atrophy.27 Nonetheless, several studies, using experimental models and established prostate cancer cell lines, have established a chemopreventive and antitumor activity of COX-2 inhibitors, implying the putative role of COX-2 in prostate carcinogenesis.28–37 A direct demonstration of potential role of COX-2 in advanced prostate cancer was recently demonstrated by one of us.38 Using an antisense COX-2 cDNA construct (tetracycline-inducible model) to suppress COX-2 expression in PC-3ML cell line (a highly metastatic androgen unresponsive prostate cancer line), we demonstrated tumor-enhancing function of COX-2. In a related study, we also showed chemosensitization of prostate tumor xenografts by a COX-2 inhibitor celecoxib, indicating a potential role of COX-2 in both carcinogenesis and resistance to treatment.39 Until recently, elevated COX-2 levels were shown to be related to carcinogenesis but not progression as illustrated by its elevated levels in preneoplastic colon polyps, inflammatory atrophy and PIN. Moreover, the significance of the elevated expression of COX-2 was believed to be secondary to the elevation of several COX-2 inducing signaling mechanisms, notably the activated levels of Akt (phospho-AKT)40,41 and nuclear-factor kappa B (NFkB) that activates COX-2 promoter and transcription.40–42 The significance of COX-2 overexpression in disease progression, especially in prostate cancer, has evaded rigorous testing until now. This ambivalence is due to earlier studies that showed uneven labeling of COX-2 in prostate cancer tissues in archival samples. However, if COX-2 expression in prostate cancer specimens correlates with disease recurrence/progression, it may explain the conflicting results reported in various studies regarding COX-2 expression.10,28 Investigating such a correlation is also important to understand prostate cancer progression because at least in experimental models of prostate cancer, COX-2 overexpression leads to increase in aggression of tumor cells, higher secretion of angiogenic factors and metastasis.43–46 In this study, we investigated COX-2 expression in archival radical prostatectomy specimens from random samples of 60 prostate cancer patients on whom a minimum 62-month follow-up was available. Our results show that COX-2 expression in prostate cancer cells is an independent prognostic indicator for predicting biochemical recurrence.
Both the authors (BLC and PG) contributed equally to this work. Grant sponsor: NIH/NCI; Grant number: 2RO1-CA061038 (BLL); Grant sponsor: NIH/NCI; Grant number: RO1 072821-06; Grant sponsor: DOD-DAMD; Grant number: 170210005 (UBL). *Correspondence to: Department of Urology (M-800), P.O. Box 016960, Miami, FL 33101, USA. Fax: 305-243-9724. E-mail:
[email protected] Received 18 August 2005; Accepted 10 November 2005 DOI 10.1002/ijc.21749 Published online 23 March 2006 in Wiley InterScience (www.interscience. wiley.com).
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COX-2 EXPRESSION PREDICTS PROSTATE CANCER RECURRENCE TABLE I – PRE- AND POSTOPERATIVE PARAMETERS OF THE STUDY PATIENTS Preoperative parameters Progression
Postoperative parameters
Age (yrs)
PSA (ng/ml)
Clinical stage
Biochemical recurrence (n 5 23)1
Median: 66 Mean: 65.0
Median: 8.9 Mean: 12.1
T1c: 11 T2a: 3 T2b: 9
No biochemical or clinical recurrence (n 5 37)
Median: 63 Mean: 62.8
Median: 6.3 Mean: 6.3
T1c: 23 T2a: 9 T2b: 2 2 T2: 3
Gleason sum
652 7 5 11 855 955 451 554 658 7 5 21 852 951
EPE
Margin
SV invasion
(1) 5 17 (2) 5 6
(1) 5 15 (2) 5 8
(1) 5 9 (2) 5 14
(1) 5 6 (2) 5 31
(1) 5 11 (2) 5 26
(1) 5 2 (2) 5 35
EPE, extraprostatic extension of tumor; SV, seminal vesicle invasion. Two patients who had biochemical recurrence had positive lymph nodes.–2Three of the patients had only designation of tumor stage as T2.
