Role of TROP2, Cyclin D1 and FOXP3 in Bladder Carcinoma in Egyptian Patients: An Immunohistochemical Study


 BackgroundIn Egypt, Urinary bladder carcinoma is a common malignancy accounting for 14.3% of total malignancies in both sexes with 3:1 male to female ratio. It comprises 88.3% of the total urinary system tumors. To reduce bladder cancer morbidity and mortality there is an urgent need to identify novel tumor marker which are specific enough for prognosis and can serve as effective anticancer targets .Therefore the purpose of this study is to evaluate the role of TROP2, CYCLIN D1, FOXP3 and their relationship with the established clinico-pathological parameters and overall survival of bladder cancer in Egyptian patientsMethodsUsing the standard immunohistochemical technique, TROP2, CYCLIN D1 and FOXP3 expression in 80 primary bladder carcinomas and 20 specimens as non neoplastic group were assessed. The bladder carcinoma cases included 50 cases with muscle invasive bladder cancer and 30 cases with non-muscle invasive bladder cancer ResultsOverexpression of both TROP2 and FOXP3 implied poor prognostic impact as significantly associated with muscle invasive bladder cancer, high grade, advanced stage, lymph node involvement and high mitotic count. Cyclin D1 displayed an inverse relation with both TROP2 and FOXP3 reflecting a favorable prognostic impact. Tumoral FOXP3 expression is directly correlated with peritumoral FOXP3+ lymphocytes expression. TROP2, CYCLIN D1, FOXP3 expression didn’t affect the overall survival of the studied sample.ConclusionsThe inverse relation between Cyclin D1 and TROP2 proposes consumption of Cyclin D1 by TROP2 as a ligand in the urinary bladder carcinogenesis. Strong diffuse overexpression of both TROP2, and FOXP3 could be promising potential biomarkers for identifying patients with poor prognostic factors in bladder cancer serving as potential targets for cancer therapy.


Abstract Background
In Egypt, Urinary bladder carcinoma is a common malignancy accounting for 14.3% of total malignancies in both sexes with 3:1 male to female ratio. It comprises 88.3% of the total urinary system tumors. To reduce bladder cancer morbidity and mortality there is an urgent need to identify novel tumor marker which are speci c enough for prognosis and can serve as effective anticancer targets .Therefore the purpose of this study is to evaluate the role of TROP2, CYCLIN D1, FOXP3 and their relationship with the established clinicopathological parameters and overall survival of bladder cancer in Egyptian patients

Methods
Using the standard immunohistochemical technique, TROP2, CYCLIN D1 and FOXP3 expression in 80 primary bladder carcinomas and 20 specimens as non neoplastic group were assessed. The bladder carcinoma cases included 50 cases with muscle invasive bladder cancer and 30 cases with non-muscle invasive bladder cancer

Results
Overexpression of both TROP2 and FOXP3 implied poor prognostic impact as signi cantly associated with muscle invasive bladder cancer, high grade, advanced stage, lymph node involvement and high mitotic count.
Cyclin D1 displayed an inverse relation with both TROP2 and FOXP3 re ecting a favorable prognostic impact.
Tumoral FOXP3 expression is directly correlated with peritumoral FOXP3+ lymphocytes expression. TROP2, CYCLIN D1, FOXP3 expression didn't affect the overall survival of the studied sample.

Conclusions
The inverse relation between Cyclin D1 and TROP2 proposes consumption of Cyclin D1 by TROP2 as a ligand in the urinary bladder carcinogenesis. Strong diffuse overexpression of both TROP2, and FOXP3 could be promising potential biomarkers for identifying patients with poor prognostic factors in bladder cancer serving as potential targets for cancer therapy.

