Background Effective TCR-directed cell therapies in Multiple Myeloma (MM) require discovery of shared, tumor-restricted antigens with matched TCRs. Cryptic peptides from untranslated regions, aberrant splicing, and retroelements are promising candidates; however, systematic mapping of their cognate TCR:antigen pairs in MM remains limited. Here, we apply high-throughput antigen prioritization with DNA barcoded (dCODE) MHC-I Dextramer technology to validate epitopes and map TCR:antigen pairs, including cryptic epitopes from untranslated regions. Methods We developed a discovery framework integrating whole exome sequencing, RNA-seq, ribosome profiling, and single-cell (sc) immunoprofiling to identify antigens that are transcribed, translated, and predicted to be antigenic. We applied this to bone marrow (BM) from 10 HLA-A*02:01 MM patient samples from the BMT CTN 1401 trial (NCT02728102). Peptides were prioritized using a machine learning model trained on validated antigens. Prioritized antigens were confirmed for plasma cell specificity via sc RNA-seq, and MHC-I presentation potential assessed using public immunopeptidomic datasets. Top antigens were validated in patient BM and matched with cognate TCRs using sc multiomics with dCODE MHC-I Dextramers. Results Of >2,000 cryptic and mutation-derived peptides, 63 cancer-restricted antigens were prioritized, including 6 shared retroelement-derived epitopes from L1, L2, and ERV1 subfamilies. The L2 element antigen was consistently detected across 10 patients, highlighting its potential as a broadly relevant target. Many additional antigens arose from non-canonical ORFs in plasma cell-associated genes, including BCMA and SLAMF7. dCODE MHC-I Dextramer profiling of patient BM samples revealed distinct phenotypic distributions: viral antigen-specific TCRs (influenza, CMV) were enriched in CD8 tissue-resident and terminal effector memory subsets, whereas cryptic tumor antigen-specific TCRs (ERV1/L2, SLAMF7, BCMA) were enriched in early effector-memory and activated states, suggesting a proliferative potential and the ability to generate terminal effectors with strong cytotoxic function. In this pilot, >6,000 viral-/cryptic antigen-TCR pairs were mapped, representing the largest dataset of its kind in MM, with additional patients ongoing. Conclusion This study provides the first single-cell antigen-TCR atlas of cryptic antigens in MM, uncovering shared, tumor-restricted epitopes from retroelements and non-canonical ORFs and their cognate TCRs. By integrating genomics-driven discovery with dCODE MHC-I Dextramer profiling, we demonstrate a scalable, clinically relevant pipeline linking cryptic antigens to functional TCR states. These findings establish cryptic antigens as a class of actionable targets and deliver a blueprint for vaccine- and T cell-based immunotherapies in MM.

Multiple myeloma (MM) remains incurable despite advances in treatment options. Although tumor subtypes and specific DNA abnormalities are linked to worse prognosis, the impact of immune dysfunction on disease emergence and/or treatment sensitivity remains unclear. We developed an Immune Atlas of MM by generating profiles of 1,397,272 single cells from the bone marrow (BM) of 337 newly diagnosed participants and characterized immune and hematopoietic cell populations. Cytogenetic risk-based analysis revealed heterogeneous associations with T cells of BM, with 17p13 deletion showing distinct enrichment of a type 1 interferon signature. The disease progression-based analysis revealed the presence of a proinflammatory immune senescence-associated secretory phenotype in rapidly progressing participants. Furthermore, signaling analyses suggested active intercellular communication involving a proliferation-inducing ligand and B cell maturation antigen, potentially promoting tumor growth and survival. Lastly, using independent discovery and validation cohorts, we demonstrated that integrating immune cell signatures with known tumor cytogenetics and individual characteristics significantly improves stratification for the prediction of survival. Pilcher et al. present a single-cell transcriptomics-based immune atlas of participants with newly diagnosed multiple myeloma, reporting on the association of cell types, gene expression and intercellular interactions with disease progression phenotypes.

