Intratumor heterogeneity is intrinsic to cancer evolution and treatment resistance, although it is typically considered distinct from the differentiation processes driving cell-type and cancer-type diversity. Nodal B cell non-Hodgkin lymphomas are tied to distinct stages of B cell maturation. Through a single-cell multi-omic and spatial atlas of diffuse large B cell, mantle cell, follicular, and marginal zone lymphomas in addition to reactive lymph nodes in 51 patients, we uncovered co-existing B cell maturation states within the same tumors. These intratumor maturation states emerged from the same clone, revealing ongoing differentiation from the cell-of-origin. Maturation state composition varied across and within disease entities, with tumors encompassing mixed cell-of-origin clinical subtypes. Intratumor maturation states inhabited unique spatial niches, maintaining their maturation-associated cellular interactions and regulatory networks while harboring distinct genetic variant expression patterns. Our findings show that cell-type differentiation remains plastic in cancer and is central to tumor variation and evolution. ![Figure][1] ### Competing Interest Statement G.P.N. is a co-founder and stockholder of Akoya Biosciences, Inc. and inventor on patent US9909167 (On-slide staining by primer extension). [1]: pending:yes

Secondary central nervous system lymphoma (SCNSL) is a rare and difficult to treat type of Non-Hodgkin lymphoma characterized by systemic and central nervous system (CNS) disease manifestations. In this study, 124 patients with SCNSL intensively treated and with clinical long-term follow-up were included. Initial histopathology, as divided in low-grade, other aggressive, and diffuse large B-cell lymphoma (DLBCL), was of prognostic significance. Overall response to induction treatment was a prognostic factor with early responding DLBCL-SCNSL in comparison to those non-responding experiencing a significantly better progression-free survival (PFS) and overall survival (OS). However, the type of induction regime was not prognostic for survival. Following consolidating high-dose chemotherapy and autologous stem cell transplantation (HDT-ASCT), DLBCL-SCNSL patients had better median PFS and OS. The important role of HDT-ASCT was further highlighted by favorable responses and survival of patients not responding to induction therapy and by excellent results in patients with de novo DLBCL-SCNSL (65% long-term survival). SCNSL identified as a progression of disease within 6 months of initial systemic lymphoma presentation represented a previously not appreciated subgroup with particularly dismal outcome. This temporal stratification model of SCNSL diagnosis revealed CNS progression of disease within 6 months as a promising candidate prognosticator for future studies.

The COVID-19 pandemic threatens patients with a compromised immune and endothelial system, including patients who underwent allogeneic stem cell transplantation (alloSCT). Thus, there is an unmet need for optimizing vaccination management in this high-risk cohort. Here, we monitored antibodies against SARS-CoV-2 spike protein (anti-S1) in 167 vaccinated alloSCT patients. Humoral immune responses were detectable in 81% of patients after two vaccinations with either mRNA-, vector-based, or heterologous regimens. Age, B-cell counts, time interval from vaccination, and the type of vaccine determined antibody titres in patients without systemic immunosuppression (sIS). Similar to a healthy control cohort, mRNA vaccine-based regimens induced higher titres than vector-based vaccines. Patients on two or more immunosuppressants rarely developed immunity. In contrast, 62% and 45% of patients without or on only one immunosuppressant, respectively, showed a strong humoral vaccination response (titre > 100). Exacerbation of cGVHD upon vaccination was observed in 6% of all patients and in 22% of patients receiving immunosuppression for cGVHD. cGVHD exacerbation and low antibody titres were both associated with higher angiopoietin-2 (ANG2) serum levels. In conclusion, mRNA-based vaccines elicit strong humoral responses in alloSCT patients in the absence of double sIS. Biomarkers such as ANG2 might help with weighing cGVHD risk versus beneficial responses.

