{"paper_id":"e939b49a-8f41-4c33-94c5-abb0bea75d27","body_text":"The cyclooxygenase (COX) family of enzymes contains two members ( COX-1  and  COX-2 ) (Chen et al., 2014).  COX-1  is expressed ubiquitously (Williams and DuBois, 1996).  COX-2  is expressed in distinct tissues and is involved in inflammatory processes (Herschman, 1996). Chronic inflammation is well known to be linked with cancer progression (Howe, 2007). Recent emerging epidemiologic, preclinical, and clinical data suggest that  COX-2  up-regulation is a fundamental step in carcinogenesis (Cao and Prescott, 2002) and tumor angiogenesis (Davies et al., 2003). Elevated  COX-2  expression has been detected in human breast tumor tissues (Ristimaki et al., 2002).\nThe age-associated increase in  COX-2  activity has been noticed in animal models (Claycombe et al., 2002). Elevated level of COX-derived products has been identified in platelets and peripheral blood mononuclear cells (PBMCs) in elderly humans (Vericel et al., 1988; Meydani et al., 1990). Furthermore,  COX-2  up regulation is associated with distinct pathological features such as large tumor size, high tumor grade and metastasis (Ristimaki et al., 2002; Wulfing et al., 2003).  COX-2  induces aromatase in breast tissue (Salhab et al., 2007). Aromatase activity increases estrogen levels (Vienonen et al., 2002). Consequently,  COX-2  expression increases the estrogen levels and subsequently tumor progression in hormone receptor positive breast cancer (Hoellen et al., 2011).\nTryptophan catabolism is linked with immunosuppression in the tumor microenvironment.  Indoleamine 2, 3-dioxygenase (IDO)  is a heme containing tryptophan-degrading enzyme (McGaha et al., 2012).  IDO  overexpression in tumors results in prompt conversion of tryptophan into kynurenine. Tryptophan depletion and enhanced levels of kynurenine play pivotal role in immunosuppression (McGaha et al., 2012; van Baren and Van den Eynde, 2015).  IDO  expression is involved in breast tumor growth and pulmonary metastasis (Levina et al., 2012). Furthermore, high  IDO  expression is significantly linked with overall decreased patient survival (Asghar et al., 2019).  COX-2  and  IDO  promote breast cancer progression (Chen et al., 2014). The current study has been conducted in Pakistan, a country with one of the high incidence of breast cancer in its region, to investigate the association between  COX-2  and  IDO  expression in breast cancer patients.\n\nTumor tissue specimens\nA retrospective study was performed on formalin-fixed paraffin-embedded (FFPE) tumor specimens of breast cancer patients (n=100). These specimens were retrieved from pathology department, at Shaukat Khanum Memorial Cancer Hospital and Research Centre (SKMCH and RC) Lahore, Pakistan. The patients selected for the current study were diagnosed with breast cancer between 2007 and 2009. All the patients were treatment naïve. Tumor grade was allocated using the Nottingham Histologic Score. IHC analysis of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expressions were conducted and interpreted using standard methods (Chen et al., 2010). The comprehensive information about the clinico-pathological characteristics was retrieved from medical records and pathology reports.\nEthical approval for retrospective study\nThe current study (#IRB-16-08) was approved by the Institutional Review Board (IRB) of the SKMCH and RC. This study was exempted for informed consent by IRB (SKMCH and RC) which is in compliance with the Declaration of Helsinki.\nImmunohistochemical staining\nSlides were stained using Bond III Leica automated system (Leica Biosystems Melbourne, Australia) as per manufacturer’s protocol. Two sections of FFPE tumor specimens of the same patients were attained, and slides were deparaffinized on the automated system with Bond Dewax solution (Leica Biosystems). Briefly, heat induced epitope retrieval was performed with Bond ER -2 (Leica Biosystems), for 20 min. Both the primary antibodies  COX-2  (abcam, # ab15191, anti- COX-2  antibody) and  IDO1  (abcam, # ab55305, anti-Indoleamine 2,3-dioxygenase antibody) were used at a 1:200 concentration. Tissue sections were incubated for 5 min with appropriate primary antibody in diluent Bond (Leica Biosystems). Antibody labeling was visualized by BondTM polymer refine detection kit. Then incubated with post primary rabbit anti mouse IgG for 8 min and subsequently, incubated with polymer anti-rabbit poly-HRP-IgG for 8 min. DAB 3, 3’-diaminobenzidine tetrahydrochloride hydrate was used as chromogen. Slides were dehydrated and cover slipped as per our earlier described laboratory protocol (Asghar et al., 2019). Slides were visualized by an optical microscope (Provis AX-70, Olympus, Melville, NY).\nEvaluation of COX-2 and IDO scoring\nIt was a blind histopathologic assessment. The slides were evaluated by pathologists. The  COX-2  and  IDO  immunostaining were examined by using the H-score, well-defined by the following equation: H-score = ΣPi (i + 1), as described previously (Chan et al., 2012), Pi is the percentage of stained tumor cells and i is the intensity of cytoplasmic staining of tumor cells (0 to 3+).\nStatistical analysis\nStatistical analysis was carried out using SPSS software (version 20.0; SPSS, Chicago, IL, USA). Percentages (proportions) were used for categorical variables while mean and standard deviation were used for continuous variables. Bivariate analysis was done using chi-square or fisher exact test (when necessary). For continuous explanatory variables such as age, independent t-test was performed. Univariable and multivariable logistic regression model was used to identify the risk factors.\nCOX-2  Immunostaining Images.  