1
Material Specimens and study patients Sixty prostate cancer specimens were obtained from patients who underwent radical retropubic prostatectomy between 1992 and 1995 at the University of Miami Medical Center, Miami, Florida. These patients did not receive neoadjuvant hormonal therapy, and 58 patients were node negative. The minimum available follow-up on all patients was 62 months. The study was conducted under a protocol approved by the University of Miami’s Institutional Review Board for human subjects’ research. Of the 60 patients, 23 patients had biochemical or clinical recurrence (mean time to recurrence: 38.2 months; range: 1–121 months), and 37 patients were free of disease recurrence (mean follow-up: 95 months; range: 62–142 months). Biochemical recurrence was defined as a PSA level 0.4 ng/ml in 2 successive measurements after radical prostatectomy, in which case the first date of elevated PSA level was considered as the date of recurrence. The patient characteristics with respect to age, preoperative PSA and tumor (i.e., Gleason sum, stage, margin, extraprostatic extension (EPE) and seminal vesicle (SV) invasion) are shown in Table I. The specimens chosen for staining were those that represent true major Gleason score recorded by the pathologist on service. Thus, if the pathologist recorded Gleason score as 314, paraffin block representing 314 was chosen. Immunohistochemistry and slide grading For all specimens, paraffin embedded blocks containing prostate cancer tissues representing the major Gleason score was selected. The block selection was made by one of the authors, a certified pathologist (FC). From each block, 6 slides were prepared and 2 were used for COX-2 staining. The other slides were used to either optimize COX-2 antibody concentration for staining, for determining nonspecific staining, for repeating the staining in case of any discrepancy or for comparing COX-2 staining, using antibodies from different companies (as discussed later). The specimen slides were deparaffinized, rehydrated and treated with an antigen retrieval solution (Dako USA, Carpentaria, CA). The slides were incubated with a rabbit polyclonal antihuman COX-2 IgG, PG27b (200-fold dilution of the antiserum; Oxford Biomedical Res., Oxford, MI) at 4°C for 16 hr. Following incubation, the slides were washed and incubated with a linking solution containing a biotinylated swine antirabbit IgG (Dako LSAB kit, Dako USA) at room temperature for 30 min. The slides were then treated with streptavidin peroxidase and DAB chromogen. The slides were counterstained with hematoxylin, dehydrated and mounted. Initially, about 20 specimens were stained with anti-COX-2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) and Cayman Chemical (Ann Arbor, MI) along with the PG-27b antibody to evaluate the specificity of different antibodies. We also evaluated 10 specimens for COX-2 staining, using a nonbiotinbased Envision kit, as per the manufacturer’s protocol (DAKO Laboratories).
Slide grading COX-2 staining of tumor cells in each slide was initially graded as 0 to 31. To account for heterogeneity in staining, the overall staining grade for each slide was assigned based on the staining intensity of the majority of the tumor tissue in the specimen. However, if ~ 50% of the tumor cells in the section were assigned 11 staining, and the other 50% as 31, the overall staining grade was 21. If the staining distribution was, ~ 50% of the tumor cells staining 21 and the remaining staining as 31, the overall staining inference assigned were 31. The staining was later grouped as low- and high-grade staining. High-grade staining represented 21 and 31 staining, whereas low-grade staining included 0 and 11 staining intensities. Normal-benign tissues near and away from tumor cells were also evaluated for staining intensity and graded as low- and high-staining. Two readers independently evaluated all slides in a blinded fashion. Out of the total 60 stained slides, there was discrepancy in 8 slides. These discrepancies were resolved by both readers reexamining those slides simultaneously. In addition, to check for the repeatability of the evaluation system, a 3rd reader, after familiarizing with the grading system, randomly picked 40 slides and graded them for staining intensity. The discrepancy in slide evaluations by the 3rd reader was
