Enhanced detection of immune and oncogenic signals in HER2-positive breast cancer by a novel data-scaling method

Background: HER2-positive breast cancer, a clinically aggressive subtype accounting for 15-20% of all cases, is characterized by ERBB2 gene amplification and overexpression of the HER2receptor, which drives oncogenic signaling through PI3K/AKT/mTOR and MAPK/ERKpathways. Accurate transcriptomic profiling of tumor tissue versus normal adjacent tissue (NAT)is key to discovering these distinct mechanisms and guiding therapy strategies. However,traditional differential expression (DE) workflows often suffer from data distortions due tooutlier, high-read samples, and normalization limitations, likely obscuring biologically relevantdifferences. Methods: This study reanalyzed bulk RNA-seq data from HER2-positive breast cancer patients(GSE292823) to compare traditional DE analysis with a novel scaling-based preprocessingmethod. Results: The scaled data method significantly improved sample clustering in PCA and enhanceddetection of differentially expressed genes, including CCL5, which is associated with resistanceto trastuzumab. Upregulation of IL18, CD86, and NOD2 indicated inflammatory signaling,antigen presentation pathways, and immune cell recruitment, all key factors that can help tumorcells in evading an immune response. Furthermore, several other immune-regulated genes weredownregulated, including CD3G and GZMA, suggesting the suppression and reduced efficiencyof cytotoxic T-cell activity. The downregulation of SATB-AS1, PICSAR and ROR1-AS1,known to be associated with altered chromatin remodeling and long non-coding RNA-mediatedregulation in lung cancers and other types, pointed to similar regulatory mechanisms in HER2-positive breast cancer. These findings demonstrate the value of the novel preprocessing methodin uncovering both activated and suppressed gene networks that shape the tumormicroenvironment and immune response in HER2+ breast cancer. Additionally, Gene ontology(GO) enrichment analysis revealed that the preprocessing method enhanced the detection ofbiologically relevant pathways compared to the original approach. While the traditional methodidentified general immune-related pathways such as leukocyte adhesion and T cell suppression,the new method uncovered several specific immune functions, including regulation of immuneeffector processes, immunological synapse formation, and further downregulation of the T cellreceptor complex activity. Moreover, the new method captured upregulation of vesicle-mediatedsignaling and granule-associated components, suggesting heightened immune cell-tumorengagement. Conclusion: The scaled preprocessing improves the detection of gene expression differences andsignificant pathways, thereby allowing for a more comprehensive view of tumor-immuneinteractions in HER2-positive breast cancer.