Classic Hodgkin lymphoma (cHL) is a B cell malignancy characterized by rare malignant Hodgkin and Reed-Sternberg (HRS) cells within an extensive inflammatory microenvironment. The mutational profile of cHL overlaps with that of related B cell lymphomas, including primary mediastinal B cell lymphoma (PMBL), and yet these are different histologically and clinically. To discover the molecular features that distinguish cHL, we deployed flow cytometric cell sorting and low-input RNA sequencing to generate full transcriptome data from viable, isolated HRS cells from eighteen primary tumors, alongside matched intra-tumoral non-neoplastic B cells and four cell lines.

To explore the pathways upregulated in HRS cells relative to intra-tumoral B cells, we performed Gene Set Enrichment Analysis (GSEA), which revealed a significant enrichment of pathways involved in biological processes not previously described in cHL, including the unfolded protein response (UPR). To assess whether this UPR signature is specific to HRS cells, we evaluated its expression in DLBCL and PMBL using publicly available data and did not observe a consistent increase in either DLCBL or PMBL. Among the top upregulated UPR genes, we identified PDIA6,a disulfide isomerase localized in the endoplasmic reticulum that plays a crucial role in protein folding and preventing aggregation by catalyzing the formation and breakage of disulfide bonds during protein folding. Immunohistochemistry (IHC) for PDIA6 in an extended cohort demonstrated strong immunoreactivity in HRS cells compared to background lymphoid cells in all 24 cases evaluated. Apart from plasma cells, which are easily recognized morphologically, PDIA6 staining was highly specific for HRS cells, and therefore may serve as a valuable diagnostic marker.

GSEA also revealed that pathways related to NK cell mediated cytotoxicity were downregulated in HRS cells in comparison to intra-tumoral B cells. One of the key regulators of NK cell cytotoxicity is the signaling lymphocytic activation molecule family (SLAMF) receptors. Six out of nine SLAM family activating receptors - SLAMF2 (CD48), SLAMF3 (LY9), SLAMF4 (CD244), SLAMF5 (CD84), SLAMF6 and SLAMF7 - were downregulated on HRS cells, suggesting that the loss of NK cell-activating receptors may contribute to immune evasion in cHL. We further validated this finding using flow cytometry, confirming the downregulation of CD48 (SLAMF2) in HRS cells from both cell lines and five primary cases. Consistent with the reduced role of NK cells in tumor immune control, we found a significantly reduced proportion of NK cells in cHL tumors (median 0.6% vs. 1.4%, p<0.01) compared to reactive lymph nodes, as assessed by flow cytometry. Immunohistochemistry confirmed the loss of CD48 in 24 of 24 cases examined

To investigate the transcriptional differences that distinguish cHL from PMBL we compared the PMBL gene signature defined by Savage et al. to gene expression profiles from intraturmoral B cells, cHL cell lines and purified HRS cells. TNFRFS17 (encoding BCMA), typically expressed in plasma cells, and LMO2, associated with germinal center B cells, were upregulated in PMBL but uniformly absent in cHL. IHC on an extended cohort confirmed expression in PMBL cases (BCMA 45/49, LMO2 50/50), but not in cHL (BCMA 0/17; LMO2 0/13).

Lastly, we examined the cHL signature genes across various B cell subsets. The expression HRS signature most closely resembled those found in plasma cell lineages, including bone marrow plasma cells. Given the overlap between HRS cells and plasma cells, we next examined the expression of the cHL signature in multiple myeloma (MM). We found that the genes upregulated in the cHL signature were positively enriched in MM vs. GCBs (NES: 1.64; p=0.002), and conversely those negatively regulated were reduced in MM (NES: -2.45, p=0.000).

Overall, our integrated transcriptomic and IHC analysis revealed distinct molecular features of cHL, evidence of abortive plasma cell differentiation, with an unfolded protein response signature, shared with plasma cell neoplasms, but not other B-cell lymphoma types. In HRS cells, we also observed a downregulation of SLAM family receptors, which are crucial for NK cell activation, providing a potential mechanism for immune evasion from NK-mediated killing.

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2025
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