Background
In Egypt, Urinary bladder carcinoma is a common malignancy accounting for 14.3% of total malignancies in both sexes with 3:1 male to female ratio. It comprises 88.3% of the total urinary system tumors according to the National cancer institute registry 2016 (1). The expected new cases are about 10.709 by 2020, 12.762 by 2025 and 28.337 by 2050 (2,3).
To reduce bladder cancer morbidity and mortality there is an urgent need to identify novel tumor marker which are speci c enough for prognosis and can serve as effective anticancer targets (4).
TROP-2 is a transmembrane glycoprotein encoded by the Tacstd2 gene (5) ,which has been actively studied as a prognostic marker and an attractive immunotherapeutic target in human cancer treatment (6).
Trop2 has several ligands, including claudin-1, claudin-7, cyclin D1, and potentially IGF-1, as for cyclin D1 is a protein encoded by CCND1 gene and it is required for progression through the G1 phase of the cell cycle (7). Trop2 forms an oncogenic fusion protein with cyclin D1 (8). This chimera is expressed by human tumors differentially (9). FOXP3 is a forkhead box transcription factor containing a DNA-binding domain (10), it is known as the most speci c marker of the regulatory T lymphocytes (Tregs) (11). FOXP3 plays a crucial role in the development and function of Tregs, it is constitutively expressed in the nucleus of human Tregs (12).
The aim of this study is to investigate the role of TROP2, Cyclin D1 and FOXP3 in bladder carcinoma and correlate their expression with the available clinicopathological parameters and overall survival.

Methods
This retrospective study included 80 primary bladder carcinoma and 20 non neoplastic bladder specimens. Mitotic and apoptotic count were counted semi quantitatively in ten randomly selected high power elds (14) Depth of invasion and staging of the tumor were assessed according to TNM staging system/American Joint Committee on Cancer (AJCC) Staging manual 8th edition . According to TNM classi cation for the stage; the malignant tumors were classi ed histologically as non-muscle invasive urinary bladder carcinoma (NMIUBC) (stage pTa and pT1) or muscle invasive urinary bladder carcinoma (MIUBC) (stage pT2, pT3 and pT4) (15).

Immunohistochemistry:
The method used for immunostaining was streptavidin-biotin ampli ed system. Sections cut from the para nembedded blocks were stained with Anti-TROP2 (cat# 241308, abbexa, UK) puri ed rabbit poyclonal antibody was received as 0.1 ml conc. and diluted by phosphate buffer saline (PBS) in a dilution of 1:100. Anti-Cyclin D1 (cat# RM-9104-R7, Thermo Fisher Scienti c, USA) rabbit polyclonal antibody was received was received as a ready to use 7 ml vial. Anti-FOXP3 (cat # ARP32743, Aviva Systems Biology, USA) rabbit polyclonal antibody was received as 0.1 ml. conc. and diluted by PBS in a dilution of 1:100.
Tissue sections prepared from, normal skin as positive control for TROP-2 (16), from normal human tonsil for Cyclin D1 (17) and from spleen for FOXP3 (18) .Negative control slides were also included in each run by omitting the primary antibody.
Cytoplasmic and/or membranous staining in any number of tumor cells for TROP2 were required to assign the positivity (16). .Nuclear staining in any number of cells for Cyclin D1 were required to assign the positivity (17) FOXP3 was assessed in malignant epithelial tissues and in the intra-tumoral and peri-tumoral in ltrating lymphocytes, cytoplasmic and nuclear staining in any number of tumor cells and tumor in ltrating lymphocytes whether (peri-tumoral or intratumoral) respectively were required to assign the positivity (18).
Regarding tumoral FOXP3 immunoreactive score (IRS), low IRS was in favor of early stage group (P=0.003) and absence of lymph node involvement (P=0.025). In contrast, high FOXP3 IRS was signi cantly in favor of presence of LVI, since 9 cases out of 17 cases of positive LVI displayed high IRS (P=0.001) . Also, high FOXP3 IRS was statistically associated with high mitotic count (P=0.008) [ Table 5].
-Correlation between Cyclin D1 H-score and both TROP2and tumoral FOXP3 H-scores in malignant cases There was a signi cant inverse relationship between Cyclin D1 H-score and both TROP2 and

