The utility of circulating tumor DNA (ctDNA) for mutational genotyping, pretreatment prognostication, and assessment of molecular response is well established in patients with aggressive large B-cell lymphoma (LBCL). Here, we have applied targeted panel and duplex sequencing of plasma ctDNA to study copy number aberrations (CNAs) along with mutational landscapes in 123 uniformly treated patients with high-risk LBCL. We find a robust correlation between targeted and whole-genome sequenced CNA landscapes (R = 0.81) and identify CNAs in the ctDNA in 76% of the patients above the limit of detection. We describe the most frequently affected genomic regions, their interactions with diagnostic and genetic subtypes, and associations with overall and progression-free survival. Specifically, we show how ctDNA profiling of TP53 loss outperforms fluorescence in situ hybridization (FISH)-based TP53/17p analysis in risk assessment, independent of clinical risk factors and ctDNA concentration. We validate key findings of prognostic tumor fraction and TP53 loss in an independent LBCL cohort. Furthermore, we detect dynamic shifts between the fractions of lymphoma clones by assessing CNAs and mutations in the ctDNA at disease progression. These findings demonstrate the potential of minimally invasive, targeted CNA analysis in resolving the molecular heterogeneity of LBCLs.

Concerns about treatment-related fertility impairment are common among young adult lymphoma survivors. Evidence on the impact of treatment for aggressive non-Hodgkin lymphoma (NHL) on future childbearing is limited, although many individuals of reproductive age are affected. This matched cohort study identified male (18-52 years) and female (18-40 years) patients with aggressive NHL between 2007 and 2018 in Sweden and the south-east health-care region in Norway, and matched lymphoma-free comparators. Adjusted hazard ratios (aHRs) comparing childbirth rates in patients treated with primary intensive chemoimmunotherapy (CIT) (e.g., R-CHOEP and R-DA-EPOCH) to standard-of-care (R-CHOP), and to comparators, were estimated using Cox regression. Among 1037 young survivors of aggressive NHL, childbirth rates did not differ significantly by treatment intensity (intensive vs. standard CIT) in females (aHR: 0.61, 95% CI: 0.32-1.13) or males (aHR: 1.20, 95% CI: 0.68-2.09), overall or when stratified by time since diagnosis (< 3 and 3-10 years). However, females treated with intensive CIT had lower childbirth rates than comparators during the first 3 years after diagnosis (aHR: 0.52, 95% CI: 0.30-0.88), but not thereafter (aHR: 1.06, 95% CI: 0.57-1.96). A similar pattern was not seen in males. In Sweden, eight children (three with support from assisted reproductive techniques) were born after stem cell transplantation. In conclusion, childbirth rates did not differ by CIT intensity among survivors of aggressive NHL. Although rates were temporarily reduced in females during the treatment and recovery period after diagnosis, subsequent rates were similar to comparators. These findings provide reassurance, while highlighting the importance of early and ongoing fertility counseling.

Abstract Immunotherapy has transformed treatment for many cancers. In the aggressive and genetically heterogeneous diffuse large B-cell lymphoma (DLBCL), CD19 CAR T-cell therapy is highly effective, whereas immune checkpoint blockade has shown limited benefit. Loss of MHC expression is a common mechanism to escape T-cell cytotoxicity, and loss of MHC class I (MHC-I) and II are frequent in DLBCL. We applied imaging mass cytometry to diagnostic biopsies from younger, high-risk DLBCL patients to map the tumor microenvironment (TME) spatial architecture in relation to tumor cell MHC expression, mutational status, transcriptomic and proteomic profiles. Neighborhood analyses identified four TME subtypes: immune-cell depleted and three immune-infiltrated types (mixed, CD4 T cell-rich, CD8 T-cell/macrophage-rich). Depleted cases had shorter overall survival ( p = 0.033) and increased expression of proteins involved in DNA replication and proliferation markers compared to infiltrated cases. Tumor cell MHC-I expression was heterogeneous. Cases with low frequency of MHC-I pos tumor cells were enriched for the depleted TME type. MHC-I pos tumor cells were surrounded by CD4 and CD8 T cells and M1 macrophages, whereas MHC-I neg tumor cells were closer to other MHC-I neg tumor cells. These findings suggest that TME-based classification incorporating tumor cell MHC-I status may improve individualized immunotherapy selection.

