The Role of Immune Subpopulations in Governing Therapeutic Response in Early HER2+ Breast Cancer

The clinical management of HER2+ breast cancer, which comprises approximately 20% of all breast cancer cases, has improved substantially by the use of targeted therapies. However, not all patients receiving neoadjuvant treatment achieve a pathological complete response(pCR), and the ability to identify responders could improve personalisation of treatment and patient stratification. In this work, specific immune cells of the tumour microenvironment are investigated in HER2+ breast tumour samples of the neoadjuvant TCHL clinical trial (NCT01485926)to evaluate their potential as biomarkers of response to neoadjuvant HER2-targetting therapy.To identify immune and stromal cells, and checkpoint proteins,in HER2+ breast tumours, three chromogenic and three fluorescent multiplex immunohistochemistry panels were successfully designed and optimised in formalin-fixed paraffin embedded tonsil and test breast tissue of the adjuvant St Vincent’s University Hospital cohort. Primary antibodies demonstrated specificity on negative control formalin-fixed paraffin embedded cell pellets. Two chromogenic panels targeting CD3+ T-cells, CD20+ B-cells, and DC-Lamp+ mature dendritic cells (tertiary lymphoid structure panel) and CD4+ helper T-cells and CD8+ cytotoxic T-cells (T-cell panel) were applied to pre-and post-treatment TCHL patient tumours. Analysis of multiplex stained patient samples was performed using Aiforia artificial intelligence software. Two separate models were trained and validated for the analysis of the tertiary lymphoid structure panel and T-cell panel staining on TCHL tumours, with error rates of 12.08% and 13.4%, respectively. Correlation of identified immune subpopulations with clinical parameters in pre-treatment TCHL patient biopsies revealed a significantly higher density of CD3+ T-cells (p = 0.043) and higher B-cell, helper T-cell, cytotoxic T-cell and dendritic cell densities in patients achieving a pCR vs. non-pCR patients. Evaluation of pre-treatment immune cell density with clinicopathological features demonstrated higher CD4+ and CD8+ T-cells in stage III vs. stage II patients, while significantly higher CD8+ T-cells (p = 0.026) and higher densities of B-cells, CD4+ T-cells, and dendritic cells were observed in estrogen receptor negative patients. In summary, this study identifies T-cells, B-cells and dendritic cells as predictors of achieving a pCR in neoadjuvant HER2+ BC, and highlights that therapeutic response may be influenced by hormone receptor status.