7081 Background: Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies associated with poor outcomes. While adding brentuximab vedotin (BV) to CHP has improved results in CD30+ PTCL, relapses remain common. Given the suggested benefit of etoposide (CHOEP) in younger patients, the prospective multicenter single-arm Phase 2 CHEPA study (NCT05006664) was conducted to evaluate the efficacy and safety of CHEPA (BV-CHEP) induction combined with circulating tumor DNA (ctDNA) analysis. Methods: Patients with previously untreated CD30+ PTCL eligible for autologous stem cell transplantation (ASCT) were enrolled. Treatment consisted of 6 cycles of CHEPA (D1: BV 1.8 mg/kg, cyclophosphamide 750 mg/m², doxorubicin 50 mg/m²; D1–3: etoposide 100 mg/m²; D1–5: prednisone 100 mg/d) every 21 days. The primary endpoint was the complete metabolic response (CMR) rate at the end of treatment (EOT). Secondary endpoints included safety, PFS, OS, ORR, and exploratory ctDNA analysis. Plasma cfDNA was profiled using CAPP-Seq (259 genes) and clonotypic VDJ sequencing at all TCR loci via SABER. Results: Between 05/2022 and 09/2025, 40 patients were screened; 33 met eligibility criteria and initiated therapy. The cohort included 14 (42%) ALCL and 19 (58%) non-ALCL cases (median age 57 years, range 26–69). Baseline characteristics showed a predominance of males (67%), advanced-stage disease (79%), elevated LDH (58%), and bone marrow involvement (30%); all pts had an ECOG PS of 0–1. All 33 pts completed 6 cycles of CHEPA. At EOT, 25/33 (76%) achieved CMR, meeting the primary endpoint. The ORR was 91% for the entire cohort (CMR: 86% for ALCL, 68% for non-ALCL). ASCT was preplanned in 21/33 pts and has been performed to date in 14 pts. At a median follow-up of 19 months, the 18-month PFS and OS probabilities were 75% and 92%, resp. Specifically, PFS was 100% for ALCL vs. 59% for non-ALCL, while OS was 100% vs. 87%. Age and bone marrow involvement were the only baseline variables significantly associated with CMR. Baseline ctDNA burden was significantly correlated with total metabolic tumor volume (r=0.68, p<0.001), LDH level (r=0.48, p=0.03), and PIT (p<0.05). Grade 3–4 adverse events occurred in 82% of pts, primarily hematological: neutropenia (79%), thrombocytopenia (18%), febrile neutropenia (15%), and anemia (15%). No grade 3–4 peripheral neuropathy or grade 5 events were reported. Conclusions: The CHEPA study met its primary endpoint with a 76% CMR rate. Despite limited follow-up and high non-ALCL prevalence of our cohort, PFS and OS results are promising. Toxicity was manageable with no treatment-related deaths even though 33% of patients were >60 years old, supporting the safety of adding etoposide to the BV-CHP backbone. Further investigation of outcomes based on EOT MRD by ctDNA is ongoing. Clinical trial information: NCT05006664 .

Mantle cell lymphoma (MCL) is a B-cell malignancy with a chronically relapsing clinical course and pronounced genetic heterogeneity. To investigate the clonal dynamics underlying early disease relapse, we performed single-cell RNA sequencing of paired tumor samples collected at diagnosis and at first relapse. Inference of copy number variants (CNVs) identified multiple subclonal clusters at both time points. In all cases, a minor subclone present at diagnosis harbored a CNV profile highly concordant with that of the dominant relapse clone, indicating the pre-existence of therapy-resistant subclones at diagnosis. Gene set enrichment analysis comparing therapy-resistant and therapy-sensitive clones revealed substantial inter-patient heterogeneity in resistance-associated transcriptional programs. Despite this heterogeneity, convergent dysregulation of cell-cycle control pathways emerged as a shared feature across patients. Furthermore, we investigated a case of SOX11-negative indolent MCL (iMCL) with late relapse characterized by extensive extranodal dissemination, including peripheral blood leukemization and intestinal tumor masses. While bone marrow- and peripheral blood-derived MCL cells maintained SOX11 negativity and a largely conserved transcriptomic state, intestinal MCL cells displayed blastoid morphology, expression of SOX11, a profoundly remodeled CNV landscape, and the acquisition of multiple additional driver mutations. Collectively, these findings indicate that early relapse in MCL originates from minor therapy-resistant subclones present at diagnosis and subsequently selected under therapeutic pressure. Moreover, disease progression in iMCL may be driven by spatially restricted clonal evolution, with the emergence of aggressive molecular features in distinct anatomical compartments. These results provide mechanistic insight into MCL clonal evolution and relapse biology.

Resistance to Bruton's tyrosine kinase (BTK) inhibition represents a major clinical challenge in mantle cell lymphoma (MCL). While ibrutinib suppresses B-cell receptor signaling, patients eventually relapse, underscoring the need to elucidate non-genetic resistance mechanisms. MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression, yet their contribution to ibrutinib resistance in MCL remains incompletely defined. To this end, we established three ibrutinib-resistant MCL cell line models through prolonged drug exposure and performed integrated miRNA and transcriptome profiling. Resistant cells exhibited a distinct miRNA signature characterized by downregulation of miRNAs targeting the MAPK-ERK and PI3K-AKT survival pathways, resulting in pathway hyperactivation. These findings were validated in patient-derived xenografts and clinical biopsy samples from ibrutinib-resistant patients. Pharmacological inhibition of these pathways effectively suppressed pathway activation, reduced mitochondrial activity, and induced apoptosis in resistant cells, with enhanced efficacy observed upon dual pathway targeting. Among deregulated miRNAs, miR-146a-5p was consistently reduced in resistant cell lines, PDX models, and patient samples. Restoration of miR-146a-5p expression resensitized resistant cells to ibrutinib. Promoter hypermethylation of miR-146a provides a potential epigenetic mechanism underlying its repression. Overall, these data identify miRNA-mediated activation of MAPK and PI3K signaling as epigenetic driver of ibrutinib resistance in MCL and support rational combination strategies to overcome therapeutic failure.

