α-Methylacyl-CoA racemase (AMACR, p504S), an enzyme involved in cellular energy metabolism by the oxidation of branched-chain fatty acids, is a biomarker that is known to be overexpressed in prostatic and colorectal carcinoma as well as in papillary renal cell carcinoma (Gunia et al., 2008). Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in proinflammatory prostaglandin E2 (PGE2) biosynthesis, is upregulated in a variety of human cancers, and multiple lines of evidence have suggested that COX-2 and COX-2-derived PGE2 are important in carcinogenesis (Hara, 2008). The active COX-2 enzyme stimulates the production of prostacyclins. Prostacyclins are fatty acids that perform a number of hormone-like tasks in the body, including promotion of inflammation, dilatation of blood vessels, and inhibition of platelet aggregation. The connection between cancer and inflammation is not new. More recently, however, multiple studies have documented overexpression of the COX-2 enzyme in several cancers including breast, colon, lung, prostate, and bladder (Maltzman, 2005).
Bladder cancer is the most common malignancy of the urinary tract in western and industrialized countries (Jemal et al., 2007). Carcinoma of the urinary bladder is the most common malignancy in parts of Africa, where schistosomiasis is a widespread problem. Considerable evidence supports the association between schistosomiasis and bladder cancer. In experimental schistosomiasis, the activities of carcinogen-metabolizing enzymes are increased soon after infection, but are reduced again during the later chronic stages of the disease. Not only could this prolong the period of exposure to activated N-nitrosamines but also inflammatory cells, stimulated as a result of the infection, may induce the endogenous synthesis of N-nitrosamines as well as generate oxygen radicals (Mostafa et al., 1999).
Materials and methods
Histopathological examination was performed by reviewing the tissue sections of the cases of transurethral resection (37 cases) as well as cystectomy specimens (nine cases), obtained from the files of Pathology Department, Tanta Faculty of Medicine, in the period from 2009 to 2010 to determine the histological type, grade, level of invasion, presence or absence of schistosomiasis, and urothelial changes in tissue adjacent to carcinoma bladder carcinomas. Lamina propria, muscle, or serosal invasion was performed in each case. Staging could not be determined because not all the specimens were cystectomies; besides, nodal block dissections were not available in all cystectomy specimens.
Immunohistochemical studies were performed on 4-µm tissue sections from formalin-fixed, paraffin-embedded tissue blocks using a streptavidin–biotin complex. The primary antibodies used in this study included prediluted rabbit monoclonal anti-AMACR IgG (clone 13H4; Labvision, California, USA) and prediluted rabbit polyclonal antihuman anti-COX-2 IgG (cat number RB-9072-R7; Labvision). A negative control was included in which the primary antibody was replaced by preimmune mouse or rabbit IgG. Positive controls were sections from prostatic adenocarcinoma for AMACR and colonic adenocarcinoma for COX-2.
For AMACR, a tumor was recorded positive if greater than 5% of the tumor cells showed cytoplasmic staining. The positivity was classified as diffuse (greater than 75% of the tumor cells stained) and focal (5–75%), which was further divided into three subgroups (5–25, 26–50, and 51–75%; Suh et al., 2005). For COX-2, the scoring was performed using both intensity score as (0, 1, 2, 3) for the negative, weak, moderate, and strong cytoplasmic staining as well as percentage of positive cells multiplied by each other, and in cases with differential expression, adding the sum of the results in different areas, providing the final result (Nasir et al., 2007).
There were 6/46 (13%) cases of low-grade papillary urothelial carcinomas, 27/46 (59%) cases of high-grade urothelial carcinomas, 12/46 (26%) cases of squamous cell carcinomas (SCCs) ranging between grades II and III, and one case [1/46 (2%)] of high-grade adenocarcinoma. Sections from cystectomy specimens were diagnosed as 3/9 high-grade urothelial carcinoma, 5/9 SCCs, and one case of adenocarcinoma.
