Correlation of Expression of Androgen Receptors and P16 in Triple Negative Breast Carcinoma - A Five Year Retrospective And Prospective Study

: AIM AND OBJECTIVE Triple negative breast carcinoma (TNBC) is an aggressive breast carcinoma, lacking estrogen receptor (ER),progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2neu receptor) amplification, thereby, unresponsive to conventional hormonal therapy. The aim of present study is to examine androgen receptor (AR) and p16 expression in TNBC cases and explore its clinical significance in view of potential AR and p16 targeted TNBC therapy.


INTRODUCTION
Breast carcinoma have spectrum of characteristics ranging from well differentiated homogenous to mixed heterogeneous entities.Studies have shown that prognosis depends on the biological or molecular subtypes of the carcinoma.That's why; immunohistochemistry plays an important role in its final diagnosis.Several molecular expressions in breast carcinoma as well as their application have been studied.Estrogen Receptors (ER), Progesterone Receptors (PR) and Human Epidermal Growth receptor (EGFR-2 or HER 2-neu) markers have been used routinely for identification of various types of luminal cell breast carcinoma.Luminal cell breast carcinoma which does not express the ER, PR and EGFR2 molecules are labeled as triple negative breast carcinoma (TNBC) [1] .Since TNBC cases don't have actionable receptors for therapeutic target, its treatment is hampered.Lacking of targeted therapy and poor prognosis as warranted a major effort to discover specific and actionable molecular targets for treating TNBC cases.Molecular studies have also led to identification of certain targetable features such expression of androgen receptor and p16 and several other genomic alterations.However, still it is not clearly established whether these alterations are molecular 'drivers' or not along with their effects in different types of breast carcinoma [2,3] .The objectives of the current study are to examine the prevalence of AR and p16 expression and their utility in triple negative breast carcinoma (TNBC) patients.

MATERIAL AND METHOD
The present study was conducted on 94 patients diagnosed as triple negative breast carcinoma for analyzing the Androgen Receptor and p16 expression in them.The work was undertaken in the Department of Pathology, Jawaharlal Nehru Medical College (JNMC), Aligarh, (UP) from 2017 to 2019.We studied 3 years of retrospective cases and 2 years of prospective cases.The surgical breast specimens received from The Department of the Surgery, JNMC, Aligarh, were studied under gross examination and characteristics were noted.Required sections were taken as par the standards and after fixation, tissue pieces were processed in Automated Tissue Processor set, LEICAM TP1020 (Germany), for a 24-hour cycle.Processed tissues then transferred from the final wax bath to the disposable embedding cassettes (rectangular moulds), filled with molten paraffin wax to form tissue blocks.After solidification, thin sections of 3-5 microns thickness were cut using a Rotary Microtome (LEICA RM 2125 RT) followed by immediate floating in a water bath at 60 0C.The sections were mounted onto clean glass slides coated with albumin and stained with routine H&E stain using Harris Hematoxylin and aqueous Eosin [4] .AR Immunostaining was done with ready to use reagents obtained from Thermmoscientific company and p16 immunostaining with ready to use reagent from Biogenexcompany as par their protocols followed by slide examination under microscope.Cervical Carcinoma and Prostate Adenocarcinoma served as positive control for p16 and AR respectively.Absence of primary antibody was taken as negative control for both the immunostains.• Email: editor@ijfmr.com

Statistical analysis
The statistical analysis was carried out using SPSS software V 20.0, for determining the statistical significance, Student's t and Fisher's exact/ chi square tests were used for continuous and categorical variables, respectively.A p-value of <0.05 was considered to be statistically significant.metastases rate was found to be more in p16 negative cases i.e. 38.5% and 68.4% respectively (Table 3).P16 expression score correlation with grade, primary tumor stage and nodal stage have been summarized in Table 5.