Topic: 20. Lymphoma Biology & Translational Research Background: Intratumor heterogeneity (ITH) is increasingly recognized as an essential factor in the variability of B cell non-Hodgkin lymphoma (B-NHL) patient outcomes. It is well established that distinct nodal B cell non-Hodgkin lymphoma (NBNHL) entities originate from different stages of the humoral affinity maturation process, but the drivers of ITH are not well understood. Aims: In this study, we aimed to characterize ITH in B-NHL and investigate its relationship with the nodal B cell affinity maturation process. Methods: 51 diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and non-malignant reactive lymph node (rLN) samples from distinct patients were sequenced with CITE-Seq (coupling single-cell RNA-seq with surface epitope profiling). Transcription factor activities and copy number variants (validated against targeted DNA sequencing) were inferred computationally from gene expression, while B cell receptor clones were determined with VDJ profiling. Spatial relationships were determined with highly multiplexed immunochemistry (CODEX). Informed by gene expression signatures from RNA sequencing of FACS-identified nodal B cell maturation states, a CITE-Seq reference map was constructed in reactive lymph nodes. This reference map was used to classify maturation states in the malignant samples with logistic regression, which were verified and refined by their maturation marker profiles. Using the shared features between CITE-Seq and CODEX panels, maturation states were similarly classified in the spatial data and their microenvironments were characterized with cellular neighborhood analysis. (Fig A) Results: As in the non-malignant context, variability in maturation states was also observed among malignant cells in most NBNHL tumors. BCR-sequencing and light chain restriction confirmed that different intra-tumor maturation states observed in samples were monoclonal, and therefore arose from the same cell of origin. Although commonalities in composition existed between samples within entities, such as an abundance of germinal center (GC) states in DLBCL and memory states in MZL, we observed high variability in maturation state proportions between samples. For example, ABC DLBCL and FL tumors frequently contained both GC and post-GC subpopulations. Transcription factor mediators of the B cell maturation process remained active in malignancy. Copy number variation was observed between maturation states of the same tumor, indicating genetic variants can arise at sequential stages of the B cell maturation process in existent malignancy. Intratumor maturation states typically occupied distinct spatial niches. Although nodal architecture disruption and microenvironmental variation were observed across tumors, tumor cells often maintained proximity with follicular dendritic and follicular helper T cells, key mediators of the affinity maturation process. Summary/Conclusion: Our findings reveal that B cell maturation is not halted during malignancy, but remains a continuous and divergent process central to inter- and intratumor variation. Intratumor maturation states can be subject to genetic variation and occupy distinct spatial niches while retaining the cellular interactions and signals which enable the maturation process. (Fig B)Keywords: B cell development, B cell lymphoma, B cell subsets, OMICS

The antibody-drug conjugate polatuzumab vedotin (pola) has recently been approved in combination with bendamustine and rituximab (pola-BR) for patients with refractory or relapsed (r/r) large B-cell lymphoma (LBCL). To investigate the efficacy of pola-BR in a real-world setting, we retrospectively analyzed 105 patients with LBCL who were treated in 26 German centers under the national compassionate use program. Fifty-four patients received pola as a salvage treatment and 51 patients were treated with pola with the intention to bridge to chimeric antigen receptor (CAR) T-cell therapy (n = 41) or allogeneic hematopoietic cell transplantation (n = 10). Notably, patients in the salvage and bridging cohort had received a median of 3 prior treatment lines. In the salvage cohort, the best overall response rate was 48.1%. The 6-month progression-free survival and overall survival (OS) was 27.7% and 49.6%, respectively. In the bridging cohort, 51.2% of patients could be successfully bridged with pola to the intended CAR T-cell therapy. The combination of pola bridging and successful CAR T-cell therapy resulted in a 6-month OS of 77.9% calculated from pola initiation. Pola vedotin-rituximab without a chemotherapy backbone demonstrated encouraging overall response rates up to 40%, highlighting both an appropriate alternative for patients unsuitable for chemotherapy and a new treatment option for bridging before leukapheresis in patients intended for CAR T-cell therapy. Furthermore, 7 of 12 patients with previous failure of CAR T-cell therapy responded to a pola-containing regimen. These findings suggest that pola may serve as effective salvage and bridging treatment of r/r LBCL patients.

