Spatially-resolved transcriptomics reveal macrophage heterogeneity and prognostic significance in diffuse large B-cell lymphoma.

Macrophages are abundant immune cells in the microenvironment of diffuse large B-cell lymphoma (DLBCL). Macrophage estimation by immunohistochemistry shows varying prognostic significance across studies in DLBCL, and does not provide a comprehensive analysis of macrophage subtypes. Here, using digital spatial profiling with whole transcriptome analysis of CD68+ cells, we characterize macrophages in distinct spatial niches of reactive lymphoid tissues (RLTs) and DLBCL. We reveal transcriptomic differences between macrophages within RLTs (light zone /dark zone, germinal center/ interfollicular), and between disease states (RLTs/ DLBCL), which we then use to generate six spatially-derived macrophage signatures (MacroSigs). We proceed to interrogate these MacroSigs in macrophage and DLBCL single-cell RNA-sequencing datasets, and in gene-expression data from multiple DLBCL cohorts. We show that specific MacroSigs are associated with cell-of-origin subtypes and overall survival in DLBCL. This study provides a spatially-resolved whole-transcriptome atlas of macrophages in reactive and malignant lymphoid tissues, showing biological and clinical significance.

Patterns of Oncogene Coexpression at Single-Cell Resolution Influence Survival in Lymphoma.

Cancers often overexpress multiple clinically relevant oncogenes, but it is not known if combinations of oncogenes in cellular subpopulations within a cancer influence clinical outcomes. Using quantitative multispectral imaging of the prognostically relevant oncogenes MYC, BCL2, and BCL6 in diffuse large B-cell lymphoma (DLBCL), we show that the percentage of cells with a unique combination MYC+BCL2+BCL6- (M+2+6-) consistently predicts survival across four independent cohorts (n = 449), an effect not observed with other combinations including M+2+6+. We show that the M+2+6- percentage can be mathematically derived from quantitative measurements of the individual oncogenes and correlates with survival in IHC (n = 316) and gene expression (n = 2,521) datasets. Comparative bulk/single-cell transcriptomic analyses of DLBCL samples and MYC/BCL2/BCL6-transformed primary B cells identify molecular features, including cyclin D2 and PI3K/AKT as candidate regulators of M+2+6- unfavorable biology. Similar analyses evaluating oncogenic combinations at single-cell resolution in other cancers may facilitate an understanding of cancer evolution and therapy resistance. SIGNIFICANCE: Using single-cell-resolved multiplexed imaging, we show that selected subpopulations of cells expressing specific combinations of oncogenes influence clinical outcomes in lymphoma. We describe a probabilistic metric for the estimation of cellular oncogenic coexpression from IHC or bulk transcriptomes, with possible implications for prognostication and therapeutic target discovery in cancer. This article is highlighted in the In This Issue feature, p. 1027.

Effective Killing of Acute Myeloid Leukemia by TIM-3 Targeted Chimeric Antigen Receptor T Cells.

Acute myeloid leukemia (AML) is an aggressive disease with poor outcomes, overwhelmingly due to relapse. Minimal residual disease (MRD), defined as the persistence of leukemic cells after chemotherapy treatment, is thought to be the major cause of relapse. The origins of relapse in AML have been traced to rare therapy-resistant leukemic stem cells (LSCs) that are already present at diagnosis. Effective treatment strategies for long-term remission are lacking, as it has been difficult to eliminate LSCs with conventional therapy. Here, we proposed a new approach based on the chimeric antigen receptor (CAR)-directed T lymphocytes, targeting T-cell immunoglobulin, and mucin domain 3 (TIM-3) to treat MRD in patients with AML. TIM-3 is selected as the target because it is highly expressed on AML blasts and LSCs in most subtypes regardless of the patient's genetic characteristics and treatment course. Moreover, it is absent in the normal hematopoietic stem cells, granulocytes, naïve lymphocytes, and most normal nonhematopoietic tissues. Using a naïve human Fab phage display library, we isolated an anti-human TIM-3 antibody and designed a second-generation anti-TIM-3. Our anti-TIM-3 CAR T cells exhibit potent antileukemic activity against AML cell lines and primary AML blasts, and in the mouse models. More importantly, we demonstrate efficient killing of the primary LSCs directly isolated from the patients. Hence, eradication of the LSCs present in the MRD by anti-TIM-3 CAR T-cell therapy following the first-line treatment may improve the clinical outcomes of patients with AML.