Posttransplant lymphoproliferative disorder (PTLD) poses a serious challenge in kidney transplant recipients. Epstein-Barr virus (EBV)-seronegative recipients have a significantly increased risk of PTLD, but few studies have investigated risk factors for PTLD in EBV-seronegative recipients in the current era of immunosuppression. This cohort study from Norway and western Denmark included first-time kidney transplant recipients between 2007 and 2021 and estimated the cumulative incidence, risk, and prognosis of PTLD. In total, 80 of 5084 recipients developed biopsy-proven PTLD (median follow-up of 6.8 years). Two-year cumulative incidence of PTLD was 7.3% in EBV-seronegative adults and 14.1% in EBV-seronegative children. The age-adjusted hazard ratio (HR) for PTLD was 30.7 (95% CI, 13.9-67.9) in EBV-seronegative vs EBV-seropositive adults and 5.4 (95% CI, 1.1-26.9) in children. Recipients receiving induction therapy with antithymocyte globulin had an increased risk of PTLD (HR, 4.4; 95% CI, 1.8-10.6), while rituximab induction was associated with a lower risk of PTLD (HR, 0.20; 95% CI, 0.03-1.49). The age-adjusted mortality rate was higher in EBV-seronegative recipients with vs without PTLD (HR, 3.3; 95% CI, 1.3-8.3). In conclusion, the risk of PTLD in EBV-seronegative kidney transplant recipients is high in the contemporary era of immunosuppression. Induction therapy should be carefully considered in this high-risk population.

Treatment failure affects one-third of patients with aggressive large B-cell lymphoma (LBCL), and current tools are inadequate in determining remission. We investigated the added value of longitudinal ctDNA monitoring in plasma by duplex sequencing. Clearance of ctDNA after 2 or 4 cycles of immunochemotherapy identified patients with excellent survival, whereas elevated interim ctDNA levels among those with measurable residual disease (MRD) predicted refractory disease. Two-year progression-free survival rates based on end-of-therapy MRD varied greatly (94.3% vs 18.2%, negative and positive, respectively), and ctDNA analysis resolved most false radiological response assessments and revealed poor targeting of post-treatment interventions to MRD-positive cases. In surveillance, ctDNA was detectable at least 3 months before clinical relapse, while patients with isolated central nervous system relapses had the shortest plasma ctDNA lead times. Overall, ctDNA monitoring with ultra-sensitive duplex sequencing offers a dynamic molecular tool that can guide clinical decision-making in LBCL.

We report the 5-year analysis of tisagenlecleucel in 115 infused patients with relapsed/refractory (r/r) large B-cell lymphoma (LBCL) from the single-arm, open-label, multicenter, global, phase II JULIET trial (ClinicalTrials.gov identifier: NCT02445248), with a median follow-up of 74.3 months. The median duration of response (DOR) was not reached; the 60-month, relapse-free probability was 61% among responders. Higher relapse-free probability (DOR >70%) was observed in females and those with less than two baseline International Prognostic Index risk factors or with baseline stage I/II disease. The estimated probability of progression-free survival at 60 months was 28%. The probability of overall survival (OS) at 60 months was 32% for all infused patients and 56% for those achieving complete or partial response. Baseline characteristics associated with achieving a response at any time after infusion included relapsed versus refractory disease, one versus two or more bridging regimens, lactate dehydrogenase level ≤upper limit of normal (ULN) versus >ULN, and C-reactive protein levels <15 mg/L versus >15 mg/L. Baseline characteristics associated with long-term OS included lactate dehydrogenase ≤ULN and C-reactive protein <15 mg/L. No new safety signals or secondary T-cell malignancies were reported. These findings continue to support the curative potential of tisagenlecleucel in a subset of patients with r/r LBCL.

ABSTRACT:Epcoritamab is a subcutaneous CD3×CD20 bispecific antibody approved as monotherapy for relapsed/refractory (R/R) follicular lymphoma (FL). We evaluated fixed-duration epcoritamab with rituximab plus lenalidomide (R2) in R/R FL in arm 2 of EPCORE NHL-2 (phase 1b/2). Patients received epcoritamab (2 step-up doses, then 48-mg full doses) for up to 2 years, and R2 for up to 12 cycles (28 days per cycle). The primary end point was overall response rate (ORR) per investigator assessment (Lugano criteria). As of 21 September 2024, 108 patients received ≥1 epcoritamab dose in expansion (median follow-up, 28.2 months). Median age was 65 years; 57% had 1 previous line of therapy. ORR and complete response (CR) rate were 96% and 88%, respectively; CR rates in patients with high-risk features were 90% (primary refractory), 82% (refractory to anti-CD20 and an alkylating agent), and 83% (disease progression within 24 months of first-line therapy). Two-year estimates for remaining in CR, progression-free survival, overall survival, and not starting next antilymphoma therapy were 82%, 76%, 90%, and 84%, respectively. Minimal residual disease negativity was observed in 86% of evaluable patients (clonoSEQ assay). Common treatment-emergent adverse events (TEAEs) included neutropenia (65%), COVID-19 (59%), and cytokine release syndrome (CRS; 51%). Grade ≥3 TEAEs occurred in 87% of patients; 5 had grade 5 TEAEs (all COVID-19). CRS events were mostly low grade (grade 1, 38%; grade 2, 11%; grade 3, 2%), all resolved, and none led to epcoritamab discontinuation. Fixed-duration epcoritamab plus R2 demonstrated deep, durable responses with manageable safety and favorable outcomes in R/R FL, irrespective of risk features. This trial was registered at www.ClinicalTrials.gov as #NCT04663347.

