Isoform-level Transcriptome Analysis of Peripheral Blood Mononuclear Cells from Breast Cancer Patients Identifies a Disease-associated RASGEF1A isoform

Breast cancer (BC) comprises multiple subtypes with distinct molecular features, which differ in their interplay with host immunity, prognosis, and treatment. The triple-negative and HER2(+) subtypes are generally more immunogenic and associated with better immunotherapy responsiveness than the luminal subtypes. Non-invasive blood analyses can provide valuable insights into systemic immunity during cancer. The aim of this study was to analyze the expression of transcriptional isoforms in peripheral blood mononuclear cells (PBMCs) from BC patients and healthy women to identify potential BC immune biomarkers. RNA-sequencing and isoform-level bioinformatics were performed on PBMCs from 13 triple-negative and 13 luminal A patients. Isoform expression validation by qRT-PCR and clinicopathological correlations were performed in a larger cohort (156 BC patients and 32 healthy women). Transcriptional analyses showed a significant (p<.001) decrease of the ENST00000374459 RASGEF1A isoform in PBMCs of BC compared to healthy subjects, indi-cating disease-related expression changes. The decrease was associated with higher ctDNA and Ki-67 values. The levels of the RASGEF1A transcriptional isoform ENST00000374459 may have the potential to distinguish between BC and healthy subjects. The downregulation of ENST00000374459 in breast cancer is associated with higher proliferation and ctDNA shedding. Specialized bioinformatics analyses such as isoform analyses hold significant promise in the detection of biomarkers, since variant-indiscriminate (standard) RNA sequencing analyses may overlook specific transcriptional changes that may be disease-associated and biologically important. Overall design: In this work, we employed transcriptional analyses to identify differences in mRNA-isoform expression in peripheral blood mononuclear cells (PBMCs) isolated from BC patients and healthy subjects. To identify isoform differences between two immunologically distinct subtypes, the more immunogenic triple-negative and the less immunogenic luminal A, RNA-seq was employed, representing the first isoform-level transcriptome analysis in peripheral blood of BC patients.