Low-grade papillary urothelial carcinomas were either noninvasive (3/6) or showed lamina propria invasion (3/6). Carcinoma in situ was detected in three cases of high-grade urothelial carcinoma. Invasion was down to the lamina propria in 6/27 of high-grade urothelial carcinoma and down to the superficial muscles in 18/27 cases, and down to the serosal layer in 3/27 cases.
SCC cases showed invasion down to the superficial muscle layer in 7/12 cases and down to the serosal layer in 5/12 cases.
The adenocarcinoma case showed true glandular formation. The adjacent bladder tissue showed cystitis cystica and cystitis glandularis. Invasion was down to the serosal layer.
Schistosomiasis was detected histologically in 18/46 (39%) cases of urothelial and SCCs and not in adenocarcinoma. All the SCC 12/12 showed schistosomiasis ova with granulomas around them. Among urothelial carcinomas, 6/27 showed schistosomal calcified eggs with schistosomal granulomas; all were of high-grade urothelial carcinoma.
COX-2 was detected as positive cytoplasmic staining in 43/46 (93%) cases, with the positivity score ranging between 30 and 270, and it was negative in 3/46 (7%). Positive cases were 3/6 low-grade papillary urothelial carcinomas, 27/27 high-grade urothelial carcinoma (Fig. 1a and b), 1/1 adenocarcinoma, and 12/12 SCC (Fig. 2). Areas of carcinoma in situ showed high positivity for COX-2; in addition, the inflammatory cells in the lamina propria showed high positivity (Fig. 3).
AMACR was detected as positive cytoplasmic staining in 12/46 (26%) and it was negative in 34/46 (74%). Positive cases were all of high-grade urothelial carcinoma (Fig. 4a and b). No positivity for AMACR was found in low-grade papillary urothelial carcinoma, adenocarcinoma, or SCC.
COX-2 and AMACR positivity were significantly correlated with tumor grading irrespective of the tumor histological type (Tables 1 and 2). COX-2 was significantly correlated with invasion (P<0.05; Table 3), whereas no significant relationship was found between AMACR expression and the presence or absence of invasion (Table 4). No significant relationship was found between the presence or absence of schistosomiasis and both AMACR and COX-2 positivity (Tables 5 and 6). There was no significant relationship between positive or negative COX-2 and AMACR expression (Table 7).
In this study, it was found that COX-2 positivity was significantly correlated with tumor grading irrespective of the tumor histological type. This is in agreement with the finding of Hara (2008), who reported that COX-2 is upregulated in human bladder urothelial carcinoma. It was found that COX-2 positivity was significantly correlated with invasion and this is in contrast to the results of Mokos et al. (2006). In their study, COX-2 immunoreactivity was not correlated with tumor grading, staging, or cancer progression, whereas the immunoreactivity in inflammatory cells was significantly related to the number of recurrences, time to appearance of the first recurrence, and disease progression. This also was the result obtained by Naruse et al. (2010) in their study; there was no correlation between COX-2 and both grading and staging. The positive correlation between grading and COX-2 expression is comparable to the study of Matsuzawa et al. (2002), who found a stepwise increase in the COX-2 immunostaining score from grades I to III. Areas showing carcinoma in situ, considered a precursor for invasive urothelial carcinoma, were found to express COX-2 strongly in this study, and this is comparable to the result obtained by Mohammed et al. (1999) in their study, in which 75% of carcinoma in situ cases were positive for COX-2 expression.
COX-2 is involved in converting procarcinogens into carcinogens, and thus plays a role in initiating tumor formation. In addition, COX-2 is a key regulating enzyme in the synthesis of PGE2, which is important in promoting carcinogenesis. COX-2 overexpression inhibits apoptosis, and it has been implicated in angiogenesis. COX-2 overexpression has been shown in a number of premalignant and malignant conditions including colorectal adenomas and colon cancer, non-small-cell lung cancer, oral leukoplakia, and head and neck cancer. In preclinical models, COX-2 inhibitors have been shown to inhibit intestinal polyps and colorectal tumors. The COX-2 inhibitor celecoxib enhanced the efficacy of the chemotherapeutic drug 5-fluorouracil in human colon cancer models, and enhanced 5-fluorouracil activity in human head and neck cancer models. Similarly, COX-2 selective, the nonsteroidal anti-inflammatory drug nimesulide inhibited the development of chemically induced SCC of the tongue and inhibited rat urinary bladder cancer (Michelle and Washart, 2002).