Background Understanding the T cell clonotypic composition within the multiple myeloma (MM) microenvironment is critical to assess the impact of tumor-associated effector T cell populations on disease outcome. We hypothesized a significant overlap between expanded clonotypes in the bone marrow (BM) and peripheral blood (PB), allowing circulating BM-infiltrating lymphocytes to serve as a window into the MM-associated T cell response. BMT CTN 1401 is a phase II trial of MM patients post-autologous hematopoietic cell transplant (HCT) randomized to a personalized dendritic cell/MM fusion vaccine plus lenalidomide maintenance or maintenance alone. Prior studies showed expansion of MM-reactive T cells in vaccinated patients. Here, we integrated paired BM and PB TCR sequencing and gene expression profiling to characterize the clonal architecture and functional features of tumor-associated T cells and their relation to clinical outcome. Methods Single-cell RNA and TCR sequencing was performed on ∼160 paired BM and PB mononuclear cell samples from 40 BMT CTN 1401 patients, collected at enrollment and 1-year post-HCT. Patients were stratified by response: stringent complete response/complete response (sCR/CR, n=26), very good partial response (VGPR, n=9), partial response (PR, n=4), and progressive disease (PD, n=1). We analyzed 178,135 T cell clonotypes (BM: n=47,790; PB: n=130,345) focusing on clonal distribution, BM-PB overlap, and association with clinical outcome. Results At 1-year post-HCT, patients in sCR/CR had higher proportions of expanded PB effector T cells than PR patients. Substantial TCR sharing was observed between PB and BM, particularly among expanded clonotypes. >70% of highly expanded BM TCRβ clonotypes (frequency > 4) were also detected in PB, and >65% of these also expanded in PB (TCR frequency >1) at baseline, indicating a robust circulating pool of MM-associated T cells and coordinated clonal dynamics across BM and PB compartments. Similarly, >70% of expanded PB TCRβ clonotypes (frequency>4) at baseline overlapped with BM clones, suggesting that a large fraction of circulating expanded T cells is MM-associated. TCR overlap between BM and PB was significantly greater in sCR/CR compared to VGPR/PR at baseline and post-HCT, with the largest differences observed post-HCT. These results were consistent across CD8 and CD4 T cell subsets. BM samples had consistently a higher CD4/CD8 ratio than PB. Conclusion High overlap between PB and BM TCR reflects robust clonal expansion and is associated with sCR/CR, suggesting that circulating T cells may serve as surrogates for tumor-infiltrating T cell dynamics. Ongoing studies are mapping dominant clonotypes to tumor antigens and evaluating the impact of vaccination on the T cell repertoire.

BACKGROUND:The production of VLDL is one of the primary mechanisms through which liver cells regulate intracellular lipid homeostasis. We hypothesize that the disease characteristics of metabolic dysfunction-associated steatotic liver disease (MASLD) differentially impact VLDL lipid composition. This study comprehensively examines the relationship between VLDL-lipidome and MASLD histology and disease-associated genetics, aiming to define MASLD-related VLDL changes. METHODS:We performed untargeted lipidomics on serum VLDL particles in a cohort of biopsy-proven MASLD patients to examine the relationship between VLDL-lipidome and MASLD disease features as well as MASLD-related genetic variants. RESULTS:Among 1514 detected lipid species in VLDL, triglyceride (TG), phosphatidylcholine (PC), and ceramide (Cer) were the top classes. Moderate to severe hepatic steatosis was associated an increase in VLDL-TG, especially those with palmitic acid (C16:0). A unified acyl chain distribution analysis revealed that steatosis was associated with increases in TGs with saturated and monounsaturated fatty acyl chains, but decreases in polyunsaturated fatty acyl chains, a pattern that was not mirrored in acyl chains from VLDL-PC or VLDL-Cer. Lobular inflammation was associated with reductions in lipids with polyunsaturated acyl chains, particularly docosahexaenoic acid (C22:6). Meanwhile, patients with advanced liver fibrosis (stages 3-4) had reductions in VLDL-TGs with both saturated and polyunsaturated acyl chains and overall enrichment in Cer species. Furthermore, MASLD-associated genetic variants in PNPLA3, TM6SF2, GPAM, HSD17B13, and MTARC1 demonstrated distinct VLDL-lipidomic signatures in keeping with their biology in lipoprotein metabolism. CONCLUSIONS:Hepatic steatosis and liver fibrosis in MASLD are associated with distinct VLDL-lipidomic signatures, respectively. This relationship is further modified by MASLD-genetics, suggesting a differential impact of pathogenic features on hepatocellular lipid homeostasis.