We developed a robust, hybrid RNA-based reporter assay for the quantification of cytidine deaminase (CDA) activity, overcoming limitations of current methods often compromised by interferences in crude biological samples. Our two-step method physically separates the enzymatic reaction in cell lysates from a highly specific detection step in a selected and validated CDA-deficient reporter cell line (143B cell line). This design confers exceptional robustness, eliminating the need for sample purification. In the first step, a cell lysate converts 5-fluorocytidine to 5-fluorouridine. After heat inactivation of lysate proteins, 5-fluorouridine is quantified by its incorporation into reporter cell RNA, yielding a highly specific fluorescent signal. The detection limit of a formed product in the assay achieves 2.7 µM. The assay shows good correlation with LC-MS data, and its adaptability to a multi-well plate format makes it an ideal and cost-effective tool for high-throughput screening. We further applied this assay to reveal that CDA-positive cells actively secrete 5-fluorouridine, providing quantitative evidence for a potential therapeutic bystander effect.

We evaluated the in vivo efficacy of polatuzumab vedotin (POLA), administered as a single agent or in combination with venetoclax (VEN), in relapsed/refractory (R/R) mantle cell lymphoma (MCL) and BCL2-positive diffuse large B-cell lymphoma (DLBCL). In addition, we investigated the mechanisms underlying acquired resistance to POLA in vivo. Experimental therapy was assessed using a panel of 8 cell line-derived xenografts (CDXs) and 16 patient-derived xenografts (PDXs) representing MCL and DLBCL. Bulk transcriptomic profiling was performed on 7 paired tumor samples obtained from POLA-resistant tumors (generated in mice following repeated POLA-based treatments) and their corresponding untreated controls. POLA demonstrated robust single-agent antitumor activity in vivo, including in PDX models derived from patients with ibrutinib-resistant MCL. In the majority of tested PDX models, the combination of POLA and VEN produced synergistic antitumor effects without evidence of measurable toxicity. CD79B expression levels did not correlate with POLA efficacy. Following POLA treatment failure, downregulation of CD79B was observed in only a minority of models, indicating the involvement of additional mechanisms of acquired resistance. Bulk transcriptomic analysis of 7 paired POLA-resistant versus untreated tumors identified a conserved gene expression signature across all models, comprising 523 downregulated and 284 upregulated genes. This signature likely reflects core pathways associated with POLA resistance and highlights potential novel therapeutic vulnerabilities. These findings strongly support further clinical investigation of POLA in combination with VEN as a BCL2- and MCL1-targeting therapeutic strategy in patients with R/R MCL and BCL2-positive R/R DLBCL.