DISCUSSION
Our study reported the TNBC incidence of 41.2% supported by other studies like Nigam and Sood,(2014) [7] , Jana et al.,(2014) [8] and Akhtar et al.,(2015) [9] with similar TNBC incidence of 39.4%,46.7%and 43.5% respectively.However, Doval et al., (2015) [10] and Patnayak et al.,(2015) [11] observed lower incidence of 23.8% and 22.7% respectively in their studies.Boyle et al., (2012) [12] from California observed 20% incidence of TNBC.We reported 60.6% of the TNBC cases were of premenopausal women with similar reporting by Rao et al.,(2013) [13] from south India and Sen et al.,(2012) [14] from east India of 67.4% and 54.1% respectively.However, Akhtar et al., (2015) [9] from west India and Nigam and Sood,(2014) [7] from north India found higher TNBC incidence in postmenopausal group i.e. 58.8% and 54.6% respectively.Our study observed age group of 40 to 49 years to be mostly affected with median age of 45 years with similar finding of median age (46.1 years) and range ( 41-51 years) in a study in west India by Singh et al., (2014) [15] .However, Jana et al.,(2014) [8] and Boyle et al.,(2012) [12] reported median age of 54.6 years and 57 years respectively indicating higher median age in western world females..We found the left breast to be slightly more affected i.e. 51.1% as also shown by Dent et al.,(2009) [16] in Toronto Canada (58.3%).However, Suresh et al.,(2013) [17] reported higher TNBC incidence in right breast i.e. 51.9% and bilaterally affecting 0.6%.Also Dent et al.,(2009) [16] showed higher bilateral breast involvement in western females (i.e.9.5%) as compare to Indian females in studies.Upper outer quadrant was mostly affected site in our study (59.6%) with similar findings of 67% in Bashir et al., (2017) [18] and 34% in Arora et al.,(2019) [19] studies.Tumor size of 2 to 5 cm was the most commonly affected size shown in our study i.e. 72.3% as well as in Nabi et al.,(2015) [20] i.e. 76.2%.However, Akhtar et al.,(2015) observed tumor size of more than 5 cm to be mostly affected(64.7%).Invasive carcinoma (NST) was the most common histo-morphological type (97.8%) of TNBC as described in other Indian studies (Mane et al., 2015 [21] and Patnayak et al., 2015 [11] ) as well as western world study (Pareja et al.,2016) [22] .Most of the TNBC cases were of higher grade with pattern of grade 3(64.5%)> grade 2 (32.3% > grade 1 (3.2%) with 1 cases of carcinoma with medullary like features always considered of high grade.Kim et al., (2017) [23] found the similar result but Rao et al.,(2013) [13] found grade 2 to be the most common grade.We received lymph nodes in 67 cases out of total 94 TNBC cases with 62.7% lymph node involvement as also reported by Nabi et al.,(2015) [20] in their study.However, Singh et al., (2014) and italian study Urru et al.,(2018) [24] found most of the lymph node to be negative.Most of the TNBC cases in our study were of TNM stage II (76.1%) supported by other studies ( Reddy et al., 2018 [25] and Kim et al., 2017).In contrast, Agarwal et al.,(2015) [26] reported TNM stage III to be the most common stage in 47.5% of the TNBC cases.We studied AR immune expression in 94 TNBC cases, considering TNBC case with >1% of tumor cells showing AR nuclear staining as AR positive.We reported 40.4% TNBC cases were AR positive in agreement with the Astvatsaturyan et al.,(2018) [27] .However, Liu et al.,(2018) [28] from China showed AR positivity only in 21.8% of the total cases considering the same cut off value for AR positivity.This shows the wide range of AR positivity in TNBC cases attributed to TNBC tumor heterogeneity and a lack of universally accepted standards and analytical protocols for determining the AR positivity.We found 38.6% of the premenopausal women and 43.2% of the postmenopausal women were AR positive without any significant association (p value = 0.6537) as also documented by Liu et al.,(2018) [28].In contrast, Arora et al.,(2019) [19] , found significant association with menopausal status (p= 0.01).We observed AR expression rate be higher in TNBC cases of age group > 60 years (66.67%)followed by 40-49 years (46.9%)without any significant difference (p value = 0.174) among different age groups similar to Arora et al.,(2019) [19] study.Astvatsaturyan et al.,(2018) [27] reported mean age of women in the AR positive group was significantly older than that of AR negative group (p value = 0.015).It indicates that AR positivity in TNBC cases may increase with age.