BackgroundThe molecular underpinnings of organ dysfunction in severe COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we perform single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents.ResultsWe identify hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells, and a central role in a pro-fibrotic TGF beta signaling cell-cell communications network. Integrated analysis and comparisons with healthy controls reveal extensive changes in the cellular composition and expression states in COVID-19 liver, providing the underpinning of hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis characteristic of COVID-19 cholangiopathy. We also observe Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition is dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells.ConclusionsOur atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

While Chimeric Antigen Receptor (CAR) T cell therapy may result in durable remissions in recurrent large B cell lymphoma, persistence is limited and the mechanisms underlying long-term response are not fully elucidated. Using longitudinal single-cell immunoprofiling, here we compare the immune landscape in durable remission versus early relapse patients following CD19 CAR T cell infusion in the NCT02348216 (ZUMA-1) trial. Four weeks post-infusion, both cohorts demonstrate low circulating CAR T cells. We observe that long-term remission is associated with elevated native cytotoxic and proinflammatory effector cells, and post-infusion clonotypic expansion of effector memory T cells. Conversely, early relapse is associated with impaired NK cell cytotoxicity and elevated immunoregulatory cells, potentially dampening native T cell activation. Thus, we suggest that durable remission to CAR T is associated with a distinct T cell signature and pattern of clonotypic expansion within the native T cell compartment post-therapy, consistent with their contribution to the maintenance of response.

Determining how immune checkpoint inhibitors (ICI) alter functional characteristics of lymphocytes can improve our understanding of ICI. HERV-H LTR-associating 2 (HHLA2, B7-H7) is an immune checkpoint expressed by many tumors. Multiple HHLA2:KIR3DL3 blocking antibodies are currently in Phase I clinical trials and enrolling cancer patients (NCT05958199, NCT06240728), but little is known about how different subsets of NK cells are affected by HHLA2 expression. Our group previously found that in addition to an activating receptor, TMIGD2, HHLA2 has an inhibitory receptor, KIR3DL3. The KIR family of receptors is expressed in NK cells as well as T cells. Through co-culture experiments of CD56+ NK cells in the presence of K562 cells, we sought to investigate the mechanisms of NK cell engagement. Additionally, we generated transgenic K562 cell lines, over-expressing HHLA2, validated through flow cytometry, to maximize our ability to glean insight into HHLA2-KIR3DL3 mediated activation of NK cells. To this end, we generated a high quality scRNA-seq dataset of more than 100, 000 cells, comprising of more than 90, 000 NK cells, with approximately 30, 000 cells either cultured alone, co-cultured with K562 or K562 over-expressing HHLA2 tumor cells, allowing unprecedented granularity. In conjunction with published NK cell states and newly identified ones through this study, we recapitulate important known cytokines, growth factors and other regulators underlying NK cell maturation and substates that are differentially affected by treatment, in abundance, expression, and regulation. Furthermore, we shed some light on the complex interplay of positive and negative regulators, including CD48 - 2B4 and CD56 related interactions in CCL4 and XCL2 high NK cells, as well as TGFb related pathways. Uncovering NK cell activity modulators, such as amphiregulin, NKG7, FAM49B, and TXNIP, we then focused on NK cells in the context of KIR3DL or TMIGD2 expression. We add to previous efforts to understand immune checkpoint inhibition by the HHLA2-KIR3DL3 axis and in a concerted effort with blocking antibodies, provide evidence of enhanced NK cell efficacy. Better understanding of how HHLA2 pathway affects the function of different subsets of lymphocytes could determine which patients may best benefit from targeting this pathway. Nahuel Perrot, Nikolaos Kalavros, Deepthi Chowbene, Shoushuo Wang, Yered Pita-Juarez, Antonella Arruda de amaral, David McDermott, Gordon Freeman, Ioannis Vlachos, Kathleen Mahoney. Investigating the transcriptomic signature of HHLA2-mediated suppression of NK cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3229.