Introduction The TRIANGLE trial (Dreyling et al, Lancet 2024) compared three treatment arms in untreated, younger patients with mantle cell lymphoma (MCL): arm I (IR-CHOP/R-DHAP + ibrutinib maintenance [Im]), arm A+I (IR-CHOP/R-DHAP + ASCT + Im), and arm A (standard-of-care R-CHOP/R-DHAP + ASCT). Ibrutinib-containing regimens demonstrated superior failure-free survival (FFS) and overall survival (OS). Prior studies (Hadzidimitriou et al, Blood 2011) have shown skewed immunoglobulin (IG) repertoires in MCL, with preferential IG heavy chain (IGH) gene usage. However, large, homogeneous cohorts have not clearly linked IGHV usage or IGHV mutational status to clinical outcomes. Moreover, the interaction between biological risk factors such as TP53 aberrations and IGH repertoire is still unknown. Here, we assess the prognostic significance of IGHV repertoire after adjustment for baseline risk factors. Methods IGH clonal rearrangement analysis for minimal residual disease (MRD) was performed on bone marrow or peripheral blood samples centralized in 7 EuroMRD Network laboratories. Samples were analyzed by Sanger or amplicon-based NGS (VH-FR1/JH-3 primers) and were processed via IMGT/V-QUEST or ARResT/Interrogate to assign IGH rearrangements and germline FR1-IGHV identity. Diagnostic lymph node biopsies were assessed for p53 immunohistochemistry expression as a surrogate of TP53 alterations, scored as low (<50%) or high (≥50%). Missing baseline values were imputed by Chained Equations (MICE) model. The prognostic impact of IGHV genes and IGHV mutational status on FFS was analyzed with Kaplan-Meier curves and multivariable Cox regression adjusted for baseline prognostic factors. Results Out of the 870 enrolled patients, 560 had an available IGHV sequence for analysis. Baseline characteristics and outcomes were comparable to the remaining TRIANGLE patients and, in the selected patients, ibrutinib-containing arms had superior outcomes compared with the control arm (3y-FFS: arm A 70% vs arm A+I and I: 85%, P=0.001). The most common IGHV families were IGHV 3-21 (n=123, 22%), 4-34 (n=59, 11%) and 1-8 (n=42, 7.5%) whereas IGHD 3-3 (n=60, 11%) and IGHJ 4 (n=230, 41%) were the most frequent IGHD and IGHJ families, respectively. Univariable Cox regression focused on IGHV family usage identified the VH 3-21, 3-30, 3-48, and 3-74 genes as associated with improved FFS which were therefore grouped together, namely VHcomb patients (N=171). VHcomb patients were younger, had lower MIPI scores, and showed superior 3-year FFS compared to other families (VHother) (87% vs. 77%, P=0.002). Stratified by treatment arm, VHcomb had significantly better FFS vs. VHother in arm A (87% vs. 62%, P<0.001), but no difference was observed in ibrutinib-containing arms (87% vs. 85%, P=0.22). Interestingly, after adjusting for p53, MIPI and Ki67, VHcomb remained associated with improved FFS in arm A (Hazard ratio [HR] 0.46 [0.23-0.92], P=0.029), while no statistically significant impact was observed in arm A+I: HR 1.08 [0.55-2.13], P=0.82; but still a trend in arm I: HR 0.51 [0.23-1.13], P=0.096. Subsequently, to investigate an optimal cut-off point for FR1-IGHV gene identity, a Cox regression with restricted cubic splines was performed, identifying 97% as the best FR1-IGHV gene homology cut-off for prognostic discrimination of FFS after adjustment for MIPI, histologic subtype, Ki67, and treatment arm. Patients with FR1-IGHV gene identity > 97% (FR1-IGHV unmutated, n=448 [80%]) showed a trend towards worse FFS at later follow-up (log rank P=0.056) compared to patients with FR1-IGHV gene identity ≤ 97% (FR1-IGHV mutated, n=110 [20%]). No significant differences in FFS were observed according to FR1-IGHV mutation status after adjusting for p53, MIPI and Ki67 in multivariable analysis both in standard and ibrutinib-containing regimens. Conclusions This is the largest study investigating IGH repertoire in a prospective phase 3 trial in MCL. FR1-IGHV unmutated patients showed inferior FFS. The IGHV 3-21, 3-30, 3-48, and 3-74 rearrangements were associated with improved FFS in the chemo-immunotherapy arm, independently of p53 alterations. On the other hand, the addition of ibrutinib may potentially mitigate the prognostic impact of the IGHV families. Although requiring validation, these findings support a BCR-related prognostic role in MCL, independent from MIPI, Ki67 and p53 alterations and potentially modulated by ibrutinib.

Introduction:Blast phase of chronic myeloid leukemia (CML-BP) represents advanced and aggressive phase of CML, marked by high genetic heterogeneity, compromised efficacy of BCR::ABL1-targeted tyrosine kinase inhibitors (TKIs), and poor clinical outcomes. Emerging evidence indicates that combination therapies may offer more effective treatment options in this setting. This European Treatment and Outcome Study (EUTOS 2024) work aims to evaluate the efficacy of allosteric BCR::ABL1 inhibitor asciminib, 2nd generation TKI nilotinib, 3rd generation TKI ponatinib, BCL2 inhibitor venetoclax, and their combinations in patient-derived xenograft (PDX) models of CML-BP. Methods:Leukemic cells from CML-BP patients were analyzed for chromosomal abnormalities and mutations in BCR::ABL1 kinase domain (KD) and 61 other leukemia-associated genes. Ex vivo drug sensitivity was conducted. CML blasts were transplanted into immunodeficient mice to establish PDX models. Animals (n=5–8 per group) were randomized into control or treatment arms and received daily oral treatment for 7 days: ponatinib (25 mg/kg b.w.), nilotinib (40 mg/kg b.w.), asciminib (30 mg/kg b.w.), venetoclax (50 mg/kg b.w.), or their combinations. Tumor growth and event-free survival (EFS) were assessed. The experimental design was approved by the institutional Animal Care and Use Committee and the Ministry of Education Youth and Sports of the Czech Republic. Results: The PDX model BC-CML2, derived from a 70-year-old male patient with myeloid CML-BP following rapid progression on imatinib, exhibited a complex karyotype with additional Philadelphia chromosomes but no detectable somatic mutations. In line with ex vivo findings, the model was poorly responsive to nilotinib, asciminib and venetoclax monotherapies but responded well to ponatinib. Combination therapies—asciminib+venetoclax and especially ponatinib+venetoclax—significantly reduced tumor growth and prolonged EFS compared to controls and monotherapies (p<0.001). Conversely, combination of nilotinib and asciminib have limited efficacy against tumor growth and have no survival benefit. The BC-CML3 model originated from a 58-year-old female patient with B-lymphoid CML-BP in 3rd relapse on ponatinib. Blasts carried BCR::ABL1 compound mutations (T315I+E255K and T315I+E459K), a RUNX1 mutation (R201Q), and a complex polyclonal karyotype. This model was resistant to venetoclax and nilotinib alone and showed only modest responses to asciminib and ponatinib monotherapies. However, combinations of asciminib+ponatinib and ponatinib+venetoclax delayed disease progression and significantly improved EFS compared to controls (p<0.001) and single agents (p<0.01 for asciminib+ponatinib; p<0.001 for ponatinib+venetoclax). The BC-CML4 model was established from a 65-year-old male patient with myeloid CML-BP relapsing after four lines of TKI therapy including ponatinib. Mutational profiling revealed polyclonal BCR::ABL1 mutations (E255K and E255V), along with NRAS (G12D) and GATA2 (R307Q) mutations, and a complex karyotype. Ex vivo testing showed resistance to venetoclax. Ponatinib and its combination with venetoclax significantly delayed tumor growth and extended EFS (p<0.001). Conclusions: Preclinical testing in CML-BP PDX models confirmed the enhanced efficacy of combination therapies. Across all models, dual targeting with ponatinib and venetoclax was most effective. In the BC-CML3 model, harboring compound BCR::ABL1 mutations linked to ponatinib resistance, the asciminib+ponatinib combination showed notable activity, consistent with previous findings in cell line-derived xenograft (CDX) models and in vitro testing. Support:EUTOS 2024, MH CZ – DRO (IHBT, 00023736)

