Additional evaluation of macrophage significance, cellular motif optimization, and macrophage-tumor cell interactions stratified by LMP1 expression in DLBCL.

Additional antibody staining specificity validation and cell phenotyping maps across DLBCL tissue sections.

Across cancer, one of the most frequent examples of histologic transformation is the evolution of follicular lymphoma (FL) to an aggressive large cell lymphoma. Despite recent progress, understanding of the molecular and cellular underpinnings of transformation remains incomplete. Here, we dissect the interplay of tumor and microenvironment cell populations across transformation through a multimodal investigation of 95 FL and transformed FL (tFL) samples, including single-cell and bulk RNA-sequencing alongside spatial transcriptomics and proteomics, and validate findings across independent FL-tFL pairs. Upon transformation, fibroblasts and GPNMB+ macrophages increase while lymph-node organizing follicular dendritic and CCL21+ fibroblastic reticular cells were lost, resulting in an altered spatial distribution of cytokines that impacts T cell infiltration and macrophage differentiation and function. Secreted stromal and macrophage signals were further evident by non-invasive plasma proteomics. Taken together, our data reveal expansion of macrophages and fibroblasts as key features of transformation with potential diagnostic and therapeutic implications.

Classic Hodgkin lymphoma (cHL) is composed of rare malignant Hodgkin and Reed-Sternberg (HRS) cells within a T-cell-rich tumor microenvironment (TME). Epstein-Barr virus (EBV) is present in ∼25% of cases, but its contribution to pathogenesis and immunomodulation remains unclear due to technical barriers. Using complementary spatial proteomics and transcriptomics across multi-institutional cohorts, we systematically map key EBV-linked TME reorganization. EBV-positive cHL exhibits distinct immunological features, including memory CD8 T cell enrichment, heightened T cell dysfunction spatially correlated with HRS proximity, and terminally exhausted T cell signatures contrasting with progenitor-exhausted patterns in EBV-negative disease. We identify EBV-encoded LMP1 as a factor in T cell dysfunction through enhanced HRS:CD8 interactions, and its expression level correlates with T cell terminal exhaustion in a distance-dependent manner. This spatial framework dissects viral-mediated immune evasion in the cHL TME, highlighting potential therapeutic opportunities to target virus-associated T cell dysfunction for precision immunotherapy in virus-associated malignancies.

Additional image quality, tissue integrity, and segmentation validation across extended CODEX cycles and multi-omics spatial workflow orders in IN-DEPTH.

Additional details on the CODEX-GeoMx spatial alignment, integration workflow, and batch effect correction for the DLBCL cohort.

Upper and lower bound values used in data processing.

Details of the antibody panel used in all IN-DEPTH experiments throughout the study including antibody clones, vendors, working concentrations, imaging channel, imaging cycle and exposure time.

Spatial transcriptomics and proteomics have enabled profound insights into tissue organization, yet these technologies remain largely disparate, and emerging same-slide multiomics approaches are limited in plex, spatial resolution, signal retention, and integrative analytics. We introduce IN-situ DEtailed Phenotyping To High-resolution transcriptomics (IN-DEPTH), a streamlined, resource-efficient, commercially compatible workflow using single-cell spatial proteomics-derived imaging to guide transcriptomic capture on the same slide without RNA signal loss. To integrate modalities beyond niche-level mapping, we developed Spectral Graph Cross-Correlation (SGCC), a proteomic-transcriptomic framework resolving spatially coordinated functional state changes across interacting cell populations. Applied to diffuse large B-cell lymphoma (DLBCL), IN-DEPTH and SGCC enabled stepwise discovery from Epstein-Barr virus (EBV)-positive and EBV-negative tumor comparisons with single-cell resolution, revealing coordinated tumor-macrophage-CD4 T-cell remodeling, immunosuppressive C1Q macrophage enrichment, CD4 T-cell dysfunction, and a candidate IL27-STAT3 signaling axis. Collectively, IN-DEPTH enables scalable spatial multiomics to uncover clinically relevant microenvironmental mechanisms and toward robust spatial multimodal AI models. SIGNIFICANCE:IN-DEPTH enables same-slide spatial multiomics across commercial platforms via a protein-first strategy preserving protein epitopes, RNA quality, and tissue integrity. Coupled with SGCC, it resolves coordinated spatial immune remodeling, revealing EBV/LMP1-driven C1Q macrophage polarization and CD4 T-cell dysfunction in DLBCL, with broad applicability to other diseases.

