Editor's Note: The conference brought together top oncology experts and research teams from around the world to deeply explore the lymphoma microenvironment and its predictive value for novel immunotherapy strategies, particularly CD19 Chimeric Antigen Receptor T-cell (CAR T) therapy response. Among them, a groundbreaking study led by Professor David Russler-Germain's team from Washington University School of Medicine, which defined Lymphoma Microenvironment Prototypes (LymphoMAPs) based on single-nucleus multi-omics data, provided critical insights for precisely identifying advantageous subgroups for CAR T treatment, drawing widespread attention from attendees.Challenges and Breakthroughs in Large B-cell Lymphoma Microenvironment Research
Large B-cell Lymphoma (LBCL), as a highly heterogeneous malignant hematological tumor, has complex pathogenesis and significant differences in prognosis. Although targeted therapy and the standard R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone) regimen have made some progress, the efficacy remains unsatisfactory. Particularly since the advent of rituximab, the biggest breakthrough in the LBCL treatment field has been anti-CD19 CAR T-cell therapy. However, unfortunately, most patients do not achieve lasting benefits from CAR T therapy. Therefore, there is an urgent need to deeply understand pre-treatment factors influencing CAR T response to identify resistance mechanisms and develop alternative therapeutic strategies. Previous studies have linked CAR T failure to infusion product characteristics, tumor somatic mutations, and lymphoma microenvironment features, but most were evaluated through bulk gene expression profiling. Early single-cell studies, while revealing the diversity of tumor-infiltrating T cells, often underestimated stromal and myeloid cell populations due to the use of single-cell suspensions. Thus, a comprehensive, large-scale single-cell characterization of the LBCL microenvironment to uncover reproducible patterns and gain deeper insights into lymphoma biology remains a pressing scientific challenge.
LymphoMAPs: Defining a New Paradigm for Lymphoma Microenvironment Based on Single-Nucleus Multi-Omics Data
To address this critical unmet scientific need, Dr. Michael Green and his team conducted a pioneering study, with Dr. Zooben Lee and Dr. CarTEX Singal as co-first authors, publishing their findings in Cancer Cell. This study utilized single-nucleus multi-omics RNA and ATAC sequencing technologies to comprehensively analyze 217 frozen Large B-cell Lymphoma samples (approximately half untreated, half relapsed or refractory) and 15 control samples, aiming to comprehensively define the cellular landscape of LBCL. The research team also performed whole-exome sequencing, low-pass whole-genome sequencing, and bulk RNA sequencing on most tumor samples. After data integration, they deeply explored the lymphoma microenvironment and its association with LBCL clinical and molecular features, as well as responses to first-line treatment and CAR T-cell therapy.
After rigorous quality control and filtering, a total of 1.8 million cells were used for downstream analysis. By integrating B-cell receptor sequences, DNA copy number variations, somatic mutations, and gene expression data, the research team identified malignant B cells in each sample with high confidence. The remaining cells were separated into major lineages based on clustering and canonical marker gene expression patterns, and further subclustered, ultimately identifying 71 transcriptionally defined cell subpopulations. Notably, this study successfully captured previously underestimated myeloid and non-hematopoietic cells, thereby constructing a comprehensive single-cell atlas of LBCL.
The research team employed Non-negative Matrix Factorization (NMF) clustering to characterize co-associated cell subpopulations and identified five cellular modules. Given the strong correlations between two pairs of modules (each pair containing one large and one small module), to avoid data overfitting, the study merged these modules, ultimately resulting in three Lymphoma Microenvironment Prototypes (LymphoMAPs): FMAC (Fibroblast and Macrophage Enriched): Accounting for approximately 37% of all tumors, characterized by significant scarcity of T cells and NK cells, and excessive enrichment of cancer-associated fibroblasts and macrophages. LN (Lymph Node-like): Accounting for approximately 33% of all tumors, characterized by the presence of naive and memory T cells, and enrichment of lymph node structural cells. TEX (T-cell Exhaustion): Accounting for approximately 30% of all tumors, characterized by an abundance of CD8 T cells expressing exhaustion markers. Although malignant B cells showed stronger proliferative capacity in FMAC-type tumors, there was no significant difference in the proportion of malignant B cells among different prototypes.
Association of LymphoMAPs with DLBCL Molecular Subtypes
To assess the association between LymphoMAPs and established DLBCL molecular subtypes, the research team used bulk RNA sequencing to determine Cell of Origin (COO) and Dark Zone characteristics, and combined whole-exome and low-pass whole-genome sequencing to determine lymphogen and DLBCL genetic subtypes. Since these molecular subtypes were primarily identified in newly diagnosed DLBCL, and more than half of the patients in this study cohort had relapsed/refractory LBCL, to overcome this limitation and explore associations in a larger cohort, the study analyzed publicly available gene expression data from over 2000 untreated DLBCL cases.
Through feature set selection from the present study’s samples, a naive Bayes classifier was developed to predict LymphoMAPs in the public data. The results showed a significant association between the TEX prototype and ABC (Activated B-cell-like) subtype, the LN prototype and GCB (Germinal Center B-cell-like) subtype, and the FMAC prototype and Dark Zone feature subtype, suggesting that the transcriptional state of tumor B cells may be related to the microenvironment prototypes. However, despite these associations between prototypes and malignant B cell transcriptional states, the study did not observe a strong association between prototypes and B-cell tumor genetic subtypes. No significant association was found in the single-cell samples of the present study’s cohort, and only very weak associations were observed in larger public datasets, including a lower proportion of TEX prototype tumors in the EZB or C3 genetic clusters.