Circulating tumor DNA (ctDNA) has emerged as a valuable tool in revealing molecular heterogeneity and risk factors of large B-cell lymphoma (LBCL). Several ctDNA estimates, such as variant profiling and minimal residual disease kinetics, have been thoroughly dissected, while multiple other molecular aspects accessible in the liquid biopsy (LB), such as DNA copy number aberrations (CNAs) and changes in serum proteome, remain to be disclosed. We studied CNAs from the ctDNA of LBCL patients treated in a Nordic phase II trial to expand the clinically applicable LB repertoire. The patients (n=123) included in the study were young (18-64 years), had clinically high-risk LBCL and were treated in a Nordic LBC-06 trial with dose-dense immunochemotherapy. A duplex adapter informed 750 kb gene panel was applied to pretherapeutic plasma ctDNA samples, and variants and CNAs were called using Illumina's DRAGEN pipeline. Clinical data, ctDNA variants (n=123), and serum protein levels, including inflammatory (PD-L1, IL10, IL18, and CXCL9) and subtype-specific (TARC and TACI) markers measured with Luminex assay (n=122) were explored in the context of CNAs. Profiling of ctDNA revealed CNAs (range 1-38) in 80 (65%) patients. The detection of CNAs was affected by ctDNA variant allele frequency. The most recurrently altered regions included gains in the REL (2:p16) and BCL6 (3:q27) genes and losses in the CDKN2A/B (9p:21) and histone (6p:22) loci. Comparison of TP53 status (loss in 11 (9%) patients) with clinical data uncovered that TP53 assessment from the LB was more predictive than routinely performed FISH-informed TP53 analysis for overall survival (OS; HR 7.23, p = 0.001 vs HR 2.84, p = 0.09). Moreover, loss of TP53 correlated with P53 immunohistochemistry positivity (p < 0.001) and remained prognostic for OS in multivariable analysis together with age-adjusted International Prognostic Index (HR 8.49, p < 0.001). Correlation with serum protein profiles showed associations between high levels of inflammatory proteins and CNAs in key oncogenic pathways. These included gains in the NFκB pathway genes MALT1 (p < 0.001) and REL (p <0.01), immune evasion genes, such as CD274 (p < 0.05), and losses in the tumor suppressor genes PRDM1 (p < 0.05) and CDKN2A (p < 0.05). Furthermore, extended LB analysis of serum TARC levels, ctDNA variants, and CNAs revealed a subgroup of patients initially diagnosed as diffuse LBCL with a primary mediastinal B-cell lymphoma-like molecular fingerprint. Together, we show that minimally invasive CNA profiling improves risk stratification and molecular characterization of LBCL subtypes. By integrating several LB approaches, we present CNA landscapes that reflect immune evasion and a more aggressive clinical course of the disease and provide potential actionable vulnerabilities for clinical decision-making. Maare Arffman, Leo Meriranta, Judit Jørgensen, Marja-Liisa Karjalainen-Lindsberg, Klaus Beiske, Mette Pedersen, Kristina Drott, Øystein Fluge, Sirkku Jyrkkiö, Peter Brown, Harald Holte, Sirpa Leppä. Copy number aberrations in the ctDNA uncover clinical and biological heterogeneity in aggressive B-cell lymphomas [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 1942.

Abstract: Molecular characterization of high-grade B-cell lymphoma, not otherwise specified (HGBCL-NOS), is hindered by its rarity, evolving definition, and poor diagnostic reproducibility. To address this challenge, we analyzed 92 HGBCL-NOS tumors collected across Lymphoma/Leukemia Molecular Profiling Project sites. Leveraging comparison cohorts of diffuse large B-cell lymphoma, NOS (DLBCL-NOS) and Burkitt lymphoma (BL), and molecular frameworks described in these entities, our analysis revealed a heterogenous molecular landscape, reminiscent of DLBCL-NOS but with an enrichment of BL features. By cell-of-origin classification, 59% were germinal center B-cell–like (GCB), and 25% were activated B-cell–like (ABC). LymphGen, a genetic classifier for DLBCL-NOS, assigned a genetic subtype to 34% of HGBCL-NOS. Although classification rate was lower than in DLBCL-NOS (66%), assigned subtypes spanned the spectrum of LymphGen classes, including 31% of ABCs classified as MCD. Features differentiating HGBCL-NOS from DLBCL-NOS included MYC rearrangement (47% vs 6%); dark zone signature (DZsig) expression (45% vs 7%); and more frequent mutation of ID3, MYC, CCND3, and TP53, all common to BL. A genetic classifier that differentiates DLBCL-NOS from BL classified 53% of DZsig+ tumors as BL-like, and those classified as DLBCL-like were frequently BCL2-rearranged. Among DZsig− GCB tumors, 95% were DLBCL-like. Centralized pathology review reclassified almost half of tumors as DLBCL-NOS but did not identify a more homogenous HGBCL-NOS population, with no difference in features between confirmed and reclassified tumors. In conclusion, molecular testing enables a subset of HGBCL-NOS to be assigned to established categories. Based on rarity and diagnostic challenges, broader inclusion of HGBCL-NOS should be considered in biomarker-driven DLBCL trials.