The link between cancer and inflammation is not new and multiple studies have documented overexpression of the COX-2 enzyme in several cancers including breast, colon, lung, prostate, and bladder. On the basis of these preclinical observations, scientists have reasoned that disruption of the COX-2 enzyme with an inhibitor may improve treatment and even prevention efforts (Maltzman, 2005).
In the study of Zhou et al. (2002), it was found that there was overexpression of COX-2 in cancers of the prostate, ovary, breast, lung, and bladder as well as melanomas and lymphomas. They also found COX-2 overexpression in the precursor lesions as prostatic intraepithelial neoplasia and colonic adenomas. Comparatively, COX-2 overexpression was detected in carcinoma in situ.
In this study, AMACR was found to be positive in 12/46 (26%) of bladder urothelial carcinomas and it was negative in low-grade urothelial carcinoma, and squamous and adenocarcinomas. Comparatively, in the study of Jiang et al. (2003), AMACR was found to be positive in 9/29 (31%) of urothelial carcinomas.
In this study, AMACR positivity was significantly correlated with tumor grading irrespective of the tumor histological type. This is in agreement with Gunia et al. (2008), who found a significant positive correlation between AMACR expression and higher tumor grades using two types of histopathologic grading schemes for noninvasive bladder carcinomas.
In this study, there was no significant correlation between AMACR positivity and invasion; this is not in agreement with the results of Langner et al. (2006), who reported that AMACR expression correlated with advanced tumor stage.
In this study, no significant relationship was found between the presence or absence of schistosomiasis and both AMACR and COX-2 positivity. The lack of correlation between positivity for COX-2 and schistosomiasis may indicate that inflammation not schistosomiasis per se is responsible for carcinogenesis. The mechanisms known to be involved in bladder carcinogenesis are known to be because of DNA damage induced by carcinogens. Inflammatory cells are known to activate bladder carcinogens as aromatic amines. Several bacterial species mediate nitrosation of amines, providing a source for N-nitrosamine. In schistosomiasis, an elevated level of enzymes is responsible for the activation of carcinogenic N-nitroso compounds, aromatic amines, and polycyclic aromatic hydrocarbons (Mostafa et al., 1999). The source of bacteria capable of activation of carcinogens may be present in the absence of schistosomal infestation as in a study on individuals with urinary incontinence showing the prevalence of bacteria with the capacity of N-nitrosation of amines (Tricker et al., 1991). COX-2 overexpression in inflammation may share other factors in carcinogenesis as well as to be targeted.
Targeting COX-2 may aid in the prevention and treatment of bladder carcinoma. Several types of COX-2 inhibitors are clinically used not only for the treatment but also the prevention of bladder cancer and may have more effective future therapeutic outcome (Gee et al., 2006; Shimada et al., 2011).
Positivity for AMACR in only high-grade urothelial carcinoma in this study and correlation with a high grade may indicate its possible role in the etiology of this type of tumors. High-grade noninvasive bladder carcinomas have now been included in the group of AMACR-positive neoplasms and might reflect a so far unknown role of AMACR in the pathobiology and tumor cell energy metabolism of the latter tumor entity (Gunia et al., 2008).
Conflicts of interest
There are no conflicts of interest.