Introduction The International Prognostic Index (IPI) is a widely used tool for risk stratification of newly diagnosed large B-cell lymphoma (LBCL) patients. However, some patients experience refractory disease or relapse even within the low or low-intermediate risk groups (IPI 0–2). To improve risk discrimination, several modifications to the IPI have been proposed. Recent data suggest that patients within IPI 1–2 with bulky disease and/or very high lactate dehydrogenase (VH-LDH) levels have outcomes similar to those with IPI 3 (Maurer, ASH 2023), and could therefore be grouped with the intermediate-high and high-risk (H-Risk) IPI categories. This approach is already being incorporated into clinical trials (NCT06356129). However, external validation outside the US cohort is limited. We aimed to independently validate whether LBCL patients with IPI 1–2 and bulky disease and/or VH-LDH have survival outcomes comparable to those with IPI 3. Methods Consecutive patients with newly diagnosed systemic LBCL registered in the NiHiL project (NCT03199066) between 2010–2020 were identified. Inclusion criteria were clinical stage II–IV, age 18–80 years, IPI 1–5, complete baseline data, first-line R-CHOP-like therapy (n=2,303). Bulky disease was defined as ≥7.5 cm, and VH-LDH levels as >1.3× upper limit normal per prior report. IPI 1–2 H-Risk was defined by the presence of bulky and/or VH-LDH within the IPI 1–2 group. The primary endpoint was event-free survival (EFS) comparison between IPI 1–2 H-Risk versus IPI 1–2 low-risk (L-Risk) and IPI 3 groups. Hazard ratios (HRs) were estimated using Cox proportional hazards models. Age-stratified subgroup analysis was performed in patients aged ≤60 years and >60 years. Results Among 2,303 included patients, the median age was 67 years (IQR 59–72), with 71% being >60 years old, 48% were female, 78% had stage III–IV disease, and 34% had ECOG performance status 2–4. The IPI distribution was 36% IPI 1–2 (n=828), 29% IPI 3 (n=664), and 35% IPI 4–5 (n=811). Among IPI 1–2 patients, 440 (53%) were classified as L-Risk and 388 (47%) as H-Risk. The comparison of H-Risk versus L-Risk IPI 1-2 groups revealed the following differences: H-Risk patients were younger (median age 59 vs 66 years, P<0.01), had more frequently elevated LDH (70% vs 21%, P <0.01) and bulky disease (77% vs 0%, P <0.01, by definition), as well as a shorter diagnosis-to-treatment interval (median 24 vs 35 days, P <0.01). With a median follow-up of 8.1 years, 2-year EFS was 86% in the IPI 1–2 L-Risk group, 75% in IPI 1–2 H-Risk, and 65% in IPI 3; corresponding 5-year EFS rates were 73%, 69%, and 54%; with no significant differences between H-Risk vs L-Risk group (HR=1.08, 95% CI 0.87–1.35, P=0.47), while the EFS of IPI 3 patients was inferior when compared to IPI 1–2 H-Risk (HR=1.58, 95% CI 1.31–1.91, P<0.01). Due to the observed age imbalance between L-Risk and H-Risk groups, we performed an age-stratified analysis. Among patients aged ≤60 years (n=659), 361 had IPI 1–2, including 150 (42%) L-Risk and 211 (58%) H-Risk, while 203 (31%) had IPI 3. The 2-year EFS was 89% in L-Risk, 74% in H-Risk, and 65% in IPI 3; 5-year EFS was 81%, 69%, and 58%, respectively. Patients in the H-Risk group had significantly inferior EFS compared to L-Risk (HR=1.53, 95% CI 1.04–2.24, P=0.03), whereas the difference between IPI 1–2 H-Risk and IPI 3 was of borderline significance (HR=1.35, 95% CI 1.00–1.84, P=0.05). Among patients between 60 and 80 years (n=1,644), 467 (28%) had IPI 1–2, including 290 (62%) L-Risk and 177 (38%) H-Risk, and 461 (28%) had IPI 3. The 2-year EFS was 84% in L-Risk, 76% in H-Risk, and 64% in IPI 3; 5-year EFS was 70%, 68%, and 52%, respectively; with no difference between L-Risk and H-Risk groups (HR=1.00, 95% CI 0.75–1.38 P=0.98), while IPI 3 was associated with worse outcomes compared to H-Risk (HR=1.59, 95% CI 1.24–2.05, P<0.01). Conclusion The external validation in the NiHiL project demonstrated that IPI 1–2 H-Risk LBCL patients had significantly better EFS than those with IPI 3, while the EFS between IPI 1–2 H-Risk and L-Risk did not statistically differ. Thus, we were unable to confirm the findings reported in the US cohort (Maurer, ASH 2023), raising questions about the clinical utility of this stratification. However, in patients aged ≤60 years, the H-Risk group had significantly worse outcomes than L-Risk, supporting the potential prognostic relevance of this stratification in younger patients. Funding: NU21-03-00411