Background:Allogeneic stem cell transplantation (ASCT) has the potential to cure patients with lymphoma when conventional treatment fails, but it is associated with a risk of serious complications. We present treatment outcomes atter two years for 120 patients who underwent ASCT in the period 1 January 2011-30 September 2021. Material and method:The study consists of patients who underwent their first transplant at Oslo University Hospital. Several changes were made to the transplantation protocols during this period, including the introduction of prophylaxis for graft-versus-host disease (GVHD) with antithymocyte globulin in October 2016. Comparisons were made of survival and complications for patients transplanted in the period 1 January 2011-30 September 2016 (group 1: 65 patients) and 1 October 2016-30 September 2021 (group 2: 55 patients). Results:After two years, 42/65 (65 %) of patients in group 1 and 39/55 (71 %) of patients in group 2 were alive. Transplant-related mortality was 18/65 (28 %) in group 1 and 8/55 (15 %) in group 2. Five patients in group 1 and seven patients in group 2 died following a relapse. One patient in group 2 died from an unknown cause. A total of 13/65 (20 %) in group 1 and 4/55 (7 %) in group 2 developed severe acute GVHD. Among patients who survived at least 100 days, 24/61 (39 %) in group 1 and 3/51 (6 %) in group 2 developed severe chronic GVHD. Interpretation:Complications after ASCT for lymphoma have been significantly reduced since 2016.

Survival rates for patients with high-risk large B-cell lymphoma (LBCL), particularly those with biological risk factors, remain inadequate. We conducted a biomarker-driven phase II trial involving 123 high-risk patients aged 18-64 with LBCL. Based on their biological risk profiles, patients received either R-CHOEP-14 (without risk factors) or DA-EPOCH-R-based regimens (with risk factors). Biological high-risk factors included C-MYC translocation, C-MYC and BCL2 co-translocation, 17p/TP53 deletion, co-expression of MYC and BCL2, and P53 and/or CD5 immunopositivity. Additionally, we evaluated circulating tumor DNA (ctDNA) kinetics during therapy. Sixty-one patients (50%) were classified into biologically high-risk group. Three-year failure-free survival and overall survival rates for the entire study population were 79% and 88%, respectively. DA-EPOCH-R did not improve survival compared to our previous trial, where patients with the same biological risk factor criteria received R-CHOEP-14-based therapy. High pretreatment ctDNA levels, 17p/TP53 deletion, and TP53 mutations were associated with worse outcomes. In contrast, ctDNA negativity at the end of therapy (EOT) was indicative of a cure and effectively addressed false residual PET positivity. The findings demonstrate promising survival for high-risk LBCL patients, aside from those with TP53 aberrations, high ctDNA levels, and/or EOT ctDNA positivity.

Nodal follicular helper T-cell (T FH) lymphoma of the angioimmunoblastic (AITL) subtype has a dismal prognosis. Using whole-exome sequencing (n = 124), transcriptomic (n = 78), and methylation (n = 40) analysis, we identified recurrent mutations in known epigenetic drivers (TET2, DNMT3A, IDH2 R172 ) and novel ones (TET3, KMT2D). TET2, IDH2 R172 , DNMT3A co-mutated AITLs had poor prognosis (p < 0.0001). Genes regulating T-cell receptor (TCR) signaling (CD28, PLCG1, VAV1, FYN) or activation (RHOA G17V ) or regulators of the PI3K-pathway (PIK(3)C members, PTEN, PHLPP1, PHLPP2) were mutated. CD28 mutation/fusion was associated with poor prognosis (p = 0.02). WES of purified, neoplastic T-cell (CD3+PD1+) demonstrated high concordance with whole tumor biopsies and validated the presence of TET2 and DNMT3A in tumor and non-lymphoid cells, but other mutations (CD28, RHOA G17V , IDH2 R172 , PLCG1) in neoplastic cells. Integrated DNA-methylation and mRNA expression analysis revealed epigenetic alterations in genes regulating TCR, cytokines, PI3K-signaling, and apoptosis. RNA-seq analysis identified fusion transcripts regulating TCR-activation (8%), revealed a restricted TCR-repertoire (α = 87%, β = 72%), and showed the presence of Epstein-Barr virus transcriptome (73%). GEP demonstrated the association of B-cells or dendritic cells in the tumor milieu with prognosis (p < 0.01). RNA-seq and WES analysis of 12 AITL-patient-derived-xenografts (PDX) showed that bi-allelic TET2 and DNMT3A mutations or sub-clonal mutations (PLCG1, PHLPP2) propagated in sequential passages, and gene signatures related to T FH and T CM (central-memory) were well-maintained through passages. Gene expression signatures associated with late PDX passages (3rd-5th) were enriched with proliferation and metabolic reprogramming-related genes and predicted prognosis in an independent AITL series. Low PHLPP2 mRNA expression predicted poor prognosis (p = 0.05) and engineered PHLPP2 or TET2 loss in CD4+ T-cells showed enhanced PI(3)K activation, thus uncovering a therapeutic target for clinical trials.

