Cyclooxygenase-2 immunohistochemical expression is associated with worse prognosis in breast cancer
A retrospective study was carried out, between September 2019 and September 2021, and included a total of 570 BC specimens surgically removed prior to radio therapeutic, chemotherapeutic and hormonal manipulation regimes, which were investigated and examined by immunohistochemical staining, along with 52 of normal breast tissues from breast specimens resected for benign lesions or reconstruction (fibroadenoma and normal breast epithelium) were used as controls. The data of patients and histopathology blocks and slides were gathered from the Pathology Department, King Abdulaziz University Hospital, Jeddah, Saudi Arabia. Tumor grade was reviewed and reclassified in line with the classification of the World Heatlh Organization (WHO). The tumor stage was reviewed and reclassified in line with the standards of the American Joint Committee on Cancer. Stage was categorized as low stage cancers (Stages 0-II) and high stage cancers (Stages III-IV). The clinical and pathological outcomes are recorded in . This study was permitted by the Biomedical Ethics Committee at King Abdulaziz University, Jeddah, Saudi Arabia. The applied practices and techniques were compliant with the revised Helsinki Declaration. The assembly of tissue microarrays (TMA) was carried out as explained in our previous published reports. , Hematoxylin and eosin-stained slides of BC, fibroadenoma, and normal breast epithelium were assessed, and chosen areas were marked. Cases that exhibited widespread necrosis, poor cells’ preservation, inadequate tumor tissue, or cellular autolysis were excluded. Paraffin blocks of chosen cases were employed to obtain 2 cores of tumor tissue and next impeded in blocks by TMA Master 1.14 SP3 - a tissue microarray machine - (3DHISTECH Ltd., Budapest, Hungary). Then sections (4 μm) were sliced and used for immunohistochemistry staining technique. Tissue microarrays blocks have been sliced at 4 μm and put on coated slides. Deparaffinization and rehydration of sections were completed using an auto-immunostainer (Ventana Medical Systems Inc., Tucson, USA). Immunohistochemistry stain was carried out utilising a diluted monoclonal antibody for COX-2 (1:50) (Dako, Glostrup, Denmark). Positive colorectal carcinomas for COX-2 have been employed as positive control. Breast cancer slides with replacement of the monoclonal antibody with Tris-buffered saline were utilized as a negative control. The fraction of COX-2 positive cells was semi-quantitatively recorded. Slides with cytoplasmic COX-2 staining were described as positive. Staining intensity was scored strong (3), medium (2), weak (1), or absent (0). Cases with positive tumor cells of less than 5% were considered negative. Statistical analysis All information was evaluated using the Statistcal Package for the Social Sciences, version 21.0 (IBM Corp., Armonk, NY, USA). All findings were recorded in numbers and percentages. Association between clinicopathological information of BC and COX-2 phenotype was examined via Chi-squared and Fisher tests. The Cox proportional hazards model helped decide if any of the clinicopathological factors have an important influence on overall survival (OS) and disease free survival (DFS). Evaluation of survival distributions for various COX-2 expression scores used Kaplan-Meier survival curve. P -values of <0.05 were counted statistically important. 

All information was evaluated using the Statistcal Package for the Social Sciences, version 21.0 (IBM Corp., Armonk, NY, USA). All findings were recorded in numbers and percentages. Association between clinicopathological information of BC and COX-2 phenotype was examined via Chi-squared and Fisher tests. The Cox proportional hazards model helped decide if any of the clinicopathological factors have an important influence on overall survival (OS) and disease free survival (DFS). Evaluation of survival distributions for various COX-2 expression scores used Kaplan-Meier survival curve. P -values of <0.05 were counted statistically important. 