Previous studies have suggested that, after acquisition of t(11;14), mantle cell lymphoma (MCL) pathogenesis may proceed via several different genetic second hits, which may shape different mutational profiles of clinically manifest lymphoma. The most prevalent second hit in MCL includes ATM aberrations, accounting for about half of patients with newly diagnosed MCL. As ATM and TP53 mutations tend to be exclusive in MCL, we retrospectively analyzed the prognostic role of ATM deletions and/or mutations in patients with newly diagnosed MCL, both in the entire cohort and in a subcohort of patients with wild-type TP53. To investigate deletions and mutations of ATM and TP53 in newly diagnosed MCL, we used fluorescence in situ hybridization and next-generation sequencing. To assess relationships between variables, non-parametric (Spearman) and chi-square tests were used. The Kruskal–Wallis test was used to analyze differences in continuous variables between two groups of patients. For survival analyses, the standard Kaplan–Meier estimator and log-rank test were employed. Univariate and multivariate Cox proportional hazard models were used to examine the prognostic value of various factors on patient survival. We analyzed 187 patients with MCL (a median follow-up of 3.6 years). Eighty-one (43

Mantle cell lymphoma (MCL) is a relatively rare B-cell lymphoma subtype, with a higher incidence among males and a median age of 70 years at diagnosis. MCL is characterized by clinically diverse behavior, from indolent disease to extremely aggressive, related to the presence of biological risk factors such as proliferation rate and TP53 mutations. Most often, patients present with disseminated disease, necessitating systemic treatment. Immunochemotherapy has historically been the mainstay of treatment, but recent data indicate that addition of novel agents, especially covalent Bruton tyrosine kinase inhibitors (cBTKi), may substantially improve outcome in younger and older patients, although a curative approach remains to be shown. In elderly patients, the standard of care is still immuno-chemotherapy such as rituximab-bendamustine, although this may be challenged by non-chemotherapeutic options, such as rituximab plus cBTKi. For patients with relapsed or refractory disease, treatment options are developing rapidly, including CAR-T cell therapy, novel BTK targeting agents, BCL2 inhibitors, and T-cell engagers. In this clinical practice guideline, we present current evidence-based recommendations for diagnosis, staging, treatment, and follow-up of MCL.

BACKGROUND:Leukemia is driven by complex interactions within the inherently hypoxic bone marrow microenvironment, impacting both disease progression and therapeutic resistance. Co-cultivation of leukemic cells with feeder cells has emerged as a valuable tool to mimic the bone marrow niche. This study explores the interplay between human commercial SD-1 and patient-derived UPF26K leukemic cell lines with feeders - human fibroblasts (NHDF) and mesenchymal stem cells (hMSCs) under normoxic and hypoxic conditions. RESULTS:Co-cultivation with feeders significantly enhances proliferation and glycolytic activity in the SD-1 cells, improving their viability, while this interaction inhibits the growth and glucose metabolism of the feeders, particularly NHDF. In contrast, UPF26K cells show reduced proliferation when co-cultivated with the feeders while this interaction stimulates NHDF and hMSCs proliferation and glycolysis but reduce their mitochondrial metabolism with hypoxia amplifying these effects. CONCLUSIONS:Cells that switch to glycolysis during co-cultivation, particularly under hypoxia, benefit most from these low oxygen conditions. Due to this leukemic cells' response heterogeneity, targeting microenvironmental interactions and oxygen levels is crucial for personalized leukemia therapy. Advancing co-cultivation models, particularly through innovations like spheroids, can further enhance in vitro studies of primary leukemic cells and support the testing of novel therapies.