We observed the AR expression rate among grade 1 TNBC tumor of 66.7% followed by grade 2 (43.3%) and grade 3(38.3%)without any significance (p value = 0.104) and similar pattern reported by McGhan et al.,(2014) [29] .Sunar et al.,(2018) also observed the similar pattern for AR expression rate with significant association between AR expression and grade (p value = 0.001), indicating AR expression rate is associated with lower TNBC grade.We found higher intensity of AR expression score in lower grade in comparison to higher grade without any significant association (p value = 0.890) (Table 4) in agreement with Arora et al.,(2019) [19] .It indicates that AR might play major role in early developmental stage of the TNBC cancers.We documented a trend of increasing AR expression rate with increasing primary tumor stage i.e.T1(20%)< T2(32.8%)< T3 (56.3%)< T4 (66.7%) without any significant difference among primary tumor stages, supported by similar pattern found in Astvatsaturyan et al.,(2018) [27] study.We found that AR positive TNBC cases were significantly associated with higher rate of lympho vascular invasion (p value = 0.0053).McGhan et al.,(2014) [29] also reported similar invasion rate but without any significance.In contrast, Teoh et al.,(2019) [30] from Malasiya, reported lympho vascular invasion rate higher in AR negative TNBC cases without any significant difference ( p = 0.056).
In our study, 78.6% of the AR positive TNBC cases were showing metastases to the lymph node(s) which was significantly higher in comparison to 51.3% of the AR negative TNBC cases found to be lymph node positive for tumor cells (p value= 0.022) as supported by other studies [ McGhan et al.,2014 [29] (p = 0.03) and Arora et al., 2019 [19] (p value = 0.013) ].However, Tang et al., (2012) [31] found more lymph node positivity rate in AR negative TNBC cases (88.1%) without any significant differences.We found higher AR expression rate in higher nodal stage without any significant difference among nodal stage.However, Hu et al.,(2011) [6] and Astvatsaturyan et al.,(2018) [27] higher AR expression rate in N0 and N1 respectively indicating variable possibilities of different AR pathways playing role in genesis of TNBC leading to its propensity to get metastasized to lymph node (s).We observed that most of the AR positive cases belonged to stage IIB (39.3%) followed by stage IIA (33.3%) with higher expression rate in stage IIIC (100%) followed by IIIB (66.7%).Overall, most of the AR positive cases were of stage II followed by stage III and I without any association between AR expression and TNM stage (p value= 0.272).Similar finding was reported by Sunar et al.,(2018) [32] .Arora et al., 2019 documented most common stage for AR positive TNBC cases to be stage I (56.7%) and highest AR expression rate in stage IIB (66.7%) with no significant association between AR expression and TNM stage (p value= 0.59).
One of the limitations to our study was that molecular testing of androgen receptor and p16 was not performed and therefore, we suggest related molecular testing in TNBC cases of our population to establish the mutation status and its correlation with their over expression immuno-histochemically.Also, our data are limited because of the smaller numbers of patients who were not equally distributed between positive and negative results for androgen receptor and p16.A larger sample size and a matched cohort is warranted for better understanding and characterization of role of androgen receptor and p16 in TNBC patients.

CONCLUSION
Androgen positive triple negative breast carcinoma (TNBC) cases was more common in older age and had high propensity for lympho-vascular invasion and lymph node metastases.AR-positive TNBC may represent a subtype of breast carcinoma, with unique features that may be amenable to treatment with alternative targeted therapy.Moreover, high expression of p16 in TNBC suggests a potential role of this biomarker protein in TNBC pathogenesis as well as in developing targeted therapy in p16 positive TNBC patients.

No. of LN (s) involved ( Out of 42 LN positive cases
*1