Gee J, Lee IL, Jendiroba D, Fischer SM, Grossman HB, Sabichi AL. Selective cyclooxygenase-2 inhibitors inhibit growth and induce apoptosis of bladder cancer. Oncol Rep. 2006;15:471–477
Gunia S, May M, Scholmann K, Störkel S, Hoschke B, Koch S, et al. Expression of alpha-methylacyl-CoA racemase correlates with histopathologic grading in noninvasive bladder cancer. Virchows Arch. 2008;453:165–170
Hara STachikawa T, Kiyoshi N, Ohmori T, Adachi M. Role of pro-inflammatory prostaglandin E2 in bladder tumor progression. In new trends in the molecular and biological basis for clinical oncology. 20081st ed. Tokyo, Japan Springer:92–95
Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin. 2007;57:43–66
Jiang Z, Fanger GR, Woda BA, Banner BF, Algate P, Dresser K, et al. Expression of α-methylacyl-CoA racemase (P504S) in various malignant neoplasms and normal tissues: a study of 761 cases. Hum Pathol. 2003;34:792–796
Langner C, Rupar G, Leibl S, Hutterer G, Chromecki T, Hoefler G, et al. Alpha-methylacyl-CoA racemase (AMACR/P504S) protein expression in urothelial carcinoma of the upper urinary tract correlates with tumour progression. Virchows Arch. 2006;448:325–330
Maltzman JD, The COX-2 story and cancer. Abramson Cancer Center of the University of Pennsylvania; 2005. Available at: http://http://www.oncolink.org/resources/article.cfm?c=3&s=38&ss=183&id=857
[Accessed 2 January 2005]
Matsuzawa I, Kondo Y, Kimura G, Hashimoto Y, Horie S, Imura N, et al. Cyclooxygenase-2 expression and relationship to malignant potential in human bladder cancer. J Health Sci. 2002;48:42–47
Washart ML. Treating and preventing cancer through COX-2 inhibition. In: 38th Annual Meeting of the American Society of Clinical Oncology. 18-21 May 2002; Orlando, Florida 2002
Mohammed SI, Knapp DW, Bostwick DG, Foster RS, Khan KNM, Masferrer JL, et al. Expression of cyclooxygenase-2 (COX-2) in human invasive transitional cell carcinoma (TCC) of the urinary bladder. Cancer Res. 1999;59:5647–5650
Mokos I, Jakić-Razumović J, Mareković Z, Pasini J. Association of cyclooxygenase-2 immunoreactivity with tumor recurrence and disease progression in superficial urothelial bladder cancer. Tumori. 2006;92:124–129
Mostafa MH, Sheweita SA, O’Connor PJ. Relationship between schistosomiasis and bladder cancer. Clin Microbiol Rev. 1999;12:97–111
Naruse K, Yamada Y, Nakamura K, Aoki S, Taki T, Zennami K, et al. Potential of molecular targeted therapy of HER-2 and COX-2 for invasive transitional cell carcinoma of the urinary bladder. Oncol Rep. 2010;23:1577–1583
Nasir A, Boulware D, Kaiser HE, Lancaster JM, Coppola D, Smith PV, et al. Cyclooxygenase-2 (COX-2) expression in human endometrial carcinoma and precursor lesions and its possible use in cancer chemoprevention and therapy. In Vivo. 2007;21:35–44
Shimada K, Anai S, Marco DA, Fujimoto K, Konishi N. Cyclooxygenase 2-dependent and independent activation of Akt through casein kinase 2 contributes to human bladder cancer cell survival. BMC Urology. 2011;11:8
Suh N, Yang XJ, Tretiakova MS, Humphrey PA, Wang HL. Value of CDX2, villin, and α-methylacyl coenzyme A racemase immunostains in the distinction between primary adenocarcinoma of the bladder and secondary colorectal adenocarcinoma. Mod Pathol. 2005;18:1217–1222
Tricker AR, Stickler DJ, Chawla JC, Preussmann R. Increased urinary nitrosamine excretion in paraplegic patients. Carcinogenesis. 1991;12:943–946
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Zhou M, Chinnaiyan AM, Kleer CG, Lucas PC, Rubin MA. Alpha-methylacyl-CoA racemase: a novel tumor marker over-expressed in several human cancers and their precursor lesions. Am J Surg Pathol. 2002;26:926–931