BMT CTN 1401 is a phase II trial of 140 multiple myeloma (MM) patients undergoing autologous hematopoietic cell transplant (HCT) followed by lenalidomide maintenance with or without a personalized DC/MM vaccine. Vaccination was associated with increased MM-reactive T cells and clonotypic expansion of activated T cells 1-year post-HCT. Here, we identify private or shared antigenic targets in pre-HCT bone marrow (BM) samples, along with corresponding T cell clonotypic patterns. We also further analyzed the peripheral blood (PB) immune landscape of the study patients, exploring the correlation between disease response and the T cell repertoire, including dominant clonotypes. Pre-HCT BM samples from 16 patients underwent bulk RNAseq and whole exome sequencing. With an in silico-experimental framework developed by our team, we predicted tumor antigens by considering factors including translation potential, RNA expression, peptide-MHC binding affinity, immunogenicity, clonality, and physicochemical properties. 10 samples were further analyzed by single cell (sc) immunoprofiling to assess the antigens expression in MM cells. Additionally, PB samples from 40 study patients underwent sc immunoprofiling to investigate the T cell repertoire in stringent complete response/complete response (sCR/CR, N=26), very good partial response (VGPR, N=9), partial response (PR; N=4) or progressive disease (PD; N=1). We observed a high prevalence of tumor-specific (neo)antigens, with 50% shared among more than 2 patients. 19 neoantigens were prioritized based on high immunogenicity and expression in multiple patients, including four expressed in immune-privileged tissues (MAGEC1, TPTE, UCHL1). UCHL1 showed an 80-fold higher median expression in MM samples than in healthy controls. We observed a significant enrichment of shared tumor antigenic peptides (n=33) across up to 10 patients, originating from regions not typically expressed or translated, such as retroelements and downstream/upstream open reading frames. PB T cells immunoprofiling revealed higher T cell clonotypic expansion in sCR/CR and VPGR, with sCR/CR associated with higher proportion of cytotoxic CD8 T cells and fewer Tregs 1-year post-HCT. A high degree of TCR sharing between clonally expanded T cells in the PB and BM was observed. To identify novel immunotherapy targets for MM, we characterized the MM antigenic landscape, identifying personal and shared tumor antigens. In addition, we demonstrated that sCR/CR is associated with clonal expansion of cytotoxic T cells with clonal dynamics in the PB reflecting those in the BM. We are correlating the dominant T cell clonotypes with the identified antigens and assessing the impact of DC/MM fusion vaccination in this setting. Giulia Cheloni, Dimitra Karagkouni, Shivani Nanda, Lina Bisharat, Isabella Saldarriaga, MA Yuling, Shuoshuo Wang, Xanti-Lida Katopodi, Yered Pita-Juarez, Jessica Liegel, Hazal Toros, Prateek Pophali, David Chung, Nina Shah, Natalie Callander, Binod Dhakal, Thinle Chodon, Nikhil Munshi, Kenneth Anderson, Yvonne Efebera, Peiman Hematti, Hillard Lazarus, Ehsan Malek, Philip McCarthy, Ajay Nooka, Krina Patel, Aaron Rapoport, Robert Soiffer, Edmund Waller, Marcelo Pasquini, Jacalyn Rosenblatt, Ioannis Vlachos, David Avigan. Neoantigen expression and expansion of cytotoxic T cell clonotypes in multiple myeloma patients receiving a personalized cancer vaccine post-transplant: Insights from the BM CTN 1401 multicenter trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7128.

