Mapping Molecular Diversity in Prostate Cancer With a Combined Multiplex IHC and RNA-ISH Assay

Molecular heterogeneity in prostate cancer is defined by distinct gene fusions and expression profiles involving ERG, SPINK1, ETV1, and ETV4. These markers are typically mutually exclusive and represent biologically distinct molecular subtypes with clinical and therapeutic relevance. Conventional methods assess these markers independently, limiting the ability to study their spatial relationships within the same tumor focus.

We developed a combined dual immunohistochemistry (IHC) and dual RNA in situ hybridization (RNA-ISH) assay for simultaneous detection of ERG, SPINK1, ETV1, and ETV4 in formalin-fixed paraffin-embedded (FFPE) prostate cancer tissues. Validated antibodies were used for ERG and SPINK1 protein detection, while RNAscope probes were employed for ETV1 and ETV4 mRNA visualization. The assay was optimized for sequential staining compatibility, chromogen contrast, and morphological preservation, and subsequently applied to prostate cancer cores representing various molecular subtypes.

The combined assay enabled clear, concurrent visualization of ERG and SPINK1 protein expression together with ETV1 and ETV4 transcripts in a single tissue section. Expression patterns were mutually exclusive across tumor foci, consistent with known molecular subtypes of prostate cancer. The method maintained histological integrity and signal specificity, providing high-resolution spatial information that could not be obtained by separate assays. This approach allowed detailed assessment of intra- and inter-tumoral heterogeneity within the same histological context.

This dual IHC and dual RNA-ISH approach represents a novel and reliable platform for multiplex detection of key prostate cancer biomarkers on a single slide. The method offers significant advantages for molecular classification, tissue-based biomarker validation, and comprehensive evaluation of tumor heterogeneity in translational and diagnostic research.