BackgroundBiological heterogeneity of large B cell lymphomas (LBCLs) is poorly captured by current prognostic tools, hampering optimal treatment decisions.MethodsWe dissected the levels of 1,463 serum proteins in a uniformly treated trial cohort of 109 patients with high-risk primary LBCL (ClinicalTrials.gov: NCT01325194) and correlated the profiles with molecular data from tumor tissue and circulating tumor DNA (ctDNA) together with clinical data.FindingsWe discovered clinically and biologically relevant associations beyond established clinical estimates and ctDNA. We identified an inflamed serum protein profile, which reflected host response to lymphoma, associated with inflamed and exhausted tumor microenvironment features and high ctDNA burden, and translated to poor outcome. We composed an inflammation score based on the identified inflammatory proteins and used the score to predict survival in an independent LBCL trial cohort (ClinicalTrials.gov: NCT03293173). Furthermore, joint analyses with ctDNA uncovered multiple serum proteins that correlate with tumor burden. We found that SERPINA9, TACI, and TARC complement minimally invasive subtype profiling and that TACI and TARC can be used to evaluate treatment response in a subtype-dependent manner in the liquid biopsy.ConclusionsAltogether, we discovered distinct serum protein landscapes that dissect the heterogeneity of LBCLs and provide agile, minimally invasive tools for precision oncology.FundingThis research was funded by grants from the Research Council of Finland, Finnish Cancer Organizations, Sigrid Juselius Foundation, University of Helsinki, iCAN Digital Precision Cancer Medicine Flagship, Orion Research Foundation sr, and Helsinki University Hospital.

Introduction Plasmablastic lymphoma (PBL) is a rare aggressive subtype of lymphoma. It is more common in HIV positive patients, and is often associated with Epstein-Barr virus (EBV) and MYC rearrangements. PBL is characterized by markers of plasmacytic differentiation including CD138 and MUM1/IRF4, and loss of B-cell markers such as CD20, which suggests a cell of origin similar to that of multiple myeloma (MM) or activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL). The standard of care for PBL is CHOP-based chemotherapy but this is frequently not curative. Other than EBV-negative status, which defines a high-risk group, no other biological features have been discovered that are associated with outcomes. Methods Here, we provide a comprehensive analysis of the genomic and transcriptomic landscape of PBL using a collection of 176 exomes and genomes (n=55 new, contributed by Lymphoma/Leukemia Molecular Profiling Project [LLMPP] sites and centrally reviewed by the LLMPP pathology panel [WHO 2016]; n=121 previously published [PMID: 33225311, 33951889, 34465776, 34714908]), 65 transcriptomes (n=44 new; n=21 published) and 37 in situ single cell transcriptomic profiles (CosMx Spatial Molecular Imager) from archival diagnostic tissue biopsies. Somatic mutations (SNVs/Indels) were identified using an ensemble of four variant callers (Strelka2, Lofreq, Mutect2, SAGE). Salmon and DESeq2 were used to identify differentially expressed genes. This multi-omics dataset was compared to other known B-cell malignancies including 92 MM, 238 Burkitt lymphomas (BL), and 208 DLBCLs. Cell segmentation of the CosMx data was performed using Baysor and downstream analyses were conducted using the Seurat workflow. For functional validation, cell viability assays (CellTiter-Glo Luminescent Assay) were conducted in the PBL cell line, PBL-1, and 4 control DLBCL cell lines. Results Within the cohort, 63% (106/169) of primary PBL cases were EBV-positive (EBV+) and 57% (82/144) harbored MYC rearrangements (MYC+). When stratifying patients by EBV and MYC rearrangement status, mutations in different genes were enriched in each group: STAT3 occurred in 55% (29/53) of EBV+/MYC+ PBLs; NOTCH1 in 19% (7/36) of EBV+/MYC-; MYC in 24% (6/25) and TP53 in 40% (10/25) of EBV-/MYC+ tumors; TET2 was mutated in 38% (9/24) and KRAS in 25% (6/24) of EBV-/MYC- PBLs. EBV-/MYC+ tumors represented a subgroup of patients with dismal outcomes. The 2-year overall survival (OS) of these patients was 22% compared to 61%, 78%, and 54% of EBV+/MYC+, EBV+/MYC-, and EBV-/MYC- PBL patients, respectively (log-rank test p-adj. < 0.05). Consistent with previous studies, we observed recurrent mutations affecting JAK-STAT (STAT3, SOCS1, SOCS3, DUSP2) in 41% of cases and RAS-RAF (NRAS, KRAS, BRAF) signaling in 38% of all PBLs. In contrast to ABC-DLBCL, very few mutations occurred in the NF-κB signalling pathway. Accordingly, PBL showed marked downregulation of genes involved in B-cell receptor and NF-κB signaling. Consistent with the lack of reliance on these pathways, functional analyses in PBL-1 showed resistance to treatment to ibrutinib as well as PI3K- and MALT1-inhibitors. Moreover, constitutive expression of the IκBα super-repressor did not affect viability of PBL-1 cells. In situ single cell transcriptomic data analyses showed a sparse tumor microenvironment (TME) in PBL, with the primary non-malignant cell types being, on average, 10% macrophages, 7% NK/T cells, and 4% stromal cells of all represented cell types. EBV+/MYC+ tumors displayed a more abundant immune cell population with higher levels of macrophages (median 12%) and NK/T cells (median 9%), whereas EBV-/MYC- tumors were composed of the lowest levels of immune cells (median 4% macrophages and 1% NK/T cells). With respect to malignant cell phenotypes, we noted a novel population of SPP1-expressing cells that were enriched in EBV+/MYC+ tumors and a population of CD44-expressing cells that were enriched in EBV+/MYC- tumors. Conclusion Here we present novel subgroups of PBL with unique biology and clinical outcomes. We show that PBL does not rely on B-cell receptor signaling, instead relying on potentially targetable vulnerabilities in JAK-STAT, NOTCH, and RAS-RAF signaling according to EBV positivity and MYC rearrangement status. Lastly, we provide insight into the TME of PBL and demonstrate a novel population of SPP1 and CD44 expressing malignant cells in EBV+ tumors.

