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

Area of Science:

• Oncology
• Molecular Pathology
• Biomarker Discovery

Background:

• Prostate cancer exhibits molecular heterogeneity driven by gene fusions and expression profiles of ERG, SPINK1, ETV1, and ETV4.
• These markers define distinct, mutually exclusive molecular subtypes with clinical significance.
• Current methods analyze these markers separately, hindering the study of their spatial relationships within tumors.

Purpose of the Study:

• To develop a multiplex assay for simultaneous detection of ERG, SPINK1, ETV1, and ETV4 in prostate cancer tissues.
• To enable spatial analysis of these key biomarkers within a single tissue section.

Main Methods:

• A combined dual immunohistochemistry (IHC) and dual RNA in situ hybridization (RNA-ISH) assay was created for formalin-fixed paraffin-embedded (FFPE) tissues.
• Validated antibodies were used for ERG and SPINK1 protein detection, and RNAscope probes for ETV1 and ETV4 mRNA.
• The assay was optimized for compatibility, contrast, and morphological preservation.

Main Results:

• The assay allowed concurrent visualization of ERG/SPINK1 proteins and ETV1/ETV4 transcripts in single sections.
• Mutually exclusive expression patterns confirmed known prostate cancer molecular subtypes.
• High-resolution spatial information on intra- and inter-tumoral heterogeneity was obtained, maintaining histological integrity.

Conclusions:

• The dual IHC/RNA-ISH platform provides a novel, reliable method for multiplex biomarker detection in prostate cancer.
• This approach facilitates molecular classification, biomarker validation, and comprehensive tumor heterogeneity assessment.
• It offers significant advantages for translational and diagnostic research in prostate cancer.