Acute respiratory distress syndrome (ARDS) is a severe complication of lung injury characterized by hyperinflammation and fibrosis. Here, we show a significant association between the monocyte-derived enzyme adenosine deaminase 2 (ADA2) and SARS-CoV-2 induced ARDS. We note an interesting link between ADA2 and the chemokine CXCL10 and its receptor CXCR3. By using published datasets of spatial transcriptomics and single-cell RNAseq, we show that ADA2 is highly expressed by inflammatory CD14+CD16+ monocytes, along with profibrotic genes, in lungs affected by COVID-19. This study reveals important associations between key pathophysiological features of ARDS, linking hypoxia, infiltrative CXCR3 monocytes, and a monocyte-derived exoenzyme ADA2.

Background: Determining how immune checkpoint inhibitors (ICI) alter functional characteristics of lymphocytes can improve our understanding of these treatments. HERV-H LTR-associating 2 (HHLA2, B7-H7) is an immune checkpoint expressed by many tumors. HHLA2 has an activating receptor, TMIDG2, and an inhibitory receptor, KIR3DL3. The KIR family of receptors is expressed in NK cells as well as T cells. We developed a series of monoclonal antibodies that block the inhibitory HHLA2:KIR3DL3 interaction. HHLA2:KIR3DL3 blocking antibodies in Phase I clinical trials are currently enrolling cancer patients (NCT05958199, NCT06240728). Method : We performed single-cell RNA sequencing on co-cultures of NK cells isolated from peripheral blood cells with or without the presence of K562 cells expressing HHLA2, which are NK cells target, to assess the effects of HHLA2 antibodies blocking HHLA2:KIR3LD3. Co-cultured cells were treated with different HHLA2 blocking antibodies or isotype control. Results : We found different subsets of NK cells in the cocultures delineated by different marker sets, including the XCL2 and CCL4 chemokines, further differentiated TXNIP positive NK cells and BCL2 positive subsets. Additionally, we observed differential abundance between the interaction blocking conditions as compared to isotypes controls. Furthermore, differential gene expression analysis after non-selective HHLA2 blockade was more pronounced in the XCL2+ subset, followed by the CCL4+ subset, and included genes implicated in phagocytosis, regulation of macrophages and chemotaxis, signaling detection, cytokine secretion, and the humoral immune response respectively for each subset. Finally, by iteratively sub-clustering our subsets based on known NK cell markers, we have discovered clusters reflecting a milieu of NK cell states and activity profiles. We will explore whether these subsets of NK cells, specifically XCL2 and+ CCL4+ subsets, are expressed in tumors and characterize the interaction in greater depth. Conclusion : Better understanding of how blocking the HHLA2:KIR3DL3 pathway affects lymphocyte function could determine which patients may best benefit from targeting this pathway. Citation Format: Nahuel Perrot, Nikolaos Kalavros, Deepthi Chowbene, Shoushuo Wang, Yered Pita-Juarez, Antonella Arruda de amaral, David McDermott, Gordon Freeman, Ioannis Vlachos, Kathleen Mahoney. Blocking the HHLA2 immune checkpoint alters transcriptomic signature in specific subsets of NK cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr A014.

The landscape of non-coding mutations in cancer progression and immune evasion is largely unexplored. Here, we identify transcrptome-wide somatic and germline 3' untranslated region (3'-UTR) variants from 375 gastric cancer patients from The Cancer Genome Atlas. By performing gene expression quantitative trait loci (eQTL) and immune landscape QTL (ilQTL) analysis, we discover 3'-UTR variants with cis effects on expression and immune landscape phenotypes, such as immune cell infiltration and T cell receptor diversity. Using a massively parallel reporter assay, we distinguish between causal and correlative effects of 3'-UTR eQTLs in immune-related genes. Our approach identifies numerous 3'-UTR eQTLs and ilQTLs, providing a unique resource for the identification of immunotherapeutic targets and biomarkers. A prioritized ilQTL variant signature predicts response to immunotherapy better than standard-of-care PD-L1 expression in independent patient cohorts, showcasing the untapped potential of non-coding mutations in cancer.