Clinical Value of LymphoMAPs in Predicting R-CHOP First-Line Treatment and CAR T Efficacy
The study further evaluated the prognostic impact of LymphoMAPs and their association with previously published lymphoma microenvironment subtypes. In a multivariate analysis including 88 untreated patients from this study’s cohort and 1000 untreated patients receiving first-line R-chemo treatment from public data, the study found that International Prognostic Index (IPI), ABC Cell of Origin, Dark Zone features, and FMAC LymphoMAP were all independently and significantly associated with poorer prognosis. This indicates that LymphoMAP prototypes are associated with transcriptional subtypes and clinical features of LBCL, with the FMAC prototype showing a moderate but significant association with poorer prognosis after first-line R-chemo treatment.
More strikingly, since macrophage abundance, T-cell exhaustion, and T regulatory cell abundance have been independently confirmed in multiple previous studies to be associated with CAR T-cell therapy failure, and these characteristics are defining features of the TEX prototype, the research team hypothesized that patients with TEX prototype tumors might have a poor prognosis after CAR T-cell therapy. To test this hypothesis, the study analyzed published gene expression and prognostic data from 256 patients in the ZUMA-7 study (a randomized Phase III clinical trial comparing Axi-Cel CAR T with standard salvage chemotherapy + autologous transplant as second-line treatment for early relapsed or primary refractory LBCL).
The results showed that in the ZUMA-7 study, LymphoMAPs were evenly distributed between the two treatment arms. A Cox proportional hazards model was used to compare event-free survival (EFS) for CAR T versus standard treatment within each LymphoMAP or other available clinical or molecular subgroup. The TEX prototype was the only subgroup that did not show significant benefit from CAR T therapy compared to standard treatment, a result also confirmed in Kaplan-Meier curves and restricted mean survival time analysis. In contrast, patients with the LN prototype derived highly significant benefits from CAR T therapy, while patients with the FMAC prototype, although showing reduced benefit, still had statistically significant results. This finding highlights the critical role of LymphoMAPs in predicting CAR T efficacy, providing new biomarkers for achieving precise application of CAR T therapy.
In-depth Biological Mechanism Exploration of TEX Lymphoma Microenvironment
To further understand the underlying biological mechanisms of each prototype, the research team performed cell-cell communication analysis. Although the total T-cell abundance in the TEX prototype was similar to that in the LN prototype, T cells in TEX tumors were enriched with exhausted CD8 T cells, which expressed high levels of co-inhibitory receptor genes, including Tim-3, PD-1, CTLA-4, Lag-3, and TIGIT, as well as high levels of exhaustion or dysfunction-associated transcription factors such as TOX, PRDM1, and NFAT components. Furthermore, the enriched macrophage subpopulations in TEX tumors included M1 polarized macrophages and hyperactivated macrophages, which highly expressed PD-L1, SLAMF7, or other markers previously studied in acute and chronic inflammation.
The study used the NicheNet tool to further explore this phenomenon, and the results showed that PD-L1 was predicted to be the major ligand regulating these exhausted CD8 T cells. Hyperactivated macrophages were the main source of PD-L1 and other important ligands (e.g., CXCL9), which may drive the recruitment of CD8 T cells and subsequently lead to their exhaustion in TEX tumors.
Considering the importance of hyperactivated macrophages in the TEX prototype, the research team delved into the factors driving the hyperactivated macrophage phenotype. The results revealed an extensive network of interactions between malignant B cells, T cells, NK cells, and other myeloid cells, including HLA-F and LILRB1-2 interactions. NicheNet also identified Interferon-gamma (IFN-γ) as a top-ranked ligand driving hyperactivated macrophage signature genes, primarily originating from exhausted T cells.
These comprehensive observations highlight that the interaction between exhausted CD8 T cells and hyperactivated macrophages is a potential driving factor of the TEX prototype: macrophages provide co-inhibitory signals to drive T-cell exhaustion, while exhausted CD8 cells produce IFN-γ to further exacerbate macrophage hyperactivation. This vicious cycle collectively shapes the TEX microenvironment and may lead to resistance to CAR T-cell therapy.
Expert Opinion and Clinical Application Outlook
Professor David Russler-Germain summarized in his report: “This study successfully defined three lymphoma microenvironment prototypes using single-nucleus multi-omics data: the LN prototype, characterized by robust antigen-presenting cells and cytokines supporting naive and memory T cells; the T-cell depleted FMAC prototype; and the TEX prototype, associated with T-cell exhaustion. We found minimal association between LymphoMAPs and Large B-cell Lymphoma genetic subtypes, but significant associations with previously described transcriptional subtypes. These findings suggest that tumor cell genetics may not be the primary contributor to these microenvironment prototypes.”
Professor Russler-Germain further pointed out: “By applying our LymphoMAPs to published datasets, we discovered differential associations between the lymphoma microenvironment and patient prognosis, depending on the line of therapy and the treatment modality employed. Since it is not yet clear whether LymphoMAPs are stable between diagnosis and relapse, or whether they may be influenced by certain treatments, evaluating LymphoMAPs in uniformly treated cohorts, preferably in randomized trials with pre- and post-treatment biopsies, is crucial for optimizing their association with prognosis in different treatment settings.”
Conclusion
The discovery of Lymphoma Microenvironment Prototypes (LymphoMAPs) not only provides a new perspective for our in-depth understanding of the complex biology of LBCL but, more importantly, it points the way to precisely predicting the efficacy of CAR T-cell therapy. By identifying TEX subtype patients who do not significantly benefit from CAR T treatment, new therapeutic strategies can be developed in the future, such as combining checkpoint inhibitors to reverse T-cell exhaustion, or exploring other cellular therapy options. This breakthrough will undoubtedly accelerate the transition of lymphoma treatment from a “one-size-fits-all” approach to a new era of “precision,” ultimately benefiting a wide range of patients.
Submission/Interview Source: Oncology Outlook – Oncology News
