Background Trastuzumab is the cornerstone of adjuvant therapy for HER2-positive breast cancer (BC), but up to 20% of patients relapse. We performed a nested case-control study comparing the gene expression profile of relapsed cases within 5 years from the start of adjuvant trastuzumab and a group of controls not relapsed, in order to understand potential resistance mechanisms and to allow developing alternative strategies.MethodsRNA was isolated from formalin-fixed paraffin-embedded primary tumors with AllPrep DNA/RNA FFPE Kit (Qiagen) and analyzed with nCounter Breast Cancer 360 Panel (Nanostring Technologies), according to manufacturer’s instructions. The analysis of differential expression was performed by DESeq2 R package, on raw counts assuming a negative binomial distribution and using the Benjamini-Hochberg procedure to control the False Discovery Rate (FDR). Pathway enrichment analysis on Differentially Expressed Genes (DEGs) was performed by STRING database v. 11.0. Fisher’s Exact test was used for categorical variables and Wilcoxon-Mann-Whitney test for the continuous ones to analyze the comparison between cases and controls for the clinical and genetic variables. ResultsConsidering a median of > 20 counts per gene as threshold, 653 genes were analyzed. Eight significant DEGs were found between cases and controls, with a FDR < 0,10: AGTR1, Receptor for angiotensin 2 (log2foldchange [log2FC]=-1.83; FDR=0.0017), PGR (log2FC=-2.41, FDR=0.016), ROCK1, Rho-associated protein kinase 1 (log2FC=1.26, FDR=0.042), ITPR1, Inositol 1,4,5-trisphosphate receptor type 1 (log2FC=-0.98, FDR=0.052), MMP3, Stromelysin-1 (log2FC=-0.58, FDR=0.097), MMP9, Matrix Metalloproteinase-9 (log2FC=1.15, FDR=0.097), TIE1, Tyrosine-protein kinase receptor-1 (log2FC= 1.0047, FDR=0.097) and GDF15, Growth Differentiation Factor 15 (log2FC= 1.30, FDR=0.099). After adjustment for multiplicity of the tests, no statistically significant associations were found between gene expression and clinical variables (e.g. tumor size, stage…). The most enriched KEGG pathways were: Estrogen signaling (3 genes out of 133, FDR=0.00088), Vascular smooth muscle contraction (3/119, FDR= 0.00088), cGMP-PKG signaling (3/160, FDR=0.00088), Proteoglycans in cancer (3/195, FDR= 0.00093), Cortisol synthesis and secretion (2/63, FDR=0.0041), Leukocyte transendothelial migration (2/112, FDR= 0.0061), TNF signaling (2/108, FDR=0.0061), IL-17 signaling (2/92, FDR=0.0061) and Calcium signaling (2/179, FDR=0.0078). Conclusions Our preliminary results indicate that altered expression of genes related to inflammation and antigen presentation, adhesion and migration are involved in trastuzumab resistance, highlighting the role of the immune system and of the tumor microenvironment in this particular subtype of BC. For HER2+ BC with extremely poor prognosis, we aim to validate these results, reproducing the genetic landscape of unfavorable prognostic factors, in order to identify new therapeutic targets and gene signatures, able to identify Trastuzumab resistance mechanisms. Citation Format: Sara Bravaccini, Sara Ravaioli, Roberta Maltoni, Elisabetta Petracci, Michela Palleschi, William Balzi, Francesca Pirini, Giovanni Martinelli, Andrea Rocca. Gene expression profile in HER2-positive breast cancer to predict outcome in patients treated with adjuvant trastuzumab [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-19.