Backgroun d: Identifying novel antigenic targets for immune-based therapy in Multiple Myeloma (MM) is a critical area of investigation. The BMT CTN 1401 (NCT02728102), a national randomized trial evaluating dendritic cell (DC)/MM fusion cells following autologous transplant in patients with newly diagnosed MM, offers a unique source of bone marrow aspirates that can be interrogated for novel antigen discovery studies. In the present analysis, we characterize the personalized and shared tumor-associated (TAA) and tumor-specific (TSA) antigens derived from MM in the bone marrow and patterns of associated T cell clonotypes at baseline (time of enrollment). Methods: We developed an in silico-experimental framework that integrates tumor bulk RNAseq, Whole Exome Sequencing (WES), and T Cell Receptor (TCR) profiling data. We established translation probabilities and assessment of tumor specificity by analyzing >6,500,000 translated regions from raw public Ribosome profiling data and >1,000,000 transcribed regions from healthy control samples across 29 tissues. We developed an AI/ML classifier to define TAAs and TSAs, ensuring each antigenic peptide is highly expressed in tumor samples, translated, presented, and has high immunogenic potential. We applied our model to bone marrow aspirate samples collected at time of study enrollment pre-transplant from 16 patients to identify private TAAs and TSAs, as well as antigens shared across patients. Plasma-specific antigen expression was further explored using single-cell immunoprofiling data from bone marrow tumor samples in a subset of 10 patients from this cohort. Results: We demonstrated a high occurrence of tumor-specific antigens and neoantigens in MM cells at the time of enrollment. Notably, 50% of the identified neoantigens exhibited shared antigenic mutations in more than 2 patients. Nineteen of these neoantigens were further prioritized and identified as highly immunogenic and highly expressed in MM malignant plasma cells from the matched single-cell experiments. Four of these neoantigens, typically expressed in immune-privileged tissues, including cancer-testis antigens (such as MAGEC1 and TPTE) and brain-specific genes (such as UCHL1), were found to be highly expressed in MM tumor cells. Notably, UCHL1 showed a median 80-fold higher expression in MM bone marrow samples compared to public healthy controls. We observed a substantial enrichment of highly shared tumor antigenic peptides (n=33) among up to 10 patients, originating from regions not typically highly expressed or translated, such as retroelements and downstream/upstream open reading frames (ORFs). A significant association was observed between TSA and TAA abundance at baseline and the extent of α/β clonotypic expansion of tumor-infiltrating T cells, as identified from bulk and single-cell immunoprofiling data. MHC I Dextramer technology with 10x Genomics is being employed to further validate the identified antigens and characterize antigen-specific expanded T cell clones in the studied MM patients post-vaccine. Conclusion: These findings highlight the role of the baseline tumor antigenic landscape in the cohort of patients subsequently randomized to undergo DC/MM vaccination or maintenance therapy alone. We identified personal and shared tumor antigens, with a minority observed in most patients studied. We will interrogate the expanded T cell clonotypes following vaccination to better understand the dominant epitopes being recognized and their association with clinical outcomes. Acknowledgments: Support for this study was provided by grants #U10HL069294 and #U24HL138660 to the Blood and Marrow Transplant Clinical Trials Network from the National Heart, Lung, and Blood Institute and the National Cancer Institute along with contributions by Celgene Corporation and the Multiple Myeloma Research Foundation Fellowship Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the above-mentioned parties.

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3' and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization. A combination of an FFPE nuclei preparation protocol and a probe-based transcriptomic profiling technique enables snRNA-seq characterization of archival human FFPE tissues, holding promise for retrospective studies involving aged clinical cohorts.