Flow cytometry is a powerful technology for high-throughput protein quantification at the single-cell level, widely used in basic research and routine clinical diagnostics. Traditionally, data analysis is carried out using manual gating, in which cut-offs are defined manually for each marker. Recent technical advances, including the introduction of mass cytometry, have increased the number of proteins that can be simultaneously assessed in each cell. To tackle the resulting escalation in data complexity, numerous new analysis algorithms have been developed. However, many of these show limitations in terms of providing statistical testing, data sharing, cross-experiment comparability integration with clinical data. We developed MetaGate as a platform for interactive statistical analysis and visualization of manually gated high-dimensional cytometry data with integration of clinical meta data. MetaGate allows manual gating to take place in traditional cytometry analysis software, while providing a combinatorial gating system for simple and transparent definition of biologically relevant cell populations. We demonstrate the utility of MetaGate through a comprehensive analysis of peripheral blood immune cells from 28 patients with diffuse large B-cell lymphoma (DLBCL) along with 17 age- and sex-matched healthy controls using two mass cytometry panels made of a total of 55 phenotypic markers. In a two-step process, raw data from 143 FCS files is first condensed through a data reduction algorithm and combined with information from manual gates, user-defined cellular populations and clinical meta data. This results in one single small project file containing all relevant information to allow rapid statistical calculation and visualization of any desired comparison, including box plots, heatmaps and volcano plots. Our detailed characterization of the peripheral blood immune cell repertoire in patients with DLBCL corroborate previous reports showing expansion of monocytic myeloid-derived suppressor cells, as well as an inverse correlation between NK cell numbers and disease progression.