Discussion
In the current study TROP2, was expressed in 85% of the non-neoplastic urothelium and in 97.5% of the malignant group (P>0.05) in agreement with (20, 21, 22, 23). Transcription factors known to be involved in cancer cell progression, such as WT1 regulate Trop-2 transcription (24), in addition, overexpression of TROP2 may be due to Trop-2's intrinsic regulatory effects on cancer cell growth, invasion, and proliferation (25). So, overexpression of Trop-2 naturally leads to tumor progression as a key driver of cancer growth (6). On the other hand, Trop2 was found to be over expressed across normal tissues in animal model, including bladder, uterus, kidney, lung, and skin (23). The expression of Trop2 in normal tissues may play an important role in normal tissue homeostasis. EpCAM, the Trop2 paralog, is thought to function as an epithelial cell adhesion molecule (26; 27). Trop2 shares a conserved cysteine-rich region in its extracellular domain that is required for EpCAM-mediated adhesions (27).
TROP2 high immunoreactive score (IRS) was signi cantly associated with poor prognostic factors as high grade, advanced stage, presence of lymph node involvement , LVI , PNI and high mitotic count. These results are in agreement with (20) who demonstrated that high expression of TROP2 and high score were signi cantly associ ated with tumor high grade, advanced stage, and recurrence in UBC who also found that TROP2 overexpression was associated with tumor stage, lymph node involvement, tumor size and distant metastases in gastric carcinoma .
Studies such as (28,29,4) found that overexpression of TROP2 enhanced cell proliferation, migra tion, and invasion in the lung cancer cells, gall bladder cancer and oral squamous cell carcinoma (OSCC) respectively, while downregulation of TROP2 triggered apoptosis and impaired prolif eration. This was explained by (30,29) who found that TROP2 lead to activation and regulation of ERK path way in cervical cancer cells and regulation of PI3K/AKT pathway inducing EMT in gall bladder cancer respectively . Moreover, studies done (31,32) found that loss of TROP2 lead to autocrine activation of the EGFR family member ErbB3 through neu -regulin-1 in the mesenchymal subtype of head and neck squamous cell cancer (HNSCC) and induced sensitivity to anti-ErbB3 antibodies, leading to reduced pro liferation and tumorigenic growth in HNSCC cells .
Regarding Cyclin D1, it was expressed in 85% of the non-neoplastic urothelium and in 76.2% of the malignant group (P>0.05), in the current study. Our results are in concordance with (33) who reported cyclin D1 immunoreactivity simi larly frequent in bladder tumors (51.6%) and normal tissue of bladder (50%). A study done in 2007 (34) agreeing, reported uniformly intense expression of cyclin D1in the non-neoplastic group. However other studies (35) reported higher cyclin D1 protein expression in UBC and in endometrial carcinoma (36) compared to the adjacent normal tissue.
While other studies (37, 38, 39) reported complete absence of cyclin D1 in normal urothelium and in other tissues as colonic and gastric mucosa (40,41,42) while only expressed in the carcinoma group.
Cyclin D1 high expression was reported in early stage group (P=0.031), absent PNI (P=0.037) and absence of lymph node involvement (P=0.001), in agreement with (43,44 ,17) who stated low level of Cyclin D1 in advanced stage, poorly differentiated tumors, vascular invasion, as well as lymph node involvement. While Lee et al., 2010 found that cyclin D1 was signi cantly higher with advanced stage and MIBC (45).
In the current study , all high IRS cyclin D1 cases (20/20) displayed absence of bilharzaial infestation (P=0.001) in agreement with which was explained by (46) . The favorable prognostic impact implied by Cyclin D1 overexpression is attributed to its evidence in the initial stages where cell proliferation is a necessary step, involving no tumor invasion or metastasis as suggested by (47) and that low cyclin D1 expression might be a surrogate of other genetic events in the same cells, which ultimately drives cell growth and leads to worse prognosis (48). Moreover, the phenotype of cyclin D1was correlated with the degree of cancer progression and invasiveness. Altered expression of cyclin D1 may lead to changes in the biological behavior of transformed cells, for instance growth, proliferation, invasion and metastasis (17) The inverse correlation between Cyclin D1expression and poor prognostic parameters was not only reported in urothelial carcinoma; but also among other tumors, as in gastric carcinoma (49), in laryngeal squamous cell carcinoma (50) and in invasive breast carcinoma (51).
Regarding the expression of FOXP3 in the studied groups, FOXP3 was expressed only in epithelium in the nonneoplastic group and was found to be expressed in both malignant cells and lymphocytes (peritumoral and intratumoral) in cases of malignant group.
In this current study, FOXP3was expressed in 85% of the normal urothelium and in 75% of the tumor cells in malignant group (P>0.05). This is in contrast to (18) and (52) who found that FOXP3 was highly expressed in cancer cells of UBC while (53) found that FOXP3 was expressed in normal breast and down-regulated in adjacent mammary cancer. Predominance of missense mutations in breast and prostate cancer patients via transcriptionally repressing ErbB2and Skp2genes in breast cancer and c-Myc gene in prostate cancer suggested the role of FOXP3 as a tumor suppressor gene (53, and 54).