Introduction The antibody-drug conjugate polatuzumab vedotin (Pola) has recently been approved in combination with bendamustine and rituximab (Pola-BR) for patients with r/r diffuse LBCL (DLBCL). Methods To characterize the efficacy of Pola-BR in a real-world setting, we retrospectively analyzed data from 97 patients with r/r LBCL who were treated with Pola in 24 German centers within the national CUP. Clinical baseline and follow-up (FU) data were collected by chart review and summarized descriptively. Progression-free survival (PFS) and overall survival (OS) were analyzed using Kaplan-Meier and Cox regression methods. Fisher's exact test was used to compare categorical factors between groups of patients. Results 97 patients with LBCL (DLBCL n=90, High-grade B-cell lymphoma n=6, Primary mediastinal B-cell lymphoma n=1) were included as of July 22nd, 2020. 49 patients were treated with Pola as bridging concept to immunotherapies (bridging cohort: chimeric antigen receptor T-cells (CART) n=39, allogeneic stem cell transplantation (alloSCT) n=9, bispecific antibodies n=1), and 48 patients were treated with Pola in palliative intention (palliative cohort). Within the bridging cohort the median age was 61 years (range: 22-82). Patients were heavily pretreated with a median of 3 treatment lines (range: 2-6). 84% (41/49 patients) had been refractory to their last treatment line, and 31% had failed an autologous stem cell transplantation. Notably, 14% and 10% of patients had failed prior CART and alloSCT, respectively, and were planned for the alternate cellular immunotherapy. Based on an individual decision, patients were treated with Pola-Rituximab (Pola-R, n=20), Pola-chemotherapy (Pola-chemo, BR n=25; R-CHP n=1) or Pola-monotherapy (Pola-mono, n=3). With a median of 2 Pola cycles (range 1-6), overall response rate (ORR) of all evaluable patients was 33% (15/46 patients) including patients with complete response (CR n=1), partial response (PR n=9) and clinical response (n=5). Although not significant, ORR tended to be better in patients treated with Pola-chemo versus Pola-R/Pola-mono (ORR: 42% versus 20%, Fishers test p=0.1). 11 of these 15 responders (24% of the entire bridging cohort) proceeded to CART or alloSCT, while 4 responders (8% of entire bridging cohort) experienced fast progression after their initial response and were referred to best supportive care. 15 of 31 non-responders (33% of entire bridging cohort) underwent immunotherapy with either stable disease (n=6), mixed response (n=2), or progression on Pola (n=7). The remaining 16 patients (35% of entire bridging cohort) were all refractory to Pola and either received alternative salvage treatments which enabled 8 further patients to proceed to the intended immunotherapy, or best supportive care. Taking the effects of CART or alloSCT into account, median OS from initiation of Pola treatment was 8.2 months (median FU 7.2 months, Fig. 1A). The palliative cohort tended to be older than the bridging cohort with a median age of 73.5 years (range: 37-86, p<0.001). Patients were pretreated with a median of 3 treatment lines (range: 2-8), and 85% (41/48 patients) had been refractory to their last treatment line. Patients in the palliative cohort were treated with a median of 4 Pola cycles (range: 1-9). 65% received Pola-chemo (BR, n=30; R-Gemcitabine, n=1) and 35% Pola-R. The CR rate and ORR was 19% (9/48) and 56% (27/48), respectively. The 6-month PFS and OS from initiation of Pola was 36% and 51%, respectively (median FU of 9.7 months, Fig. 1B). Again, ORR and OS tended to be better in patients treated with Pola-chemo versus Pola-R (ORR: 61% versus 47%, Fishers test p=0.4; median OS 7.2 versus 4 months, HR 0.8, 95%CI 0.4-1.9, p=0.7). In univariate analysis, failure to respond to the last treatment line predicted inferior PFS (HR 2.4, 95%CI 1.2-5.0 p=0.02) and OS (HR 2.5, 95%CI 1.2-5.4 p=0.02). Patients with more than two prior treatment lines in total tended to have a shorter PFS (HR 2.0, 95% CI 0.9-4.5, p=0.1) and OS (HR 1.8, 95% CI 0.8-4.0, p=0.2), although significance was not reached. Conclusion Pola permits effective bridging to CART and alloSCT in r/r LBCL. In the palliative setting, Pola represents an effective salvage option for patients with transplantation-ineligible r/r LBCL. Compared to the approval study, the inferior outcome of the patients of this real-world analysis might be explained by their more advanced disease course. Disclosures Duell: Morphosys: Research Funding. Kerkhoff:BMS: Honoraria. Leng:Roche: Other: lecture fee; Celgene: Other: traveling expenses and congress attendance fee. Holtick:Miltenyi Biotec B.V. & Co. KG: Honoraria. Mayer:Amgen: Honoraria, Other: travel grants; Abbvie: Other: travel grants; Novartis: Honoraria; Roche: Honoraria. Hüttmann:Celgene: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Gilead: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Roche: Other: Travel expenses; Seattle Genetics: Research Funding; University Hospital Essen, University of Duisburg-Essen, Essen, Germany: Current Employment; Lead Discovery Center GmbH: Consultancy. Brunnberg:Gilead: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: travel grants; MSD: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Amgen: Other: Travel grants. Bullinger:Menarini: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Hess:Roche: Research Funding; Celgene: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Genmab: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; EUSA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Morphosys: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Mueller-Tidow:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Deutsche Krebshilfe: Research Funding; BMBF: Research Funding; Wilhelm-Sander-Stiftung: Research Funding; Jose-Carreras-Siftung: Research Funding; Bayer AG: Research Funding; Daiichi Sankyo: Research Funding; BiolineRx: Research Funding; Janssen-Cilag Gmbh: Membership on an entity's Board of Directors or advisory committees; Deutsche Forschungsgemeinschaft: Research Funding. Lenz:Verastem: Research Funding; AQUINOX: Research Funding; BMS: Consultancy; AstraZeneca: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Agios: Research Funding; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy; Morphosys: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Dreger:Roche: Consultancy, Speakers Bureau; Neovii: Research Funding; AbbVie: Consultancy, Speakers Bureau; AstraZeneca: Consultancy; Gilead: Consultancy, Speakers Bureau; Janssen: Consultancy; Novartis: Consultancy, Speakers Bureau; Riemser: Consultancy, Research Funding, Speakers Bureau. Dietrich:Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees.