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Introduction: Androgen receptor (AR) expression has been shown to correlate with improved survival in luminal A, B and triple negative (TN) breast cancer (BCa). Cancer stem cells (CSC) with CD44+/CD24- and ALDH1+ phenotype is associated with pro-invasive gene signature in BCa. Aim of our study was to evaluate prognostic significance of AR expression in relation to CD44, CD24 and ALDH1 in invasive BCa. Methodology: Immunohistochemical expression of AR, CD44, CD24 and ALDH1 was evaluated in 180 FFPE blocks of BCa cases from one institution. Clinico-pathological details and follow-up data were collected from medical charts. Data was analyzed on SPSS 19. Five-year survival was estimated by Kaplan Meier. Results: Median age of patients was 55 years (range: 28-87). Fifteen percent of patients were diagnosed with stage I, 52.2% with stage II, 25% with stage III and 7.8% with stage IV disease. Mean duration of follow-up was 4.5 years (SD+ 2.7) and 29% mortalities were attributed to BCa. AR was expressed in 63.9%, ER in 61%, PR in 51% & HER-2/neu in 30% of tumors. CD44, CD24 and ALDH1 were expressed in 60%, 43.3% & 28.3% of tumors respectively. AR expression was associated (p-value Five year survival of patients with AR+ versus AR- expressing tumors, showed that patients with AR+ tumors had significantly better outcome (p-value=0.03) with survival advantage of 39.6 months. Survival advantage decreased by 9.6 months in tumors expressing CD44+/CD24-/AR- (p-value=0.01) phenotype and by 12 months in patients whose tumors were phenotypically ALDHI+ /AR- (p-value=0.12). We stratified TN tumors into 2 groups: A. TN/CD24+/AR+; B. TN/CD24-/AR- and found that group A tumors had a trend towards better 5 survival (p-value=0.09). Conclusions: Three individual markers (CD44, CD24, AR) may afford prognostic information. Combinations of CD44, CD24 and AR permitted patient stratification into 3 risk categories: poor risk CD24-/AR-; intermediate risk CD44+/CD24-/AR- and good risk CD24+/AR+. Stratification of TN group showed that loss of AR and CD24 was associated with poorest outcome. These results underscore the relevance and importance of AR and CD24 as prognostic markers in invasive BCa. Citation Format: Nazia Riaz, Romana Idress, Sadia Habib, Azhar Hussain, El-Nasir Lalani. Expression of androgen receptor and CD24 correlates with improved 5-year survival in Pakistani patients with invasive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3822. doi:10.1158/1538-7445.AM2017-3822

Introduction: Metaplastic carcinoma (MCa) is a rare and aggressive subtype of invasive breast carcinoma exhibiting epithelial to mesenchymal transition (EMT) where tumor of epithelial origin manifests differentiation into non-glandular, mesenchymal phenotypes such as spindloid, chondroid or osseous components. Acquisition of EMT is associated with low expression of e-cadherin, claudins and high expression of vimentin. Biological mechanisms for acquisition of metaplastic components within MCa are not well understood. The present study was undertaken to establish and characterize a cell line from a patient diagnosed with MCa and to evaluate the role of mesenchymal stem cells (MSC) in attainment of metaplastic phenotype. Methodology: AKU-BC-42 was established from a 65 years old Pakistani female patient diagnosed with T4N1M0 MCa. Specimen was procured and processed under sterile conditions and cultured in DMEM supplemented with 10% FBS. Epitheloid colonies were visualized after 3 weeks of culture, which were passaged and propagated. AKU-BC-42 was phenotypically and genotypically characterized using karyotyping, gene expression analysis, immunocytochemistry and florescent insitu hybridization. MSC marker expression was assessed by flow-cytometry. AKU-BC-42 was cultured under appropriate conditions for assessment of differentiation along osteogenic, adipogenic and chondrogenic pathways. Lineage differentiation was evaluated by special immunohistochemical stains and induction of lineage differentiation genes was assessed by Q-PCR. Results: AKU-BC-42 was found to have a human karyotype with multiploidy and population doubling time of 60 hours. Gene expression profiling revealed negative expression for estrogen and progesterone receptors and positive expression of androgen receptor (AR) and HER-2/neu. FISH analysis was negative for HER-2/neu amplification. Basal (5, 14 & 19) and luminal cytokeratins (8 & 18) were expressed at mRNA level along with myoepithelial markers (CD10, S100A7, p-cadherin, desmin, S100A4, S100A2 & á-SMA). AKU-BC-42 demonstrated MSC pool with expression of CD73 (79.2%), CD90 (10.3%) and CD105 (30.2%) as revealed by flow-cytometry. Osteogenic differentiation was assessed by von kossa stain for mineralization with up-regulation of ALPL and OPN genes. Similarly, differentiation of AKU-BC-42 into mature adipocytes was evaluated with Oil red O staining of lipid droplets and expression of FABP4 gene. Chondrogenic induction was achieved by performing pellet cultures. Collagen synthesis in extracellular matrix of chondrogenic beads was assessed by mason trichrome staining with up-regulation of ACAN, COL10A1 and COMP. Conclusions: We report a novel MCa cell line containing a MSC pool. MSC pool within MCa may be responsible for acquisition of metaplasia in this rare subtype of breast cancer. Further studies on this cell line may reveal the biological mechanisms of MCa of the breast. Citation Format: Nazia Riaz, Sadia Habib, Azhar Hussain, El-Nasir Lalani. Role of mesenchymal stem cells in acquisition of metaplasia in AKU-BC42 breast metaplastic carcinoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5802. doi:10.1158/1538-7445.AM2017-5802