Cyclooxygenase-2 was expressed in the cytoplasmic part of malignant epithelial cells as brown granular staining in 239 (42.7%) cases of BC  and was detected in 14 (26.9%) of normal breast tissue . The present data show an important association between COX-2 phenotype and BC compared to benign breast tissue ( p =0.034). Fibroblasts and other interstitial cells were infrequently stained with COX-2. displays the status of COX-2 phenotype in BC cases and its correlation with several pathological and clinical factors. The percentage of positive malignant cells varies between 5-100% in BC of the current study. Almost half of BC cases revealed positive COX-2 staining in greater than 50% of their malignant epithelial cells. Stage of BC is correlated significantly with escalated COX-2 immunoexpression ( p =0.001). A substantial portion of high-stage cases was observed to be frequent with positive COX-2 staining. Considerably, more tumors with distant metastases were detected with positive COX-2 staining ( p =0.003). Almost half of the cases with lymphovascular invasion showed positive COX-2 staining ( p =0.034). Local recurrence of the tumor was marginally correlated with positive COX-2 immunostaining cases ( p =0.072). Recurrence is less common in negative COX-2 staining cases. No association was detected among COX-2 immunohistochemical phenotype and age, gender, size, lymph node metastasis, or margin status. The results of Log Rank test showed that substantial diverse survival distributions are found for various scores of COX-2 staining. The statistics reveals that COX-2 immunoexpression is correlated with the probabilities of DFS (log rank: 5.968, p =0.015) and OS (log rank: 4.136, p =0.042) . Positive COX-2 immunostaining is related to poor survival significantly. 

Although the immunoexpression of COX-2 has been widely studied in BC tissues . Over the past decade, 17 reports employed immunohistochemistry staining to describe the phenotype of COX-2 in BC and to associate this expression with the clinicopathological parameters of BC cases, but the results showed a considerable controversy. Four of these studies showed statistically significant overexpression of COX-2 in BC tissues compared to that in benign tissues of breast, and potential clinical use of COX-2 in prognosis prediction. - These results are consistent with our report that addressed a significant increase ( p =0.034). While only 2 studies found opposite results and stated that COX-2 expression is more common in normal and benign lesions of the breast compared with BC, and the remaining studies did not investigate COX-2 immunoexpression in normal tissue and benign lesions of the breast. , A total of 17 studies demonstrated an important relationship between the phenotype of COX-2 and one or more of the histopathologic parameters of BC cases such as age, tumor size, histological type, tumor grade, advanced stage, lymph nodes metastasis, lymphovascular invasion, distant metastasis, surgical margins, and disease recurrence or shorter DFS. - , - On the other hand, 3 studies found an inverse association with grade and DFS, and few other reports could not find such relationships. , , - Here we detected an important relationship between increased COX-2 immunohistochemical expression and advance stage, metastases and lymphovascular invasion, which characterizes aggressive types of BC. In respect of the association with tumor stage, our outcomes are in line with the following earlier studies and contradict the following reports, which could not find such association. - , - , , Only 3 studies examined the relationship among COX-2 immunoexpression and distant metastases, of which 2 studies found statistical significance and were in line with the current report, while only one study opposed these results. , , A total of 7 studies attempted to link the immunoexpression of COX-2 with lymphovascular invasion, the results of 4 studies supported our finding and showed significant association with COX-2 expression while the other 7 reports failed to do so. , , - , Regarding DFS, 6 studies used a log-rank comparison test to reveal substantial different survival distributions for several scores of COX-2 immunostainings, of which 3 studies showed that COX-2 immunohistochemical phenotype is allied with bad survival significantly. , , Our results support these 3 studies and contradicted the remaining 3, which one of them found an inverse association. , , Study limitations Although our findings are encouraging, our investigation and the other 17 reports have several limitations such as the sensitivity of utilized techniques, populations diversity, sample size variations, inconsistent scoring methods, and the semi-quantitative evaluation of staining. Still, multicentre research with a larger number of cases is positive and huge value for evaluating the clinical importance of COX-2 staining in the detection and prognoses of BC. In conclusion, COX-2 is a valuable marker that could support BC diagnosis and prognosis. Its expression associated with several clinicopathological factors which distinguish aggressive subtypes of BC, such as advanced stage, distant metastases, lymphovascular invasion, and poor survival. 

Although our findings are encouraging, our investigation and the other 17 reports have several limitations such as the sensitivity of utilized techniques, populations diversity, sample size variations, inconsistent scoring methods, and the semi-quantitative evaluation of staining. Still, multicentre research with a larger number of cases is positive and huge value for evaluating the clinical importance of COX-2 staining in the detection and prognoses of BC. In conclusion, COX-2 is a valuable marker that could support BC diagnosis and prognosis. Its expression associated with several clinicopathological factors which distinguish aggressive subtypes of BC, such as advanced stage, distant metastases, lymphovascular invasion, and poor survival. 

