Understanding which biomarkers are present in a tumor is critical for diagnosing cancer and deciding which treatment may be most effective. Traditionally, doctors use a method called immunohistochemistry, or IHC, to identify these biomarkers. However, IHC can sometimes be subjective, depending on how the results are interpreted under a microscope.

A team led by researchers at BostonGene Corporation set out to see how well RNA sequencing, a technique that measures gene activity, could match the results of IHC across different types of solid tumors. They analyzed tissue samples from 365 patients with cancers such as breast, lung, and gastrointestinal tumors. The team measured the activity of nine important genes, including ESR1, PGR, and PD-L1, and compared these levels to standard IHC results.

They found that RNA sequencing results strongly matched IHC results for most of the biomarkers, with correlation scores as high as 0.89. Even for PD-L1, which is known to be influenced by the surrounding tumor environment, the RNA data still showed a moderate correlation. The researchers also established clear thresholds for RNA expression that could reliably distinguish between positive and negative IHC classifications.

Clinical applicability of RNA-seq cut-offs in comparison with IHC thresholds

(A) RNA-seq cut-off effectively identified breast cancer subtypes in the validation dataset (stages III and IV, TCGA-BRCA, n = 237, multivariate logrank test), yielding overall survival (OS) results similar to IHC. Hormone receptor (HR) status corresponded to ER/ESR1 and PR/PGR expression. Negative IHC annotation for ER, PR, and HER2, as well as the negative mRNA cut-offs for the corresponding genes (ESR1, PGR, and ERBB2) were used to define triple-negative breast cancer (TNBC). (B) Disease-free intervals in ESR1+ ERBB– MKI67-high versus ESR+ ERBB– MKI67-low breast cancer patients (Stages I–III) classified by the elevated RNA cut-off of 4 log₂(TPM + 1). Statistical analysis was performed using the multivariate logrank test. (C) The CD274 (TC) RNA-seq threshold of 3.5 log₂(TPM + 1) showed similar differences in survival between the PD-L1–low and PD-L1–high groups as the 50% TC IHC threshold in lung cancer (SU2C-MARK lung cohort, n = 64, multivariate logrank test). (D) The CD274 (CPS) RNA-seq threshold of 2.5 log₂(TPM + 1) significantly diversified responders from non-responders to pembrolizumab in metastatic gastric cancer (PRJEB25780, n = 42). SD: stable disease; PD: progressive disease; CR: complete response; PR: partial response.

These findings show that RNA sequencing can be a valuable companion to IHC, providing fast, objective, and scalable insights into tumor biology. Especially in complex cases where IHC might be less reliable, RNA sequencing can help doctors make more precise decisions about diagnosis and treatment.