Chimeric antigen receptor T-cell (CAR-T) therapy has transformed the management of relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). Initially approved for use in the third line (L3) setting and beyond, CAR-T has increasingly been adopted earlier in the second line (L2). However, many patients continue to receive CAR-T as L3 therapy due to prior treatment choices or L2 CAR-T ineligibility (e.g., late relapses). The aim of this analysis is to compare survival outcomes between CAR-T and alternative L3 therapies using propensity score matching (PSM) to adjust for key clinical differences. Methods We analyzed 240 consecutive r/r DLBCL patients prospectively enrolled in the NiHiL project (NCT03199066), all initially treated with R-CHOP-like regimens and receiving L3 therapy between 2020–2023. Baseline characteristics, diagnosis(dg)-to-L3 interval, clinical parameters at L3 (age, ECOG performance status [PS], and year of L3), L3 and L4 treatment patterns and outcomes were collected. Patients with ECOG PS 0–2 at L3 (n=163) were included; those with prior L2 CAR-T failure (n=6) were excluded, resulting in 157 eligible patients. PSM (1:1, caliper 0.2, nearest neighbor) was performed between patients with the intention of receiving CAR-T and those treated with alternative L3 therapies. The matching was based on age, ECOG PS, and dg-to-L3 interval. The endpoints were event-free survival (EFS) and overall survival (OS), both calculated from the L3 initiation (for CAR-T patients defined as the start of bridging [holding] therapy, or apheresis if no bridging). Results Of the 157 included patients, 75 were intended-to-CAR-T and 65 received CAR-T (axi-cel n=26, or tisa-cel n=39), while 82 received alternative L3 treatments (novel therapies n=27, salvage chemotherapy n=14, other therapy n=41). PSM yielded 104 matched patients (52 per group). We first compared the outcomes of patients included in the PSM (i.e., matched; n=104) versus those excluded (n=53). Among patients intended for CAR-T (n=75), individuals excluded from PSM (n=23) were younger (P<0.01), and had longer dg-to-L3 interval (P<0.01); these unmatched CAR-T patients had better EFS at L3 (P=0.05; OS P=0.29) compared to matched CAR-T patients (n=52). In alternatively treated patients (n=82), those who were not matched by PSM (n=30) were older (P<0.01), had longer dg-to-L3 interval (P<0.01), but did not show survival differences compared to matched patients (n=52; EFS P=0.48, OS P=0.59). PSM (n=104) achieved good balance across key covariates at L3 initiation between CAR-T and alternatively treated cohorts (both n=52): median age 64 vs 66 years (SMD 0.180), ECOG PS 1–2 in 35 (67%) vs. 37 (71%) cases (SMD 0.083), median dg-to-L3 interval of 19 vs 15 months (SMD 0.148). Disease characteristics at initial diagnosis were also comparable between groups: clinical stage III–IV (81% vs 83%, ns), elevated LDH (69% vs 71%, ns), extranodal involvement >1 site (38% vs 48%, ns), IPI score 3–5 (60% vs 60%, ns), and primary refractory or early relapsing disease (46% vs 57%, ns). Of alternatively treated patients included in the PSM (n=52), 20 (38%) received novel therapies (Pola-BR n=14, bispecific antibodies n=4, lenalidomide plus rituximab n=1, ibrutinib n=1), 12 (23%) salvage, and 20 (38%) received other treatments. After median follow-up of 20.9 months (living patients), EFS was significantly improved in the matched CAR-T group (43.5% vs 24.7% at 18 months, HR=0.62, 95% CI 0.40–0.98, P=0.04) compared to matched alternatively treated patients, with a trend towards prolonged OS (66.9% vs 43.1% at 18 months, HR=0.72, 95% CI 0.43–1.21, P=0.22). Among the matched patients who ultimately received CAR-T (n=48 out of 52), 18-month EFS was 47.2% and 18-month OS was 68.2%. Of these, 21 (44%) required subsequent L4 therapies (novel agents n=11, salvage n=1, other n=9). In the alternatively treated group (n=52), 31 (60%) required L4 therapy (CAR-T n=8, novel agents n=8, salvage n=4, other n=11). Conclusion After adjusting for key clinical covariates, L3 CAR-T therapy was associated with significantly improved EFS compared to alternative L3 treatments in r/r DLBCL. Despite its increasing use in earlier lines, CAR-T remains a valuable and effective treatment option in the L3 and beyond for eligible patients not previously exposed to this treatment modality. NU21-03-00411, Charles University Haematology-Oncology Cooperatio Program.

Despite therapeutic advances, a significant proportion of patients with diffuse large B-cell lymphoma (DLBCL) experience disease relapse (R). Previously, we reported a 5-year cumulative incidence of systemic relapse (Sy-R) and central nervous system relapse (CNS-R) of 21.25% and 3.76%, respectively (Klanova et al., ASH 2023). Compared to Sy-R, CNS-R is generally associated with worse outcomes. Most CNS-Rs present with isolated CNS involvement (CNS-I-R), while a smaller subset develops concurrent CNS and systemic R (CNS+Sy-R). Based on localization, CNS-R can affect parenchymal (Par), leptomeningeal, or both sites (Lep/Comb). This study compared clinical outcomes of Sy-R, CNS-I-R (further divided into CNS-I-Par and CNS-I-Lep/Comb), and CNS+Sy-R in a large real-world DLBCL cohort. Methods Using the prospective NiHiL project (NCT03199066), we identified 1,364 patients with systemic DLBCL who experienced their first R between 2005 and 2023. Patients with CNS involvement at initial diagnosis or incomplete staging at R were excluded. Among the included patients (n=1,351), 1,186 (87%) had Sy-R, 127 (9%) had CNS-I-R (CNS-I-Par n=92; CNS-I-Lep/Comb n=34; unknown localization n=1), and 38 (3%) had CNS+Sy-R. In total, 1,153 patients received chemotherapy (CTx), including 901 treated with curative intent. We first compared outcomes across Sy-R, CNS-I-R, and CNS+Sy-R. Subsequently, CNS-I-R cases were stratified by localization (CNS-I-Par vs CNS-I-Lep/Comb). The primary endpoint was progression-free survival (PFS); overall survival (OS) was the secondary endpoint, both measured from the date of R. Univariate and multivariate Cox regression analyses were performed. Results When comparing Sy-R, CNS-I-R, and CNS+Sy-R, the median time from diagnosis to relapse was 12.3, 14.2, and 9.3 months, respectively (P=0.01); median age at R was 68, 68, and 64.5 years, respectively (P=0.04). ECOG performance status (PS) 2–4 was more frequent in R involving the CNS (CNS-I-R 65%, CNS+Sy-R 63%) than in Sy-R (32%; P<0.01). In contrast, elevated LDH was more common in relapses with Sy involvement (Sy-R 67%, CNS+Sy-R 61%) compared to CNS-I-R (43%; P<0.01). With a median follow-up of 7.6 years, median PFS decreased across Sy-R, CNS-I-R, and CNS+Sy-R: 8 vs 6 vs 3 months (P<0.01), and median OS was 13, 6, and 3 months, respectively (P<0.01). We then focused on CNS-I-R and compared outcomes by localization. Patients with CNS-I-Par (73%) were older (median age 70 vs 62.5 years; P=0.048) and had longer time from diagnosis to relapse (15.75 vs 8.15 months; P<0.01) than those with CNS-I-Lep/Comb (27%). Median PFS and OS for CNS-I-Par vs CNS-I-Lep/Comb were 7 vs 4 months (HR=0.49, P<0.01) and 8 vs 4 months (HR=0.48, P<0.01), respectively. When comparing all four relapse types (Sy-R, CNS-I-Par, CNS-I-Lep/Comb, CNS+Sy-R), Sy-R and CNS-I-Par had the most favorable outcomes with similar PFS (median 8 vs 7 months; HR=0.98, P=0.87) and modestly better OS for Sy-R (median 13 vs 8 months; HR=0.77, P=0.04). CNS-I-Lep/Comb and CNS+Sy-R had similarly poor outcomes (median PFS 4 vs 3 months, HR=0.99, P=0.95; median OS 4 vs 3 months, HR=0.85, 95% CI 0.52–1.38, P=0.51). After adjusted for age, ECOG PS, LDH, and diagnosis-to-relapse interval, survival was similar between CNS-I-Par and Sy-R (PFS P=0.63; OS P=0.15). However, both CNS-I-Lep/Comb (PFS HR=1.82, P=0.01; OS HR=1.55, P=0.05) and CNS+Sy-R (PFS HR=1.60, P=0.01; OS HR=1.69, P=0.01) remained independently associated with worse survival compared to Sy-R. These findings were consistent in the curatively treated subgroup (n=901): median PFS 10, 8, 4, and 3 months, median OS 19, 10, 4, and 4 months, resp., for Sy-R, CNS-I-Par, CNS-I-Lep/Comb, CNS+Sy-R resp. (all P<0.01). Conclusion While all DLBCL relapses are associated with poor prognosis, clinical outcomes vary by relapse pattern. Sy-R and CNS-I-Par showed comparable PFS, and better survival compared to other subtypes,. In contrast, CNS-I-Lep/Comb and CNS+Sy-R were associated with worst outcomes. The OS in CNS-R cases closely mirrors the PFS, suggesting that treatment remains challenging, and therapeutic failure often represents a terminal event. Novel approaches, including CNS-penetrating targeted agents and CAR-T, which show early promise in CNS-Rs, are needed for these patients. Grant No. NU23-03-00127.