Background: Metaplastic carcinoma (MCa) is a rare entity of breast cancer (BCa) where tumor of epithelial origin manifests differentiation into non-glandular, mesenchymal phenotype. These tumors exhibit heterogeneity with spindle, squamous, chondroid or osseous differentiation. Majority of these tumors do not express estrogen or progesterone receptors and are negative for HER-2/neu gene amplification, limiting use of available targeted therapies. This may translate into poor clinical outcome in this subset of patients as opposed to triple negative subtype of breast cancer. Cancer cell lines have been proved to be a valuable tool in understanding pathogenesis of BCa and development of appropriate drugs. Considering tumor heterogeneity, selecting an appropriate cell line model is imperative to understand signaling mechanisms of each of the distinct sub-types of BCa.Aim of this study was to establish and characterize a BCa cell line from a non-caucasian Pakistani patient. Methodology: Cell line was established from primary culture of tumor tissue from a 65 year old Pakistani female patient diagnosed with T4N1M0 MCa of breast. Specimen was procured in Dulbecco's Modified Eagle's Medium and processed under sterile conditions by mincing and gentle pipetting followed by culturing in complete medium supplemented with 10% fetal bovine serum. Epitheliod colonies were visualized after 3 weeks of culture, which were subsequently passaged and propagated. This cell line has been phenotypically and genotypically characterized using karyotyping, gene expression analysis, immunocytochemistry and florescent insitu hybridization (FISH). Results: Cell line has a human karyotype with multiploidy and population doubling time of 60 hours. Gene expression profiling revealed negative expression of ER, PR and positive expression of androgen receptor (AR) and HER-2/neu. FISH analysis was negative for HER-2/neu amplification. Both basal (cytokeratins 5, 14 & 19) and luminal markers (cytokeratin 8 & 18) are expressed at mRNA level along with myoepithelial markers (CD10, S100A7, p-cadherin, desmin, S100A4, S100A2 & α-SMA). In accordance with mesenchymal phenotype, it has low expression of e-cadherin and high expression of vimentin. Invasion assay revealed this cell line to be non-invasive forming spheroids. CAG repeat length of AR is 22 and AR has been found to be functional by luciferase reporter assay in response to treatment with dihydrotestosterone. Conclusions: We report a novel metaplastic carcinoma cell line from a nan-caucasian female patient with functional AR. Targeting AR signaling may be a promising therapeutic target in this rare sub-type of BCa. Citation Format: Nazia Riaz, Zulfiqar Naqvi, Anwar Ali Siddiqui, Shaista Masood Khan, ElNasir Lalani. Androgen receptor expressing metaplastic carcinoma cell line established from non-caucasian female patient. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1208. doi:10.1158/1538-7445.AM2014-1208

Androgen inhibits growth of malignant breast cancer (BCa) cells by mechanisms that remain poorly defined. Here, we have investigated how androgen suppress proliferation and induces apoptosis in BCa cells. Treatment of MCF-7 cells with 1nM 5α-dihydrotestosterone (DHT) inhibited cell proliferation as well as endogenous HER2 expression, both at mRNA and protein levels, suggesting a link between androgen receptor (AR) and HER2 signaling pathways. Expression of HER2 and AR was also determined in patients with BCa. We found a survival advantage of AR expression in patients with HER2 positive BCa, whereas, loss of AR expression was associated with poor survival. This finding would suggest that AR loss may provide a growth advantage to HER2 over-expressing tumors. We showed the functional consequence of the stimulation of AR-signaling and inhibition of HER2-signaling on breast epithelial cell growth and on expression of 84 genes involved in BCa and estrogen-mediated signaling pathway. Exposure to a combination therapy of DHT and Herceptin resulted in additive anti-proliferative effect in human MCF-7 cells as compared to either agent alone. Furthermore, combined treatment of DHT with Herceptin reduces the expression of genes predominantly involved in proliferation, invasion, anti-apoptosis and cell cycle regulation. These findings provide the key insights into how Herceptin and DHT produce anti-proliferative effect in BCa cells. In conclusion, adding DHT to Herceptin may provide a rational treatment option for BCa, not only by direct inhibition of cell proliferation, but also causing changes in the expression of many genes that are critically involved in the control of proliferation. Establishment of any interaction between AR and HER2 may improve the understanding of carcinogenesis and will provide helpful information in the development of novel preventive and therapeutic strategies. Citation Format: Aisha Sultana, Zulfiqar Ali Naqvi, Shaista Khan, Anwar Ali Siddiqui, El-Nasir Lalani. 5α-dihydrotestosterone down-regulates HER2 and sensitizes breast cancer cells to Herceptin-induced apoptosis in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4230. doi:10.1158/1538-7445.AM2014-4230