Additional reproducibility assessment of IN-DEPTH in tonsil benchmark tissues across serial tissue sections, including antibody validation, spatial registration, and multi-modal consistency across protein expression, transcriptomics, and deconvolution methods.

Mean expression, statistical metrics, and Log2fold change of ligand-receptor interactions.

Enrichment pathways in LMP1-positive and LMP1-negative tumors and their neighboring macrophages.

Details of batch effect correction for the DLBCL IN-DEPTH datasets.

Clonal mature plasmacytoid dendritic cell proliferations (MPDCP) are a recently recognized entity in the WHO fifth edition, but their pathologic spectrum remains poorly characterized. Cases with extensive MPDCP in the bone marrow (BM) are rare and can exhibit overlapping features with blastic plasmacytoid dendritic cell neoplasm (BPDCN). We compared clinicopathologic and genomic features of 11 patients with myeloid neoplasm-associated MPDCP to 5 patients with secondary BPDCN arising from clonally-related myeloid neoplasms. MPDCP exhibited variable degrees of BM involvement (5% to 50%) and architectural patterns, were uniformly positive for CD123, CD4, TCF4, and IRF8, variably positive for TCL1 (7/11), CD5 (7/11), and CD7 (2/11), and negative for SOX4, CD56, and TdT. MPDCP skin involvement was rare (1/11), with no CNS involvement. MPDCP heralded myeloid disease progression in a subset of patients, with increased blasts or progression to AML (4/11). In contrast, secondary BPDCN was uniformly positive for SOX4 and TCL1, with frequent CD56 (4/5) and subset/weak TdT (3/4). All BPDCN patients had characteristic skin lesions, and a subset with CNS involvement (2/5). The underlying myeloid neoplasms associated with MPDCP or BPDCN were enriched in TET2 , SRSF2 , ASXL1 , RUNX1 , and RAS pathway mutations. While karyotypic abnormalities were uncommon in MPDCP, all BPDCN showed chromosomal structural abnormalities and copy number variants, including deletions of 3p, 9p, and 12p. Our findings expand the histopathologic, immunophenotypic, and genetic characterization of MPDCP, and highlight pathologic features that distinguish it from BPDCN. Utilization of SOX4 immunohistochemistry, combined with careful clinical and molecular correlation, can aid in resolving these diagnostic challenges.

OBJECTIVES:Plasma cell neoplasms (PCNs) harboring IGH::CCND3 (t(6;14)) are rare (<1% of PCNs). We sought to study these cases, as they have not been studied as a distinct entity, and their clinicopathologic features are not well characterized. METHODS:Cytogenetics databases were searched for positive IGH::CCND3 interphase fluorescence in situ hybridization (FISH) results in plasma cell neoplasms. We analyzed the clinical, morphologic, immunophenotypic, and cytogenetic features of these cases and evaluated the utility of cyclin D3 immunohistochemistry (IHC) for identifying these neoplasms. RESULTS:Sixteen patients were identified, presenting with multiple myeloma (n = 9), monoclonal gammopathy of undetermined significance (n = 3), smoldering myeloma (n = 2), plasma cell leukemia (n = 1), and amyloid light chain amyloidosis (n = 1). Median age at diagnosis was 68.9 years. Most cases showed lymphoplasmacytoid morphology (63%, 10/16), with frequent CD20 expression (53%, 8/15) and aberrant expression of CD56 (71%, 10/14) and CD117 (69%, 9/13). Monosomy 13 was the most frequent concurrent cytogenetic abnormality (50%, 7/14). Cyclin D3 IHC showed nuclear expression in plasma cells in all tested patients (11/11). Background erythroid precursors and megakaryocytes also showed variable positivity. When performed as a dual immunostain with CD138, cyclin D3 served as a highly sensitive and specific method for detecting these neoplastic plasma cells. CONCLUSIONS:IGH::CCND3 PCNs frequently exhibit lymphoplasmacytoid features and CD20 expression, resembling the more common t(11;14) PCNs. Cyclin D3 IHC is an effective surrogate for the t(6;14) translocation in PCNs. We recommend using cyclin D3 IHC in the workup of PCNs with lymphoplasmacytoid morphology but negative cyclin D1 to appropriately trigger reflex FISH testing in positive cases and rescue them from the "partner-negative" category.

File containing extended details on SGCC method development, key resource table and Supp Table legend

Additional benchmarking of IN-DEPTH against existing spatial-omics platforms across performance, cost, and data quality metrics is presented.

Additional supporting data for multicellular SGCC analysis in the DLBCL study.