The Clinical Value of VDR and CTLA 4 in Evaluating the Prognosis of Invasive Duct Carcinoma of Egyptian Patients and their Benefit as a Target Therapy

Objective: Breast cancer represents the second most common female malignancies worldwide and the most common in Egypt. The nuclear vitamin D receptor plays a role in the biology of cancer by affecting inflammatory microenvironment. The aim of this study is to evaluate the role of VDR and CTLA 4 in invasive duct carcinoma of Egyptian patients. Methods: This is a retrospective study that included 70 invasive duct carcinoma specimens retrieved from the archival material of Pathology Department, Faculty of medicine, Menoufia University, Egypt, spanning the period between January 2010 and December 2017. All cases were stained for VDR and CTLA 4 antibodies. Results: There is significant association between high VDR expression in tumor cells and parameters of good prognosis as low tumor stage (T1) and (N0) stage. On the other hand, there is significant association between low CTLA4 tumor expression and good prognostic parameters as low tumor stage (T1) and absent vascular invasion. Regarding lymphocyte expression, there is significant association between positive CTLA4 expression in lymphocytes and parameters of good prognosis as absent metastasis. High VDR tumor expression is the most independent prognostic factor on overall survival of breast carcinoma patients. Conclusion: high VDR expression in tumor cells is associated with good prognostic parameters and is the most independent prognostic factor on overall survival so it might be of benefit as a target therapy for Egyptian invasive duct carcinoma patients and VDR might augment the expression of CTLA-4, So tailored immunotherapy might have an impact on invasive duct carcinoma patients.


Introduction
Breast cancer represents 24.5% and is considered the first most common female malignancies worldwide (GLOBOCAN, 2020), while in Egypt it represents 38.8% and is considered the most common female cancer and the second cause of female cancer death after lung cancer (Ibrahim et al., 2014;El Bolkainy et al., 2016).
The source of vitamin D in the body may be endogenous through sun exposure (up to 90%) or exogenous through dietary and supplemental intake (up to 10%) (Holick, 2006). There are many controversies between the relation between vitamin D and reduced breast cancer risk ( Robien et al., 2007;Wang et al., 2014).
The active form of vitamin D (1,25(OH)2D) binds to the vitamin D receptor (VDR), a ligand-dependent transcription factor, that regulates transcription of a number of genes involved in apoptosis, growth factor signaling, cell proliferation, differentiation and immunomodulation (McCullough et al., 2007). Presence of VDR in normal breast epithelial cells and breast cancer cells suggests a relation between it and breast cancer risk Egyptian females and despite the advances in surgery and diagnosis, the prognosis and survival of the patients are still unsatisfactory, so it is mandatory to search for different parameters related to prognosis and therapy. Also studies of tumor cells showed the important regulatory effect of vitamin D on the inflammatory microenvironment, but the evidence linking vitamin D and immune response in cancer is still scarce (Liu et al., 2018). The aim of this study is to evaluate the immunohistochemical expression of VDR and CTLA4 in invasive duct carcinoma of Egyptian patients and their relation to the available clinicopathological data.

Materials and Methods
This retrospective study was conducted on 70 breast invasive duct carcinoma cases obtained from the archival cases of Pathology Department, faculty of medicine, Menoufia University, in the period between January 2010 and December 2017. Clinical and survival data were retrieved from medical patients' files.
Inclusion criteria: 1-The type of breast carcinoma: we selected only invasive duct carcinoma not otherwise specified type 2-The type of surgery: we selected radical mastectomy specimens only.
3-Therapy: cases that didn't receive any neoadjuvant therapy received Exclusion criteria: 1-The type of breast carcinoma: we exclude any type other than invasive duct carcinoma not otherwise specified type 2-The type of surgery: we excluded core, incision and excision biopsies.
3-Therapy: we exclude any case that received any neoadjuvant therapy.

Histopathological Evaluation
From each representative paraffin block, 4-μm thick serial sections were cut and stained with haematoxylin and eosin stain.
The clinicopathological data as histological grade, hormonal status, vascular, perineural invasion, extracapsular nodal invasion, Multicentricity and metastasis were obtained from the patients' sheets and TNM staging system (2010) is used for staging of the tumor according to size into T1, T2, T3 and T4 and to nodal status into Nx, N0, N1, N2 and N3 (Edge, 2010).
A positive reaction was revealed using the streptavidinbiotin-peroxidase technique (cat. #TP-015-HD) (Lab Vision Cooperation) with chromogen DAB. The sections were then counterstained with Mayer's haematoxylin (cat. No. 94583;Bio Genex) for 30 to 60 s to stain nuclei. Sections were washed in tap water for 5 min. Placental tissue and human tonsil tissue were used as positive controls for VDR and CTLA4 respectively and normal breast tissue was used as positive control for ER, PR and HER2 neu.