Introduction: In recent years, multiple myeloma (MM) research has increasingly focused on unraveling the complex interactions between malignant plasma cells and their surrounding bone marrow microenvironment (BMME). To further elucidate these interactions, we developed a comprehensive single-cell atlas of the MM BMME, integrating both coding and noncoding (ncRNA) transcriptional profiles. By correlating these profiles with specific cytogenetic abnormalities in myeloma cells, we aim to uncover how ncRNAs regulate the phenotypic and functional states of myeloma cells and, consequently, shape the cellular landscape of the BMME. A detailed understanding of how the noncoding transcriptome affects the cellular components and immune landscape of the BMME is essential for advancing our knowledge of MM pathogenesis and identifying novel biomarkers and therapeutic targets. Methods: Our atlas was constructed using single-cell RNA sequencing data from 481 samples of CD138neg cells sorted from bone marrow aspirates of MM patients enrolled in the MMRF CoMMpass study. This dataset includes accompanying whole-genome sequencing and survival data, enabling correlative analysis between transcriptional profiles, cytogenetic abnormalities, and patient outcomes. To generate a combined coding and non-coding transcriptome, we developed an expanded human reference genome by systematically merging mRNA and ncRNA transcripts from the LncBook2.0 and GENECODEv42 reference genomes. The sequencing data was aligned to this expanded genome, followed by quality control processing, batch correction, clustering, and supervised analysis. Results: The resulting coding and non-coding atlas comprises over 1.9 million cells, spanning immune, plasma, and stromal compartments. Remarkably, ncRNAs constituted 46% of the associated genes, including 22,102 long ncRNAs (lncRNAs) and 3,112 small ncRNAs. Subclustering analysis of the major immune compartments (B lymphoid, myeloid, and NK and T lymphoid) yielded 70 subclusters, with ncRNAs representing 24-38% of the top 100 differentially expressed genes within each compartment. This underscores the significant role of ncRNAs in distinguishing immune subpopulations. Moreover, examining the association between specific cytogenetic abnormalities and the immune composition of the BMME, 30 subclusters were found to be differentially abundant (P < 0.05) between cytogenetic abnormalities, supporting our hypothesis that myeloma cell genotypes significantly shape the immune microenvironment. For instance, patients with CCND1 amplification displayed an increase CD4+ central memory T cells with a decrease in CD8+ cytotoxic T cells. Similarly, patients with 1q21 gain exhibited an increase in TGFβ-stimulated monocytes, while a decrease in cytotoxic CD8+ T and CD56dim NK cells was observed in patients with 17p13 deletion. Next, examining which ncRNAs are associated with specific cytogenetic abnormalities, we identified 14 ncRNAs significantly differentially expressed across cytogenetic abnormalities. Increased expression of six of these ncRNAs were associated with poor overall survival both independently and in combination (P < 0.05, HR = 3.7). Notably, three of these ncRNAs have documented roles in cancer progression but have not been previously studied in the context of MM. Subsequently stratifying patients based on their enrichment of these outcome-associated ncRNAs revealed even stronger associations with immune composition, including an increase in CD4+ regulatory T cells (P < 0.05). This further supports our hypothesis that myeloma cell genotypes shape the BMME. Lastly, by constructing a gene regulatory network to predict the interactions between coding and noncoding RNAs, we illustrate the regulatory networks of ncRNAs modulating the phenotypes of myeloma and immune cell subpopulations. Conclusion: In summary, we present the first high-resolution transcriptomic atlas of the MM BMME, integrating both coding and noncoding RNAs. This study reveals distinct immune subpopulations associated with specific myeloma cell genotypes and highlights the central role of ncRNAs in modulating the phenotypic and functional states of both malignant and non-malignant cells in the MM BMME. Our findings emphasize the potential of ncRNAs as therapeutic targets and prognostic markers for MM, offering new avenues for research and clinical intervention.