Introduction: Epcoritamab, a CD3xCD20 bispecific antibody, has been approved as a single agent for the treatment of relapsed or refractory (R/R) diffuse large B-cell lymphoma and follicular lymphoma (FL) after ≥2 lines of systemic therapy based on results from the phase 1/2 EPCORE® NHL-1 trial (NCT03625037). For patients (pts) with R/R FL, rituximab + lenalidomide (R2) is an approved and widely accepted regimen based on results from the AUGMENT trial (overall response rate [ORR], 78%; complete response [CR] rate, 34%; estimated 2-y progression-free survival [PFS], 58%). There exists a clear need for improvement of outcomes, especially for pts with high-risk features, including disease progression within 24 mo of first-line chemoimmunotherapy (POD24) or primary/double-refractory disease, for whom there is no established standard of care. Previously, in arm 2 of the EPCORE NHL-2 trial (phase 1b/2; NCT04663347), fixed-duration epcoritamab + R2 showed encouraging antitumor activity and a manageable safety profile in a large population of pts with R/R FL, including high-risk pts (Merryman et al, ASCO 2023). Here, we present long-term follow-up beyond 2 y and minimal residual disease (MRD) analysis for the first time. Methods: Adults with R/R CD20+ FL received subcutaneous epcoritamab + R2 for up to 12 cycles (Cs; 28 d each). Epcoritamab was administered with a 2-step (0.16 and 0.8 mg) step-up dosing (SUD) regimen in C1 and 48-mg full doses either QW in C1-3, Q2W in C4-9, and Q4W in C≥10 (arm 2a) or QW in C1-2 and Q4W in C≥3 (arm 2b) for up to 2 y. MRD analysis was performed on peripheral blood mononuclear cell samples collected at prespecified time points (clonoSEQ® assay, Adaptive Biotechnologies) and quantified as tumor clones detected per 1 x 106 nucleated cells. The primary endpoint was ORR per Lugano criteria. Results: As of May 15, 2024, 111 pts with R/R FL had received epcoritamab 48 mg + R2. Median age was 65 y, 61% had stage IV disease, and 57% had only 1 line of prior treatment. Most pts had received alkylating agents (93%) and anthracyclines (64%); 3% had received prior CAR T. At a median follow-up of 25.3 mo (range, 2.4+ to 34.1), 17 pts (15%) were still on treatment, 41 (37%) completed treatment per protocol, and 53 (48%) discontinued treatment (progressive disease, n=18; AEs, n=22; pt withdrawal, n=7; death, n=1; COVID-19 control measure, n=1; and other reason, n=4 [investigator decision]). The ORR was 96%, and CR rate was 87%. CR rates were similarly high regardless of high-risk features: primary refractory (n=39)/non-primary refractory (n=72), 90%/86%; double refractory (n=39)/non-double refractory (n=72), 82%/90%; POD24 (n=42)/non-POD24 (n=69), 79%/93%. Estimated 24-mo PFS and overall survival rates were 70% and 90%, respectively. At 24 mo, an estimated 69% of responders remained in response (duration of response; DOR), and an estimated 75% of complete responders remained in CR (duration of CR; DOCR). MRD status was assessed in 73 evaluable pts; of these, 64 (88%) became MRD negative. With the majority of pts being enrolled and treated during the global COVID-19 pandemic, COVID-19 was reported in 57% of pts and led to epcoritamab discontinuation in 11% of pts; COVID-19 events included 5 grade (G) 5 treatment-emergent AEs (TEAEs; COVID-19, n=3; COVID-19 pneumonia, n=2). The other most common TEAEs were neutropenia (62%) and CRS (51%). CRS events with this 2-step SUD regimen were mostly low grade (38% G1, 12% G2, 2% G≥3) and primarily occurred following the first full dose on C1D15; all resolved, and none led to epcoritamab discontinuation. ICANS occurred in 1 pt (G1) and resolved. Conclusions: With more than 2 y of follow-up, fixed-duration epcoritamab + R2 continued to show deep and durable responses (CR rate, 87%; estimated 24-mo DOCR, 75%) in pts with R/R FL, irrespective of high-risk features. Considering limitations of cross-trial comparisons, these results (estimated 2-y PFS, 70%) compare favorably with those reported for R2 alone in the AUGMENT trial (estimated 2-y PFS, 58%). The depth of responses was underscored by MRD negativity in 88% of evaluable pts. There were no new safety findings, and the safety profile remained consistent with previous reports. Epcoritamab + R2 is being studied further in the ongoing, randomized, phase 3 EPCORE FL-1 trial (NCT05409066).

The tumor microenvironments (TME) of diffuse large B-cell lymphoma (DLBCL) subgroups have remained poorly characterized. Here, we dissected the composition and spatial organization of the TME in germinal center B-cell (GCB), activated B-cell (ABC), and testicular (T-) DLBCL using gene expression profiling and multiplex immunohistochemistry. We found that high proportions of M2-like tumor-associated macrophages (TAM) and cytotoxic tumor-infiltrating T cells (TIL) were characteristic of ABC DLBCL TME. Furthermore, high CD8+ TIL content translated to favorable outcomes. In contrast, GCB DLBCL TME was enriched in CD4+ TIL, regulatory TIL, and a higher M1-like: M2-like TAM ratio, and high proportions of TAM and Granzyme B+ cells associated with worse survival. TIL and TAM interacted more frequently with M2-like TAM and cytotoxic TIL in the ABC DLBCL in contrast to GCB subtype, where the interactions were more abundant with other TIL and CD4+ TIL. In T-DLBCL, TME resembled that of ABC DLBCL with a higher proportion of M2-like TAM and cytotoxic cells, except that checkpoint-positive TIL were less prominent compared to DLBCL NOS. Cytotoxic TIL also interacted more with TIL and TAM. A large number of CD163+ TAM interactions with distinct TIL translated to unfavorable survival both in GCB DLBCL and T-DLBCL, whereas a high number of interactions between TIL and TAM, CD4+ TIL and TAM, and CD4+ TIL and other TIL were associated with favorable outcomes only in T-DLBCL. Together, our data demonstrate biologically and clinically relevant differences in the composition of and cellular interactions in the TME between various DLBCL entities.