Introduction The LEO Comorbidity Index (LCI), comprising ten equally weighted comorbidity categories (respiratory, cardiovascular, digestive, hepatic, renal, autoimmune, diabetes, cancer history, HIV, and stroke), has demonstrated prognostic utility in patients with non-Hodgkin lymphomas. However, its predictive accuracy may vary with increasing age, due to age-related rises in comorbidity burden and physiological vulnerability. To investigate these dynamics, we evaluated the age-stratified predictive performance of LCI in patients with large B-cell lymphoma (LBCL), with a focused analysis on individuals aged 80+ years. We also examined which specific comorbidities most strongly influenced overall survival (OS) and developed a simplified, clinically applicable index tailored to this population. Methods We first included adults ≥40 years with newly diagnosed LBCL and recorded comorbidities from the LEO registry (USA, 2015–2020; n=1,652) to assess age-stratified predictive performance of LCI using the C-index. Next, we focused on patients 80+ years (n=263), pooled from the LEO cohort (n=147) and NiHiL registry (restricted to General Hospital, Prague, Czech Republic, 2010–2023; n=116). Comorbidities were collected from patient self-reports (LEO) and/or medical records (LEO/NiHiL). Univariate and multivariate Cox models were used. The primary endpoint was OS. Results Among the total LBCL population (n=1,652), LCI predictive performance improved by age category with C-index for patients <65 of 0.584, 65–79 years 0.579, and ≥80 years 0.647, indicating stronger discrimination in the 80+ group. Subsequent analysis focused on patients aged 80+ years (n=263; median age 83). Within this cohort, 34% had ECOG performance status (PS) 2–4, 66% stage III–IV disease, 59% elevated lactate dehydrogenase, 35% >1 extranodal site involved, and 58% International Prognostic Index (IPI) score of 3–5. A total of 377 comorbidities were reported, averaging 1.43 per patient (1.22 in LEO; 1.61 in NiHiL). LCI distribution was: 0 points in 24% (n=62), 1 point in 33% (n=88), and 2–5 points in 43% (n=113). Higher LCI scores had worse ECOG PS 2–4: 19% (LCI=0), 33% (LCI=1), and 43% (LCI ≥2; P=0.01), and inferior OS (median 6.9 years LCI=0, 3.0 years LCI=1, and 2.8 years LCI≥2, resp.; P=0.02). Presence of ≥1 comorbidity was associated with shorter OS compared to no comorbidities (HR=1.67, P=0.01). The LCI was an independent predictor of OS after adjusting for age (HR=1.18, P=0.01, C-index 0.623), IPI (HR=1.16, P=0.03, C-index 0.628) as well as for recently proposed SENIOR-IPI (HR=1.14, P=0.049, C-index 0.665). When evaluating individual comorbidities, heart disease (HR=1.54, P=0.01), stroke (HR=1.89, P=0.03), and recent malignancy (HR=1.99, P=0.02) were independent predictors of OS. Based on these findings, we developed a simplified LCI (sLCI) for use in 80+ patients incorporating only the three comorbidities (1 point each). Patients were categorized as sLCI: 0 (49%, n=129), 1 (44%, n=115), and 2–3 points (7%, n=19). sLCI scores 0, 1, 2–3 were associated with ECOG PS 2–4 27%, 37%, and 63%, respectively (P=0.01) and inferior OS: median 5.3 years (0), 2.5 years (1), and 1.4 years (2–3; P<0.01). sLCI remained an independent and strong predictor of OS after adjusting for age (HR=1.71, P<0.01, C-index 0.636), IPI (HR=1.66, P<0.01, C-index 0.635), and SENIOR-IPI (HR=1.60, P<0.04, C-index 0.659). The results were consistent in both NiHiL and LEO cohorts and remained significant among patients treated with anthracyclines (sLCI adjusted for age: HR=1.58, P<0.01, C-index 0.607; for IPI: HR=1.53, P=0.01, C-index 0.623; for SENIOR-IPI: HR=1.51, P=0.01, C-index 0.633). Conclusion Comorbidity burden becomes increasingly prognostic with higher age in LBCL patients. In the 80+ individuals, comorbidities – particularly heart disease, recent cancer, and stroke – have an independent impact on OS. While the original LCI captures the impact of broadly defined comorbidities, its prognostic utility attenuates in the 80+ patients, likely reflecting limited relevance of indolent chronic conditions in this population's constrained survival horizon. The sLCI, focused on the most prognostically relevant comorbidities, offers practical risk stratification. Incorporation of sLCI into clinical decision-making – alongside other prognostic indices – could better identify high-risk individuals and guide treatment strategies. Funding: NU21-03-00411, U01 CA195568.