COX-2  expression in breast cancer patients (n=100) tissues was assessed by immunohistochemistry (A) Low  COX-2  expression was observed in ductal carcinoma. (B) High  COX-2  expression was detected in invasive ductal carcinoma. The staining was cytoplasmic. Images were captured at x 40 magnification\nDemographic Characteristics versus Low and High  COX-2  Scores\nvalues indicate statistical significance p< 0.05; SD, Standard deviation\nClinicopathological Characteristics versus Low and High  COX-2  Scores\n*values indicate statistical significance p< 0.05; IDO, Indoleamine 2,3-dioxygenase\nCOX-2  and  IDO  co-Expression. (A) Strong  COX-2  expression was detected in invasive ductal carcinoma. Sections from the same breast cancer patients were stained for  IDO  (B) Strong and diffuse  IDO  staining in invasive ductal tumor cells. Both staining were cytoplasmic. Images were captured at x 40 magnification\nRisk Factors of  COX-2  High Expression\n*values indicate statistical significance p< 0.05; IDO, Indoleamine 2,3-dioxygenase\n\nDemographic characteristics versus low and high COX-2 scores\nDemographic characteristics of 100 breast cancer patients are summarized in  Table 1 . Overall the mean age at breast cancer diagnosis was 48.28 ± 11.83, and there was a mean difference of age in low and high  COX-2  expression (p=0.02). Majority of patients belonged to the Punjab region (88%).\nClinicopathological characteristics versus low and high COX-2 scores\nIn order to examine the association between  COX-2  and IDO, we categorized the patients into  COX-2  low versus high. There was a statistically significant association of  COX-2  with  IDO  expression (p = 0.004), ER (p = 0.003), PR (p = 0.002) and tumor grade (p= 0.02) respectively ( Table 2 ).\nCOX-2 and IDO immunostaining\nTo investigate the expression of  COX-2  and  IDO , FFPE tumor specimens (n=100) of same patients were selected. Out of 100 tumor specimen,  COX-2  high, and low scores were 59%, and 41%, respectively ( Figure 1 ).  IDO  positivity was observed in all breast tumor specimens.  COX-2  and  IDO  co-expression is shown in  Figure 2 .\nRisk factors of COX-2 high expression\nTable 3  summarizes various clinical and pathological factors that were included in the univariable and multivariable analyses to identify the  COX-2  association with  IDO  expression. In multivariable analysis, three variables were identified as significant independent risk factors for high  COX-2: IDO  expression high; [adjusted odds ratio (AOR) 6.51; 95% confidence interval (CI) (2.00- 21.20), p=0.001], ER; [AOR 5.62; 95% CI (1.80- 17.84), p=0.002] and age [AOR 1.04; 95% CI (1.00- 1.10), 0.05.\n\nPrevious studies established that  IDO  overexpression is involved in tumor immune escape in various cancers (Muller et al., 2005; Mansfield et al., 2009). It has been documented that  IDO  and  COX-2  promote breast cancer progression (Chen et al., 2014). To the best of our knowledge, this is first study which revealed that high  IDO  expression is associated with high  COX-2  expression in Pakistani breast cancer patients; coherent with the findings demonstrated by Mei et al., (2012). It is well established that  COX-2  expression induces constitutive expression of  IDO  in human tumor cells (Hennequart et al., 2017). But interestingly Mei et al., (2012) demonstrated that IDO inhibitors suppressed the  COX-2  expression and  IDO  may be involved in endometriosis pathogenesis via promoting  COX-2 . Our data showed as well that high  IDO  expression is associated with  COX-2  expression which might play a role in breast cancer pathogenesis.\nCOX-2  upregulation is involved in age-related dysregulation of the immune responses (Wu and Meydani, 2004). Siironen et al., (2004) demonstrated that an increase in the  COX-2  expression is associated with age in papillary thyroid cancer patients. Our data revealed that increase in the  COX-2  expression is associated with age in Pakistani breast cancer patients. In addition, it has been established that ER-positive breast cancer may evolve from low to high grade (Lopez-Garcia et al., 2010; Natrajan et al., 2010).  COX-2  expression upregulates transcription of aromatase and consequently stimulates tumor cell progression in ER-positive breast cancer (Diaz-Cruz et al., 2005). Our results are in agreement that high  COX-2  expression is associated with ER-positive breast cancer.\nAs we aimed to investigate the association between  COX-2  and  IDO  expression in breast cancer patients from Pakistan, a country with high incidence of breast cancer cases, our data showed the high  IDO  expression is associated with high  COX-2  expression. The co-expression of both enzymes may suggest their role in disease pathogenesis. Basu et al., (2004) suggested that  COX-2 -mediated immunosuppression through IDO regulation may act as therapeutic target for the development of future cancer vaccines. Chen et al., (2014) demonstrated that stromal  IDO  upregulation is associated with overexpression of  COX-2 . Previous studies established that increase in  COX-2  expression lead to increase in  IDO  expression. Our data is demonstrating that an increase in  IDO  expression is associated with an increase in  COX-2  expression. A significant association is present between  IDO  and  COX-2  expression in Pakistani breast cancer patients. The data presented in this study will certainly serve as a useful addition to the already available knowledge, while the molecular mechanisms underlying  IDO  mediated  COX-2  expression need further investigation. Hence the simultaneous targeting of  IDO  and  COX-2  may be a promising therapy for breast cancer.","source_license":"CC-BY-4.0","license_restricted":false}