Multiplex immunohistochemistry in multiple myeloma: principles, spatial profiling applications, and emerging frontiers in immuno-oncology

Multiple myeloma (MM) progression and therapeutic resistance are dictated by the bone marrow microenvironment (BMME). Conventional single-marker immunohistochemistry (IHC) cannot resolve the complex cellular neighborhoods, non-cellular signaling networks, and spatial exclusion patterns that characterize this disease. Multiplex immunohistochemistry (mIHC) and allied high-plex spatial imaging technologies now enable simultaneous visualization of 6 to over 60 protein targets on a single formalin-fixed paraffin-embedded (FFPE) section while preserving tissue architecture. This review first establishes a rigorous technical foundation by reclassifying mIHC and related high-dimensional imaging platforms according to their detection readout modality—fluorescence spectral unmixing/tyramide signal amplification (TSA) cyclic staining, DNA-barcode cyclic hybridization, metal-isotope mass spectrometric imaging, and regional-photolysis digital spatial profiling—and by clarifying several long-standing technical misconceptions (e.g., the historical relationship between PhenoCycler and Vectra Polaris, the cyclic nature of the Opal/TSA workflow, and the true elution chemistry of CODEX and t-CyCIF). We then present an expanded, bone-marrow-specific horizontal comparison of mIHC against flow cytometry, mass cytometry (CyTOF), imaging mass cytometry (IMC)/multiplexed ion beam imaging (MIBI), and spatial transcriptomics. Building on this technical scaffold, we organize the biological and clinical applications of mIHC along the natural disease continuum—monoclonal gammopathy of undetermined significance (MGUS) → smoldering myeloma (SMM) → newly diagnosed MM (NDMM) → post-treatment residual disease → relapsed/refractory MM (RRMM) → extramedullary disease (EMD)—integrating T-cell, stromal, myeloid, and NK-cell compartments together with cytokine networks, metabolic competition, and bone-remodeling signals. We further delineate realized multimodal integration paradigms (mIHC combined with spatial transcriptomics, single-cell RNA sequencing, digital spatial profiling, and artificial intelligence) and, separately, the technical, economic, and workflow bottlenecks—including spectral overlap, antibody cross-reactivity, turnaround time, cost, and the choice between bone marrow clot and decalcified trephine specimens—that currently constrain clinical translation. As methodological workflows for decalcified bone marrow specimens mature, mIHC is transitioning from a research tool to a critical component of precision immuno-oncology, offering actionable insights for overcoming CAR-T resistance and improving risk stratification.