Clinical impact of the cell-of-origin classification based on immunohistochemistry criteria and Lymph2Cx of diffuse large B-Cell lymphoma patients in a South-east Asian population: a single center experience and review of the literature.

Background: Previous studies in Western populations, using immunohistochemistry (IHC) methods to subtype diffuse large B-cell lymphoma (DLBCL), suggest that germinal center B-cell lymphomas (GCBs) have improved outcomes. However, data in Asians have been limited and conflicting. This study aims to evaluate the prognostic impact of cell-of-origin (COO) subtyping by IHC and Lymph2Cx in South-East Asian (SEA) DLBCL patients, and to summarize the existing literature.Methods: A single-center retrospective analysis of 384 DLBCL patients diagnosed 2013-2018 who received Rituximab-based chemotherapy was performed. Hans and Lymph2Cx were used to assign COO and correlated with outcomes.Results: International Prognostic Index (IPI) score was associated with overall survival (OS) and progression-free survival (PFS). The 5-yr-OS for non-GCB versus GCB for COO by Hans was 70% versus 71% p=0.39, while 5-yr-OS for ABC versus GCB for COO by Lymph2Cx was 74% versus 92% p=0.19. The 5-yr-PFS for non-GCB versus GCB for COO by Hans was 65% versus 70% p=0.26, while 5-yr-PFS for ABC versus GCB for COO by Lymph2Cx was 64% versus 86% p=0.07.Conclusions: IPI is reaffirmed to be relevant in the rituximab era. COO by Hans has no prognostic significance, while subtyping by Lymph2Cx trends toward GCBs having better PFS and OS.

Real world experience of R-CHOP with or without consolidative radiotherapy vs DA-EPOCH-R in the first-line treatment of primary mediastinal B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMBCL) is a distinct clinico-pathological subtype of diffuse large B-cell lymphoma with unclear prognostic factors and limited clinical data. Optimal treatment and role for radiotherapy is not fully defined. We performed a multicenter retrospective review of 124 patients with newly diagnosed PMBCL between 2001 and 2016. Treatment regimens were R-CHOP (n = 41), R-CHOP + RT (n = 37), and DA-EPOCH-R (n = 46). 6% (n = 3) in the DA-EPOCH-R group received RT. With a median follow up of 45 months, the overall 5-year OS and PFS was 89.4% and 82.4%, respectively. The type of chemo-radiotherapy regimen, B symptoms and Ann-Arbor staging showed a significant association with OS on univariate analysis but only B symptoms remained prognostic (P = 0.012) after multivariate analysis. The chemo-radiotherapy regimen, Japanese IPI and Ann-Arbor stage was significantly associated with PFS in univariate analysis, but only chemo-radiotherapy regimen remained significant (P = 0.02) after multivariate analysis. Patients who received R-CHOP + RT or DA-EPOCH-R had better PFS than those receiving R-CHOP alone, with 5-year PFS of 90% vs 88.5% vs 56%, respectively (P = 0.02). In the subgroup analysis of patients with bulk (n = 71), R-CHOP alone (n = 21) had inferior 5-year PFS 56.6% compared to those who received R-CHOP + RT (n = 23) 91.3% or DA-EPOCH-R (n = 27) 92.6% (P = 0.007). In contrast, in patients without bulk (n = 42), there was no impact of treatment regimen on PFS (P = 0.25). In conclusion, R-CHOP + RT and DA-EPOCH-R provide excellent outcomes in patients with PMBCL. In patients with bulky disease, the use of DA-EPOCH-R may be preferable as it allows omission of RT without reduction in efficacy.