Breast cancer is oneofthemostcommonmalignanciesacross the worldwithabout2,261,419newcasesand 6,84,996deaths as per the GLOBOCAN 2020 data. It is the fifth majorcauseofcancermortalityworldwide,and is thesecond leading cause of cancer related mortality in females. Breast cancer incidence in India has surpassed cervical cancer, and the mortality rate is higher when compared to western population which is mostly due torecurrenceandmetastasis. Currently, the treatment of breast cancer depends predominantly on histological type, microscopic grading, tumor size, nodal status, metastasis, and immunohistochemistry-based molecular subtypes such as luminal A, luminal B, HER2/neu and triple negative breast carcinoma (TNBC). However, a deeper understanding of the process involvedin thetransitionofnon invasive breast cancer cells intomore aggressive clones with metastatic potential isimportant, in order to stratify patients and render specific treatment. In addition, it has been documented that the immune reaction to the tumor cells plays a vital role in tumor behaviorand its response to treatment.
In this work, an attempt has been madeto analyze Epithelial Mesenchymal Transition(EMT) related markers (EpCAM and SNAIL1) among the molecular subtypes of breast carcinoma and adjacent normal breast tissue, and to comprehensively correlate the expression of these markers with tumor characteristics. Further, tumor infiltrating lymphocytes (TILs) and CD8+ (Cluster Differentiation) T lymphocytes in breast carcinoma tissue were evaluated using specifically-designed computational algorithms. ImmunohistochemicalstainingforEpCAMandSNAIL1 was performedon 200 radical mastectomyspecimenswithhistopathologicaldiagnosisofcarcinoma breast. Tumor cells showed strong expression of EpCAM and SNAIL1 whencomparedtonormal breast tissue. EpCAM expressionpattern was diffuse and membranous in tumor cells, and a differential expression was observed in the various molecular subtypes of breast carcinomawithstrongandsignificantEpCAMexpressioninTNBC. EpCAM expression correlated significantly with increasing tumor grade and a higher expression was seen in node positivetumorsand in advanced stage tumors, although it was notstatistically significant. SNAIL1 expression varied significantly among intrinsic subtypes of breast carcinoma with TNBC and HER 2/neu showing higher expression. SNAIL1 expression correlatedsignificantly withincreasingtumor grade and pathological TNM stage (Tumor size, Nodal involvement andMetastatic status). The expression analyses of EpCAM and SNAIL1 performed on breastcancercell lines, derived from the molecular subtypes, confirmed higher expression in breast cancer cells than in normal breast cell lines.
In order to assess the immune response around the tumor cells, Tumor InfiltratingLymphocytes (TILs) in the stromal area were evaluated on Hematoxylin &Eosin-stained sections in 50 cases of carcinoma breast of different molecular subtypes. High TILs were observed in TNBC, high grade tumors and those with nodal metastasis. A subset of TIL, the CD8+T cells,were evaluated in biopsies of TNBC cases using immunohistochemistry. Cases with high CD8+T lymphocyte density in biopsy showed fairly good pathologicalresponse in the mastectomy specimen, following platinum-based chemotherapy.
The findings of the current study show that EMT related markers (EpCAM and SNAIL1) can be used for identifying aggressive behavior, and the strong expression of both the markers in tumor cells indicate their potential role in tumorigenesis. Furthermore, the evaluation of tumor infiltrating lymphocytes gave an insight into the potential role of immune response to tumor cells. The analysis of its subset, the CD8+ T cells aided in assessing the effectiveness of neoadjuvant chemotherapy in breast carcinoma, particularly in TNBC.
These findings, taken together, will help to stratify patients according to tumor behavior, provide risk assessment, design targeted and immune mediated therapies and in addition predict response.

Publications:
1. Sandhya Sundaram, Christian SD, Krishnakumar R, Ramya R, Ramadoss M,KarunagaranD.Clinicopathologicimplicationsofepithelialcelladhesionmolecule expression across molecular subtypes of breast carcinoma. (2020) Journal of Cancer Research and Therapeutics,16(6),1354-9.
2. SSabhariPriya,SandhyaSundaram,D. Karunagaran,V. Pavithra. ImmunohistochemicalanalysisofKi-67inbreastcarcinoma-AsurveyinIndian population. (2020) IndianJournalofPathology&Oncology,7(4),513–518
3. Sandhya Sundaram, Mahalakshmi Ramadoss, Krishnakumar Ramanathan, Devarajan Karunagaran. (2021) Expression of EMT Marker SNAIL1 Across Molecular Subtypes of Breast Carcinoma and its Clinicopathological Correlation. Modern Pathology 34,154-154 (Conference proceedings).