The prognostic value of androgen receptor and cyclin D1 in infiltrating duct The prognostic value of androgen receptor and cyclin D1 in infiltrating duct carcinoma of the breast carcinoma of the breast

Objective : Breast cancer (BC) tissue is heterogeneous with a number of cellular pathways involved in cell growth and proliferation. Activation of androgen receptors (AR) signaling pathways plays a role in BC. On the other hand, cyclin is a regulatory subunit of cyclin-dependent kinases that affect cell cycle G1/S transition. This study aimed at investigating the relationship between the expression of AR and cyclin D1 and the clinicopathological details of BC patients registered in the archive of our department within a speci ﬁ ed period and determining the prognostic impact of such expression. Methods : The study included 182 IDC patients aged from 20 to 65 whom were registered in the archive of the Clinical Oncology & Nuclear Medicine Department from January 2013 to December 2015. All clinicopathological data were obtained from patient records. Immunohistochemistry study for AR and cyclin D1 was done for the pathologic specimens. Results : The expression ratio of AR in 182 specimens was 43.4% (79/182). AR positivity was signi ﬁ cantly associated with estrogen receptor (ER) and progesterone receptor (PR) positivity and negative HER2 status, lower tumor grade, smaller tumor size, and negative lymph node involvement ( P values < 0.05). Cyclin D1 positivity was reported in 116/182 (64%). There was positive correlation between cyclin D1 and ER, PR positivity, triple negativity, small tumor size, and negative lymph node involvement ( P value < 0.05). The


Introduction
B reast cancer (BC) is indeed heterogeneous clinically, histologically and genetically (Ricciardelli et al., 2018).Biologically, androgen expression exists in many tissues, including BC (Barton et al., 2015).Consequently, antiandrogens could be part of personalized BC therapy (Ricciardelli et al., 2018;Barton et al., 2015;You et al., 2022).The cyclin D1 and cyclin-dependent kinase 4 and 6 (CDK4/6) complex play a role in cell cycle regulation and several downstream signals.During cell cycle progression, the cyclin D1-CDK4/6 complex encourages the phosphorylation and inactivation of the retinoblastoma protein (pRb), allowing cells transition from G1 phase to S phase.Dysregulation of the cyclin D1-CDK4/6 complex is an important step in the genesis of breast cancer (Mohammadizadeh et al., 2013;Ahlin et al., 2017).Cyclin D1 also has CDK-independent functions and may stimulate ERmediated transcription independently of estrogen and thereby potentially affects the estrogen response (Ortiz et al., 2017).Ethnicity affects BC molecular biology (Hirko et al., 2022).Consequently, the aim of this study was to investigate the association between the expression of AR and cyclin D1 and the clinicopathological details of BC patients registered in the archive of our department from Jan 2013 to Dec 2015 and defining the prognostic impact of such expression in comparison with the literature of other ethnic groups.

Study design
One hundred and eighty-two cases of primary infiltrating duct carcinoma (IDC) of breast registered in the archive of the Clinical Oncology& Nuclear Medicine Department from January 2013 to December 2015 were enrolled.
Inclusion criteria included female patients aged 20e65 years old with histologically confirmed IDC and ECOG performance status > or ¼ 2. Exclusion     criteria were existing of multiple malignancies, major morbidities, receiving neoadjuvant chemotherapy and pathologies other than IDC.
All the demographic and the clinicopathologic data of the patients were collected.The median follow-up was 62 months (range: 2e132 months).
Slides were stained with hematoxylin for 1 min Applications of primary and secondary antibodies followed the manufacturer protocols.Cut off values of ER, PR, tumors were considered positive if at least 1% of the tumor cells showed unequivocal nuclear staining of tumor cells.For HER2 neu, membranous staining was scored for as follows: score 0, no staining or faint incomplete staining in <10% cells; score 1þ, faint incomplete membranous staining in >10% cells; score 2þ, weak to moderate complete or moderate incomplete membranous staining in >10% of cells; score 3þ, strong complete membranous staining in >10% cells.Only score 3þ was considered as positive (Deyarmin et al., 2013;Xie et al., 2014).AR and cyclin D1 positivity was when at least 10% of tumor cell nuclei stain positive (Kensler et al., 2019a;Huang et al., 2016).

Statistics
The Statistical Package for the Social Sciences, SPSS version 20 was used.Comparison of continuous variables was performed through independent t tests.Categorical variables were investigated by the c2 test.The Cox proportional hazard analysis was applied to define the prognostic factors.The KaplaneMeier method helped assessing the cumulative survival rates.Local progression-free survival (LPFS) and distant progression-free survival (DPFS) were calculated from date of diagnosis till date of local and distant progression, respectively.While the overall survival (OS) was calculated from the date of diagnosis till the date of death or last follow-up.Approval of this work by our Institutional Review Board was obtained (MD.18.09.92 in Nov. 2018).