Interpretation of VDR and CTLA-4 immunostaining
Expression: VDR was assessed in tumor cells and positive expression was considered if any tumor cells showed positive brownish nuclear staining in any number of cells. While CTLA4 was assessed in tumor infiltrating lymphocytes as well as tumor cells. Positive expression was considered if any tumor cells and/or lymphocytes showed positive brownish cytoplasmic staining in any number of cells (Adisa et al., 2017;.
Percentage: In both markers the percentage of positive cells was counted. The median percentage of positive cells was used as a cutoff point and the cases were divided into two groups: • Low percentage: ≤ the median • High percentage: > the median .

Interpretation of ER and PR Immunostaining
Bothe ER and PR showed nuclear staining and we assess the percentage and the intensity of the tumor cell, regarding percentage, both ER and PR are considered positive when more than 1% cells showed nuclear staining. The intensity of the staining was graded as weak, moderate and strong. It was considered negative when internal control cells present showed no nuclear staining (Longacre et al., 2017).

Interpretation of Her 2 neu Immunostaining
Only membranous staining was considered and Her 2 neu positivity was assessed using the following scoring system: (Yan et al., 2014). 0 : No membrane staining or less than 10% of cells. 1+: Partial membrane staining in more than 10% of cells.
2+: Weak, circumferential membrane staining in more than 10% of cells, or intense membrane staining in less than 30% of cells.
The relationship of VDR and CTLA4 expression in lymphocytes and tumor cells of the studied invasive duct carcinoma cases : 3+: Intense membrane staining in more than 30% of cells.
*Score 0,1&2 were considered negative for HER2 and score 3 was considered positive for HER2.

Overall survival data
Overall survival time is the length of time from either the date of diagnosis or the start of treatment for a disease, such as cancer, that patients diagnosed with the disease are still alive. In a clinical trial, measuring the overall survival is one way to see how well a new treatment works.
By revision of patients' files for breast carcinoma cases ranged from 2010 to 2017, overall survival time was available for 68 out of 70 patients.

Statistical analysis
The statistical analysis was conducted using SPSS "Statistical Package for the Social Sciences″ program for windows, version 20, SPSS Inc., Chicago, Illinois, USA. Mann-Whitney U and Kruskal-Wallis tests were used to compare nonparametric data, the chi-square test was used to assess the association between the clinicopathologic parameters and VDR and CTLA 4 expression and Kaplan-Meier test was used for survival analysis. P≤0.05 was considered to indicate statistical significance in all tests.

Results
Clinicopathological data of the studied invasive duct carcinoma are shown in Table 1 The age of the studied breast carcinoma cases was ranged from 27-83 year with a median of 48 and mean ± SD of 48.228 ± 12.53.
The Immunohistochemical results of VDR and CTLA4 antibodies are shown in Table 2 and plate 1 The relationship of VDR tumor expression in the studied invasive duct carcinoma cases and the clinicopathological parameters: There is significant association between high VDR tumor expression and good prognostic parameters as low tumor stage (T1) and (N0) nodal stage (P value= 0.02 and 0.01 respectively). Moreover, there is a trend of significance between positive VDR in the tumor cells and absent vascular invasion and absent metastasis (P value= 0.06 and 0.09 respectively). Also between high VDR expression in tumor cells and low grade (P= 0.09) ( Table 3 and 4).
The relationship of CTLA4 tumor and lymphocyte expression in the studied invasive duct carcinoma cases and the clinicopathological parameters: There is significant association between positive CTLA4 expression in lymphocytes and good prognostic parameters as absent metastasis (P= 0.018), and a trend of significance with low tumor stage (P= 0.08) (Table 5) On the other hand there is significant association between high CTLA4 expression in tumor cells and poor prognostic parameters as advanced tumor stage (T4) and presence of vascular invasion (P= 0.01 and 0.05 respectively), also a trend of significance with advanced nodal stage (N2 and N3) (P= 0.07) ( There is a direct association between positive VDR expression in tumor cells and positive and high CTLA4 expression in lymphocytes (P= 0.000 and 0.09 respectively) ( Table 7).
On the other hand there is inverse association between positive VDR expression in tumor cells and negative and low CTLA4 expression in tumor cells (P= 0.03 and 0.01 respectively) (Table 8).
Furthermore, there is inverse association between positive and high CTLA4 expression in tumor cells and negative and low expression in lymphocytes (P= 0.001 and 0.000 respectively for positivity and 0.005 and 0.009 respectively for high score of expression) (Table 9).