Introduction: The prevalence and clinical impact of clonal hematopoiesis (CH) mutations in peripheral blood (PB) of patients with aggressive large B-cell lymphomas (LBCL) has remained poorly characterized. Moreover, CH mutations partly overlap with lymphoma driver mutations, potentially compromising the specificity of emerging biomarker assays for LBCLs based on plasma circulating tumor DNA (ctDNA). Hence, we set out to discover the impact of CH mutations in PB of young high-risk LBCL patients and correlated the results with clinical characteristics, outcome, and ctDNA features. CH and lymphoma mutations were traced longitudinally in PB and cell-free DNA (cfDNA) compartments. Methods: We sequenced 65 myeloid driver genes in PB samples of 163 healthy donors and 247 primary high-risk (94% with age-adjusted IPI ≥ 2) LBCLs patients treated in Nordic Lymphoma Group (NLG) phase II protocols with anthracyclin-based intensified immunochemotherapy and central nervous system prophylaxis (NLG-LBC-04, -05 and -06). The patients were young (median age, 55 years; range, 19-64) and the median follow-up was 47 months. We included CH mutations with variant allele frequency (VAF) of 0.5% for single-nucleotide mutations and 1% for frameshift variants. ctDNA data was available from 227 patients (Meriranta et al. 2022, Leppä et al. 2024), and CH mutations were evaluated in pretreatment and follow-up plasma cfDNA of 126 patients with targeted sequencing incorporating duplex error correction. Associations between PB and cfDNA-derived VAFs were analyzed using linear regression. Uni- and multivariable analyses were performed to evaluate associations between CH mutations, patient phenotypes, and outcomes. Results: CH mutations were found in 38% (95/247) of the pretreatment PB samples from lymphoma patients with a median VAF of 1.2% (range 0.5-41%). Correspondingly, 37% (61/163) of the healthy donors had a CH mutation. Lymphoma patients were more likely to have a CH mutation compared with healthy donors when adjusting for age and sex (OR=1.7, P=0.023). Of the most recurrently mutated genes, TET2 mutations were enriched in LBCL patients compared with healthy donors both in univariable (P=0.016) and multivariable models (OR=4.4, P=0.004). All 62 CH mutations with cfDNA available were also detected in cfDNA using force genotyping. Notably, two variants in EZH2 and one in TP53 showed strikingly high cfDNA VAFs compared with whole PB VAFs reflecting lymphoma driver mutations, and were excluded from the subsequent analyses for the clinical associations with CH mutations. For the remaining CH mutations, VAFs in PB showed a strong association with cfDNA-derived VAFs (R2=0.74, P<0.001). The presence of CH mutations was associated with older age (median, 58 vs 52 years, P<0.001). We observed no differences in other clinical characteristics, such as sex, symptom burden, IPI score, or ctDNA burden, between patients with and without CH mutations. Furthermore, we observed no difference in overall survival (OS) based on the presence of CH mutations in the univariable (P=0.87) or the multivariable model adjusting for age, sex, and IPI score (P=0.66). Also, we detected no difference in progression-free survival by CH mutation status (P=0.84). Patients with TP53 mutations in PB at diagnosis had inferior OS compared to patients without TP53 mutations (3-year OS 50% vs 88% P=0.04). Out of 43 LBCL patients with follow-up PB samples available, seven (16%) showed the emergence of new TP53/PPM1D clones (median VAF, 1.6%) following therapy. DNMT3A and TET2 mutations showed stable clones during follow-up (median VAF change, 0.1% and -0.1%, respectively). In 25 patients with pretreatment and end-of-therapy cfDNA samples available, DNMT3A, TET2, and ASXL1 mutations remained stable or showed decrease in clone size (median VAF change, 0.9%, -0.7%, -3%, respectively), whereas TP53 mutation VAFs increased in cfDNA (median VAF change, 1.7%) despite the clearance of the lymphoma ctDNA. Conclusion: Collectively, our results suggest that CH mutations may influence cfDNA characteristics, with potential implications for longitudinal use of cfDNA-based analyses in clinical contexts. CH mutations were not associated with survival; however, TP53 mutations in PB conferred worse OS. Additional studies are warranted to assess the long-term impact of CH mutation dynamics on outcomes, such as the risk of therapy-related myeloid malignancies.

Mantle cell lymphoma (MCL) is clinically and biologically heterogeneous. While various prognostic features have been proposed, none currently impact therapy selection, particularly in older patients, for whom treatment is primarily dictated by age and comorbidities. Herein, we undertook a comprehensive comparison of clinicopathological features in a cohort of patients 60 years and older, uniformly treated with bendamustine and rituximab, with a median survival of >8 years. The strongest prognostic indicators in this cohort were a high-risk call by a simplified MCL international prognostic index (s-MIPI) (HR: 3.32, 95% CI: 1.65-6.68 compared to low risk), a high-risk call by MCL35 (HR: 10.34, 95% CI: 2.37-45.20 compared to low risk) and blastoid cytology (HR: 4.21, 95% CR: 1.92-9.22 compared to classic). Patients called high risk by both the s-MIPI and MCL35 had the most dismal prognosis (HR: 11.58, 95% CI: 4.10-32.72), while those with high risk by either had a moderate but clinically relevant prognosis (HR: 2.95, 95% CI: 1.49-5.82). A robust assay to assess proliferation, such as MCL35, along with stringent guidelines for cytological evaluation of MCL, in combination with MIPI, may be a strong path to risk-stratify older MCL patients in future clinical trials.