Results
Clinicopathological characteristics of 182 patients are shown in (Table 1).The majority of the cases were premenopausal, T2, showing positivity for ER, PR and cyclin D1 and negativity for HER2 and AR.
Detailed management is presented in (Table 2).Modified radical mastectomy was the commonest surgery, while anthracyclin based regimen was the commonest adjuvant chemotherapy protocol.Tamoxifen was the commonest adjuvant hormone used.
The median follow-up of the 182 patients was 62 months (range, 2 to 132).At the end of the follow up, 87 patients were living, 18 patients had local recurrence and 46 patients developed distant metastasis.4).IHC showing positivity for AR is shown in (Fig. 7).
There was significant association between cyclin D1 and favorable factors except triple negativity (Table 5).IHC showing positivity to cyclin D1 is revealed in Fig. 8.
Univariate and multivariate analysis proving the favorable prognostic impact of AR on LPFS and  DPFS are shown in (Tables 6 and 7).Cyclin D1 was proved not to be an independent prognostic factor by multivariate analysis (Table 7).

Discussion
Oncologists in different geographic areas need to document the molecular biology of cancers diagnosed in their region as molecular biology is a fundamental player in making treatment decision.
The biologic roles of AR in development and progression of breast cancer are under investigation (Kensler et al., 2019a).These investigations can pave the way for new therapeutic strategies targeting AR in breast cancer.Our AR expression was positive in 43.4% cases similar to the Chinese report of Hwang et al. (Huang et al., 2016) While, publications from Australia, Belgium, Sweden, Vietnam and turkey reported higher levels (57%, 58,6% and 76% and 65%, respectively) (Peters et al., 2012;Bozovic-Spasojevic et al., 2017;Nim eus et al., 2017;Phung et al., 2022;Arici et al., 2020).
Although our AR expression was generally lower than many literature, still AR could be a hopeful therapeutic target.This issue is under investigation in several phaseII/III trials (Venema et al., 2019;Christenson et al., 2021;Ferrari et al., 2022).
Our AR expression was significantly associated with favorable prognostic factors similar to several publications from India, USA and China (Kensler et al., 2019a;Huang et al., 2016;Vellaisamy et al., 2019).
Multivariate analysis revealed that AR expression in our cases (with a majority of ER positivity) had a significant positive impact on LPFS and DPFS similar to literature from Spain, China and USA (Gonzalez et al., 2008;Jiang et al., 2016;Kensler et al., 2019b).In the ER þ AR þ breast cancer cells, AR-ligand complex leads to apoptosis through a process involving estrogen-related element in the nucleus.On the other hand, in the ER-AR þ breast cancer cells, AR-ligand complex leads to proliferation through a process involving androgen-related element in the nucleus (Ricciardelli et al., 2018;Robinson et al., 2011).Difference in ER expression in different tumors is not the only factor affecting the published AR prognostic power as there are emerging new molecular breast cancer subtypes that vary in levels of AR expression (Barton et al., 2015).Furthermore, there might be variable micro-RNAs regulatory mechanism of AR expression in breast cancer (You et al., 2022).
Cyclin D1 dysregulation in human breast cancer cells in vitro potentiates progression to G1⁄S transition, with disturbed growth control.By contrast, in normal breast tissue, cyclin D1 overexpression causes growth inhibition rather than growth and helps differentiation (Ortiz et al., 2017).Cyclin D1 expression ratio was 64% in our cases similar to Mohammad izadeh et al. (Mohammadizadeh et al., 2013) from Iran and Ahlin et al. from Sweden (Ahlin et al., 2017).In the current study, cyclin D1 expression by univariate analysis was significantly associated with ER, PR positivity, small tumor size, and negative lymph node involvement.Similar reports from China and Sweden (Ahlin et al., 2017;Huang et al., 2016) and contradictory results from Iran were reported (Mohammadizadeh et al., 2013).
By multivariate analysis, our cyclin D1 was not of prognostic value similar to Diest et al. from Netherlands (Diest et al., 1997).On the other hand Ahlin et al. from Sweden (Ahlin et al., 2017) and Ortiz et al. (2017) from Spain reported poor prognosis with cyclin D1 over expression in ER-positive breast cancer cases and not the negative cases.Bilalovic et al. from Bosnia (Bilalovi c et al., 2005), Wen Cheng et al. from Taiwan (Cheng et al., 2012) and Sirag et al. from Saudi Arabia (Siraj et al., 2021), clarified that cyclin D1 was a favorable prognostic factor.The reason for such contradictory results might be the different number of patients included, different techniques of IHC, and different ratios of molecular subtypes involved in the different studies.
Limitation of this study: short follow up period.

Conclusion
Our results differed from literature in that AR expression was lower and that cyclin D1, unlike AR, was not of prognostic value.Difference could be attributed to different number of patients included, different techniques of IHC, and different ratios of molecular subtypes involved in the different studies.Proving the ethnicity effect needs a large Arabic study.Searching for new biomarkers that can detect patients who can benefit most from targeting AR and cyclin D1 is needed.

Fig. 2 .
Fig. 2. LPFS curves in AR-positive and negative cases with significant difference.

Fig. 3 .
Fig. 3. DPFS curves of AR-positive and negative cases with significant difference.

Fig. 5 .
Fig. 5. OS curves of cyclin D1 positive and negative cases with significant difference.

Fig. 6 .
Fig. 6.OS curves of AR-positive and negative cases with significant difference.

Table 4 .
The statistical relationship between AR expression and the clinicopathological features.

Table 5 .
The statistical relationship between cyclin D1 expression and the clinico-pathological features.