Overall survival data
By revising the files for breast carcinoma patients from 2010 to 2017, overall survival time was available for 97% of patients and with the range of 1 to 121 months, mean ± standard deviation of 44.94 ± 35.953 and a median of 26.5 months.

Univariate survival analysis for breast carcinoma cases
Univariate survival analysis revealed that positive VDR tumor expression (P-value = 0.001) and high VDR tumor expression (p-value = 0.001) have a good prognostic impact on the outcome of patients, but CTLA4 and other variables weren't significant (Figures 1 and 2).

Multivariate survival analysis for breast carcinoma cases
From multivariate survival analysis, high VDR tumor expression was proved to be the most and the first independent prognostic factor on overall survival of breast carcinoma patients i.e VDR is a favorable prognostic indicator for breast carcinoma. (Table 10) (Figure 3and 4)

Discussion
Breast cancer is the most common malignancy in Egyptian females and despite the advances in surgery and diagnosis, the prognosis and survival of the patients are still unsatisfactory, so it is mandatory to search for different parameters related to prognosis and therapy (GLOBOCAN, 2020) .  I n t h i s s t u d y w e t r i e d t o e v a l u a t e t h e Immunohistochemical expression of VDR and CTLA4 antidodies and their role in breast cancer of Egyptian patients as there is a great controversy about their role in suppression or promotion of breast cancer (Zhang et al., 2014;Murray et al., 2017;Tavera-Mendoza et al., 2017;Xu et al., 2018).
In the present study 58.2% of the studied cases showed moderate and strong VDR expression in tumor cells and this comes in line with Al-Azhri et al., (2016) who found 58% of the cases showed moderate and strong   In the current study there is significant association between high VDR tumor expression and parameters of good prognosis as low tumor stage (T1) and (N0) nodal stage, Moreover, there is a trend of significant between positive VDR in the tumor cells and absent vascular invasion and metastasis. Also between high VDR expression in tumor cells and low grade and these results come in line with Huss et al., (2019) who found that high expression of VDR is associated with favorable prognosis and low death rate in invasive duct carcinoma cases.
These results may be explained by the inhibitory intranuclear action of VDR as it inhibits breast cancer cell line growth (Murray et al., 2017) and induce autophagy in breast cancer cell line (Tavera-Mendoza et al., 2017). Also these results agreed with Zheng et al., (2017) who detected that VDR overexpression is associated with good prognostic parameters as small tumor size and low nodal stage and results may be explained as VDR inhibits breast cancer growth as it has pro-apoptotic and anti-proliferative effects through inhibition of the Wnt/β-catenin signaling pathway. Also regarding lung cancer, Increased VDR expression in lung adenocarcinoma is associated with improved survival. This may relate to a lower proliferative status and G1 arrest in high VDR-expressing tumors (Kim   DOI:10.31557/APJCP.2021.22.4.1183VDR and CTLA4 in Breast Carcinoma. et al., 2012. Moreover, regarding prostate cancer, high VDR expression is also associated with good prognostic parameters as lower Gleason score and early tumor stage (Hendrickson et al., 2011) On contrary to the present study El-Shorbagy et al.,    (2017) found that no association between breast cancer and VDR BsmI pleomorphism also other studies come in line with this opposite opinion (Chen et al., 2005;Wang et al., 2013;Zhang et al., 2014). These differences might be explained by different techniques used in different studies.  DOI:10.31557/APJCP.2021.22.4.1183 VDR andCTLA4     Regarding survival VDR is a favorable prognostic indicator for breast carcinoma and this come in line with Huss et al., (2019) but was opposite to that of Al-Azhri et al., (2016).
From the present study as high VDR is good prognostic factor for breast carcinoma as it associated with low tumor and nodal stage together with low grade, absent vascular invasion and metastasis. So It may be used to personalize the adjuvant therapy used in treatment of breast carcinoma as Mastectomies were performed more often on VDRnegative tumors (55%) compared to VDR-positive tumors (41%). The postoperative treatment conference recommended adjuvant endocrine therapy for a smaller proportion and chemotherapy for a larger proportion of patients with VDR-negative tumors compared to VDRpositive tumors (Tavera-Mendoza et al., 2017). Also, vitamin d may be used to decrease the risk of breast carcinoma in Egyptian patients.
