Molecular and immune profiling of breast cancer from pregnancy to postpartum: Insights into the tumour-immune landscape during breastfeeding from GEICAM EMBARCAM study.

530 Background: Breast cancer (BC) is the most common malignancy among young women of childbearing age, with a rising incidence in this population. Pregnancy-associated breast cancer (PABC) is an aggressive entity linked to poor prognosis and elevated metastatic risk. Despite advances in BC research, the impact of pregnancy, breastfeeding, and postpartum mammary gland remodeling on tumor biology remains unclear, highlighting the need to explore their interaction with the tumor microenvironment for novel therapies. Methods: A gene expression and immune cell profiling analysis was conducted using the nCounter Breast Cancer 360 panel (NanoString) and CIBERSORTx (Newman 2019, Nat Biotechnol) on FFPE tumor samples from GEICAM/2017-07 EMBARCAM study (NCT04603820) PABC patients during the gestation (PABC_GS, n=21), breastfeeding (PABC_BF, n=21), and first-year postpartum (not during lactation period, PABC_FY, n=15) vs non-PABC tumours (n=49). Differential expression analysis per gene and biological signature was performed using the limma package. P-values were adjusted with the Benjamini-Yekutieli false discovery rate (FDR) method. Additionally, the LM22 matrix from CIBERSORTx was employed to quantify 22 immune cell types from normalized NanoString data. Statistical significance was set at 5%. Results: PABC clinical subtypes were 46% HR-positive/HER2-negative, 21% HER2-positive, and 33% triple-negative. Differential gene expression analysis identified similar significant enrichment pathways across all PABC groups compared to non-PABC: DNA repair-related signatures (HRD, BRCAness, BC p53) and BC proliferation (adj p<0.05), along with higher CDK4 expression and genomic risk (p<0.05). Conversely, key regulatory pathways such as apoptosis, TGF-Beta, and PD-L1 were downregulated (adj p<0.05). Nonetheless, it is noteworthy that the PABC_BF group showed a unique profile marked by increased immune activity and cell abundance (cytotoxic cells, CD8 T cells, T-reg, cytotoxicity), elevated SOX2 expression (adj p<0.05) and inflammatory chemokines levels (p<0.01) compared to non-PABC. The CIBERSORTx analysis supported these findings, demonstrating a significantly higher abundance of several immune cells in PABC_BF, remarkably CD8+ T-cells and T-regs, compared to all other groups (p<0.05). Conclusions: This GEICAM EMBARCAM sub-study reveals that PABC tumors display aggressive molecular features across all subtypes, contributing to poor prognosis. Notably, the breastfeeding-associated subset (PABC_BF) exhibits a highly active tumor-immune microenvironment with robust immune cell infiltration and inflammatory signalling, highlighting potential for targeted immunotherapy. These findings underscore the need for further clinical research to optimize immune-based strategies in PABC patients’ management. Clinical trial information: NCT04603820 .