Low tumoral FOXP3 immunoreactive score (IRS) is in favor of early stage group (P=0.003), absence of lymph node involvement (P=0.025), absence of LVI (P=0.001) and low mitotic count (P=0.008). Association of tumoral FOXP3 with poor prognostic factors is in agreement with (55) who demonstrated that expression of tumoral FOXP3 was associated with lymphatic metastasis, advanced stage and high proliferative index (Ki-67 ≥ 14%) in cancer breast .
The prognostic role of FOXP3 in tumor cells has been studied for many years. In vitro, FOXP3 represses the transcription of the HER2, SKP2, MYC, MMP2, and UPA genes and induces the expression of p21 and LATS2. Thus, inhibited cell growth, cell migration and cell invasion which have been observed in cell lines derived from breast, prostate and ovarian cancers that overexpress FOXP3 (56).
This poor outcome in cancer may be due to variable mechanisms. Regulatory T cells inhibit many adaptive and innate immune cells, including CD4+ T cells, CD8+ T cells, dendritic cells, macrophages, and B cells. It has been shown that Treg cells also inhibit NK cells in a TGF-b dependent manner (57). Most of FOXP3+ Treg cells are CD4+ T cells that express CD25 can suppress the activation, proliferation and effector functions of a wide range of immune cells displaying a central role in the prevention of immune diseases (65,66).
In this current study, tumoral FOXP3 H. Score was signi cantly associated with increase in peritumoral positively stained FOXP3+ lymphocytes H. score (p=0.001) and this agreeS with (67) who reported that a high in ltration of FOXP3+ lymphocytes was accompanied by FOXP3+ tumor expression in cancer breast . FOXP3 expressed in the malignant cells succeeded to recruit lymphocytes in ltration into tumor microenvironment .Most of FOXP3+ Treg cells are CD4+ T cells that express CD25 suppressing effector functions of a wide range of immune cells.
Regarding overall survival, in current study, presence of bilharziasis, advanced pathological T stage and presence of lymph node invasion showed poor impact on patient's outcome. Furthermore, by multivariate COXregression analysis, pathological T stage was the most independent prognostic factor affecting patient's overall survival agreeing with (13).
On contrary with several studies which found that TROP2 expression had been associated with poor survival in various cancers including; CRC (68, 69 and 70), breast cancer (8), gastric cancer (71; 72; 73) and cancer cervix (30), our study failed to revealed signi cant association between the TROP2 immunoreactivity and patient's survival. On the other hand, studies as (74) found that Trop2 overexpression was associated with better survival in non small cell lung cancer (NSCLC) in patients with adenocarcinoma and may be a better prognostic marker in advanced stage adenocarcinoma .
Similarly, our study failed to reveal signi cant association between the Cyclin D1 immunoreactivity and patient's overall survival. This was similar to (75), while (33) reported that decreased expression of Cyclin D1 was associated with poor prognosis.
Furthermore, our study failed to reveal signi cant association between the tumoral FOXP3 immunoreactivity and patient's survival. This was contrary to (76, 52; 77; 55) who found that increased expression of FOXP3 was associated unfavourable prognosis in pancreas, bladder and breast carcinoma respectively which is in contrast to (63,78) who demonstrated a positive correlation between tumoral FOXP3 and survival in patients with HER2+ tumours who have received neoadjuvant therapy and in gastric carcinoma respectively. These ndings might be explained by few number of studied cases.

Declarations
Ethics approval and consent to participate: The ethics committee in the faculty of medicine Menou a University approved the purpose of this retrospective study and the use of archival para n blocks to evaluate the expression of the studied primary antibodies.

Consent for publication:
Not applicable Availability of data and materials: The data that support the ndings of this study are available from [third party name] but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of [third party name].
Competing interests: The authors declare that they have no competing interests Funding: No fund was contributed to the current study Authors' contributions: Dalia Rifaat Al-Sharaky -participated in histopathological assesement of the stained slides, interpretation of the results, analysis and a contributor in writing the manuscript.
Moshira Mohammed Abdelwahed -participated in interpretation of the results, analysis and writing the discussion.
Hend Ahmad Abdou Kassem-participated in histopathological assesement of the stained slides, interpretation of the results, analysis and writing the manuscript.
Abdelnaby Saied Abdelnaby -participated in histopathological assesement of the stained slides All authors read and approved the nal manuscript.