Background: Increasing evidence shows the ability of RNA sequencing (RNA-seq) to detect expression of multiple biomarkers with improved sensitivity and scalability, offering a more objective and reproducible interpretation of data compared to pathological assessment methods by immunohistochemistry (IHC). Further, ongoing research demonstrating strong correlation between RNA-seq gene expression values and IHC values suggests the utility of RNA-seq as an objective diagnostic approach. Here, we highlight 4 cases where discordance in expression levels measured by IHC and RNA-seq resulted in re-evaluation of IHC values and a subsequent change in the clinical course. Methods: All patients underwent RNA and DNA sequencing of tumor tissue as part of a standard-of-care workflow. Sequencing was performed and reported with the BostonGene Tumor Portrait test, utilizing whole-exome sequencing (WES) and transcriptome sequencing data for tumor analysis. Gene expression levels were evaluated by comparison to a reference cohort of patients with a similar diagnosis. Results: In one case, a 53-year-old female was diagnosed with metastatic cholangiocarcinoma. While IHC of her biopsy reported positive HER2 (+3) expression, RNA-seq revealed low expression. A repeated IHC assay confirmed negative HER2 expression, leading to a change in therapy options. Another 61-year-old female was diagnosed with triple negative breast cancer (TNBC) (IHC: ER 0%, PR 0%, HER2 1+). Integrated WES and RNA-seq analysis detected HER2 amplification (+8 copies), ERBB2-activating mutation, high ERBB2 (HER2) expression and a HER2-enriched PAM50 subtype. IHC was repeated, now showing positive HER2 (+3) expression. In order to target HER2, systemic treatment was changed to a combination containing trastuzumab and pertuzumab, resulting in pathological complete response. In the third case of an 85-year-old female with relapsed TNBC, IHC demonstrated 1% PDL-1 staining. In contrast, RNA-seq detected high PD-L1 expression leading the treating physician to repeat IHC. Staining showed high PDL-1 (CPS50), prompting a change in therapy to an anti-PDL-1 (pembrolizumab) therapy. Lastly, while IHC of a 21-year-old female diagnosed with diffuse large B-cell lymphoma (DLBCL) showed negative CD30, subsequent RNA-seq detected relatively high CD30 expression that prompted a second IHC evaluation confirming positive CD30 expression. Eventually, the diagnosis was changed to primary mediastinal B-cell lymphoma and DA-EPOCH was selected as the therapy. Conclusions: RNA-seq is emerging as an objective tool to evaluate key diagnostic and targetable events in multiple cancer types. The use of RNA-seq can also add value as an objective measurement to confirm critical IHC findings, potentially changing clinical care.