THE IMMUNE MOLECULAR LANDSCAPE OF BREAST CANCER DURING PREGNANCY

Introduction: Breast cancer during pregnancy (PrBC) is a rare and aggressive type of early onset breast cancer (EOBC) with only partially known biologic determinants. The interplay between the anti-tumor immune response and the mechanisms of maternal-fetal immune is not clear at all, underscoring the complexity of PrBC biology. We hypothesize that a deeper understanding of the molecular processes of immune pathways interplay is required for refining the clinical management of PrBC. This project aimed to profile the immune molecular landscape of PrBC, with the goal of identifying potential actionable biomarkers. Methods: We selected n=83 PrBC and n=89 pregnancy-unrelated EOBC cases from our institutional registry and assessed tumor-infiltrating lymphocytes (TILs) as well as CD4, CD8, forkhead box P3 (FOXP3), and programmed death-ligand 1 (PD-L1) (clone 22C3) expression by immunohistochemistry. Additionally, RNAs extracted from n=75 PrBC and n=67 EOBC cases were subjected to gene expression analysis using a next-generation sequencing assay (Oncomine™ Immune Response Research Assay, ThermoFisher) targeting 395 immune-related genes. The expression of the identified differentially expressed genes (DEGs), was further validated by immunohistochemistry (IHC) in all cases with available material (n=70 PrBC and n=65 EOBC). The statistical approach encompassed Fisher’s and Chi-squared tests, with appropriate adjustments for multiple comparisons, logistic regression models, and survival analyses based on the Kaplan–Meier method. The sequencing output yielded read counts, which served as input for the DEGs analysis by the R Statistical Software package DeSeq2 version 1.42.0. Gene Ontology Enrichment analysis of DEGs was conducted utilizing Enrichr and TopGene software packages. Results: In PrBC, a lower frequency of hormone receptor (HR)-positive tumors were noted, alongside a higher prevalence of low/null PD-L1 and CD8+TILs, particularly in HR+/HER2- cancers. PrBC exhibited significantly elevated risks of relapse and disease-related mortality compared to EOBC, with CD8+TILs significantly associated with disease relapse and higher mortality rates in PrBC. Poorly differentiated (G3) neoplasms increased with advancing gestational age. Histologic subtypes, HR, and HER2 status remained stable across pregnancy trimesters. Notably, higher proportions of tumors with high/moderate TILs were observed in early pregnancy, along with increased FOXP3 expression in HR+/HER2- subtype. PD-L1 negativity was more common in relapsed cases during the first trimester, without significant differences in survival outcomes across trimesters. Comparison between PrBC and EOBC unveiled a total of n=7 DEGs. Notably, the Melanoma Antigen Gene Family A (MAGEA) gene family exhibited consistent upregulation across all cohorts, irrespective of molecular subtypes. Classification of DEGs into superfamilies unveiled distinct genetic characteristics for the HR-/HER2- subgroup, including interleukins (ILs), lipoxygenases (Lox), and Toll-like receptors (TLRs) expression. While TILs density did not impact the expression of the DEGs in PrBC, notable associations were observed between the differential expression of MAGEA and CD4 and CD8 positivity. A significant positive correlation was observed between MAGEA genes read counts (specifically the immune response activator MAGEA1) and protein expression by IHC. Conclusion: The TME in PrBC exhibits distinct TILs subpopulation patterns, bearing significant biological and prognostic implications. Routine evaluation of TILs and their subtypes in these patients could enhance pathology reports, aiding in the identification of clinically relevant subsets of women with PrBC. The TME dynamics of HR+/HER2- PrBC vary according to gestational age, implying that immune tolerance expression during later stages of pregnancy may account for the heightened aggressiveness of tumors. The dysregulation of MAGEA family genes in PrBC, particularly MAGEA1, indicates its possible role in PrBC tumorigenesis and tumor progression.