In the past, CTLA-4 has been found to be limited to T cells but nowadays, evidence reveals CTLA-4 expression in tumor cells. CTLA-4 is also expressed in non-lymphoid cells including placental fibroblasts, muscle cells and monocytes, suggesting that this molecule might be involved in controlling functions other than the widely described T-cell response inactivation (Wang et al., 2002). Surface expression of CTLA-4 is detectable by reverse transcriptase-PCR in all cell lines derived from a variety of human malignant solid tumors including carcinoma, melanoma, neuroblastoma, rhabdomyosarcoma and osteosarcoma (Contardi et al., 2005) In the present study 40% of the lymphocytes and 81.4% of the studied invasive duct carcinoma cases show CTLA 4 positivity and these results are near those of Lan et al., (2018) regarding lymphocyte expression but not tumor expression as they found positive CTLA 4 in 41.2% of tumor and 46.1% of lymphocytes. Also, these results are in contrary with those of Mao et al., (2010) who found CTLA4 expression in 55% of lymphocytes and 100% of the studied breast carcinoma cases. These differences may be explained as the current study assessed CTLA4 expression only through semiquantitative immunohistochemistry, which may be less reliable than the other techniques used in different studies and due to heterogeneous study samples and different cut-off values of the CTLA-4 expression.
The present study showed that CTLA4 tumor expression was associated with poor prognostic parameters as there is significant association between low CTLA4 expression in tumor cells and low tumor stage (T1) and absent vascular invasion, also a trend of significance with (N0) nodal stage and these results come in line with Yu et al., (2015) and Wang et al., (2007) who found that the CTLA-4 gene may be associated with the progression of breast cancer in the Chinese Han population.
Also, some studies indicate that breast cancer patients with higher CTLA-4 mRNA levels had obvious axillary lymph node metastases and a higher clinical stage. Patients with high tumor CTLA-4 expression in mesothelioma, nasopharyngeal carcinoma and melanoma had a poorer prognosis than those with low expression, which suggested CTLA-4 as a potential target for tumor immunotherapy (Salvi et al., 2012;Huang et al., 2016;Roncella et al., 2016).
This inhibitory effect might potentially occur through the interaction between CTLA-4 expressed by the tumor cells and B7 ligands expressed by the tumor microenvironment cells including the antigen presenting cells (APC), such as dendritic cells (DCs), or antitumor activated T cells. This interaction might result in delivering CTLA-4-mediated negative signals into tumor cells leading to inhibition of their proliferation rate and/or induction of apoptotic cell death (Contardi et al.,3005) Also, agreed with Zhao et al (2018) who demonstrated that persistent expression of CTLA-4 on tumors contributed to the progression of both hematological and solid tumors, which produced inhibitory signals to weaken the immune response.
On the other hand, previous studies found that patients with positive tumor CTLA 4 expression had a better prognosis in NSCLC and gastric cancer (Kim et al., 2016). Moreover, other studies demonstrated that there is no association between CTLA4 expression in tumor cells and any of the clinicopathological parameters either in breast carcinoma or esophageal squamous cell carcinoma (Lan et al., 2018;Zhang et al., 2019).
This discrepancy whether CTLA-4 expression in tumor cells has a good prognostic impact or a bad impact is due to much less data known about its expression and function in tumor cells (Salvi et al., 2012). Moreover, there is increasing evidence that ligands of the immune checkpoint pathways could also trigger a receptor independent signal inside the cells in which they are expressed and that these signals could be different depending on the specific cell types. Therefore, it is still crucial to identify biomarkers that could predict these phenomena and to develop novel preclinical models suitable to investigate the underlining molecular mechanisms (Lecis et al., 2019) Regarding CTLA 4 expression in lymphocytes the current study demonstrated significant association with good prognostic parameters as absent metastasis, and a trend of significance with low tumor stage. These results agreed with Hu et al., (2017). These results may be explained as CTLA-4 exerts distinct independent effects during different phases of T cell responses, including setting the threshold for T cell activation, suppression of T cell proliferation, and induction of apoptosis in activated T cells (Denkert et al., 2015). On contrary, these results disagreed with Lan et al., (2018) who reported that there is no association between interstitial CTLA 4 expression and any of the clinicopathological parameters and also disagreed with  who reported that CTLA-4 lymphocyte expression showed an association with the presence of vascular emboli. This discrepancy may be explained by Sun et al., (2017) who reported that higher Tregs levels showed an association with poor tumor differentiation and metastasis.
Furthermore, There is a direct association between positive VDR expression in tumor cells and positive and high CTLA4 expression in lymphocytes and these come in line with Sheikh et al., (2018) who observed that the stimulation of CD4+ T cells with vitamin D suppresses proliferation capacity; enhanced the expression of PD1, PD-L1, and CTLA-4 inhibitory markers on CD4+ T and these results explained by Jeffery et al., (2009) who observed that stimulation of CD4+ CD25− T cells in the presence of calcitriol inhibits production of pro-inflammatory cytokines, induced high levels of CTLA-4 and FoxP3, but does not substantially affect T cell division.

Author Contribution Statement
None.