Circulating tumour DNA (ctDNA) is a promising biomarker for diffuse large B-cell lymphoma (DLBCL) risk stratification and treatment response assessment, but real-world studies were limited. Using a targeted sequencing approach (521-gene panel), we showed that (1) baseline ctDNA level correlated with tumour burden and was an independent predictor of treatment outcome, (2) achievement of minimal residual disease (MRD) negativity was associated with a better treatment outcome and (3) interim MRD-positivity combined with positron emission tomography/computed tomography scan-positivity identified a high-risk subgroup of DLBCL patients. Baseline ctDNA level and treatment related achievement of MRD negativity are valuable prognostic tools in DLBCL to improve risk stratification in routine clinical practice.

Introduction The International Prognostic Index (IPI, NEJM, 1993) has guided risk stratification in large B-cell lymphoma (LBCL) for over three decades. Its simplicity and clinical utility have driven its widespread adoption, even in the era of precision medicine. However, the prognostic value of the IPI and its components may not be uniform across the age spectrum of LBCL patients. We evaluated the contemporary prognostic performance of the IPI in a large international cohort, focusing on age-stratified performance and the relative contributions of its individual components, grouped as patient- and disease-related factors. Methods We harmonized and pooled data from 6,941 patients with newly diagnosed systemic LBCL treated with R-CHOP-like regimens (R-CHOP, R-miniCHOP, DA-EPOCH-R, R-CHOEP, R-CHOP+X) with complete IPI data from three prospective cohorts: NiHiL (Czech Republic, n=4,590; 2010–2023), LEO (USA, n=1,789; 2015–2020), and MER (USA, n=562; 2010–2015). IPI components included age, ECOG performance status (PS) as patient-related, and Ann Arbor clinical stage, serum lactate dehydrogenase (LDH), and extranodal (EN) involvement as disease-related factors. Prognostic performance was evaluated using multivariable Cox regression models and C-statistics. Age-stratified analysis was performed for: ≤40 years (7%; n=492), 41–60 years (28%; n=1,909), 61–80 years (59%; n=4,080), and >80 years (7%; n=460). The primary endpoint was overall survival (OS). Results In the pooled cohort, the median age was 66 years (range 18–95). Compared to the original IPI cohort (n=3,273), our cohort was older (age >60 years: 66% vs 41%) but otherwise comparable: ECOG PS 2–4 (25% vs 24%), clinical stage III–IV (63% vs 66%), elevated LDH (60% vs 52%), and >1 EN involvement (32% vs 30%). In multivariable analysis, age (≤60 vs >60 years) was the strongest predictor of OS (HR=2.78; vs HR=1.96 in original IPI report), followed by ECOG PS (HR=2.07; vs 1.80), clinical stage (HR=1.50; vs 1.47), LDH (HR=1.41; vs 1.85, all P<0.01); EN involvement was not significant (P=0.50, HR=1.03 vs 1.48). Risk group distribution shifted from the original IPI: 28% were low-risk (0–1 factor; vs 35%), 23% low-intermediate (2; vs 27%), 24% high-intermediate (3; vs 22%), and 25% high-risk (4–5; vs 16%). Corresponding 2-year OS rates were 95%, 86%, 78%, and 63%, superior to those of the original IPI cohort (87%, 67%, 55%, 44%; pre-rituximab era). Age was associated with an increasing risk of mortality, with a more pronounced rise beyond 60 years. In a piecewise Cox model, the HR per year was 1.04 for ages ≤60 (linear), 1.05 for 61–69 (accelerated), and 1.06 for ≥70 years (exponential, all P<0.01). ECOG PS retained prognostic significance across age groups: HRs were 1.87 (P=0.06) for ≤40 years, 1.63 (P<0.01) for 41–60, 2.22 (P<0.01) for 61–80, and 1.43 (P<0.01) for >80 years. The strength of other IPI components declined with age. Clinical stage showed decreasing impact: HR 2.08 (P=0.09), 1.97 (P<0.01), 1.49 (P<0.01), and 1.31 (P=0.05). LDH was significant only up to age 80 years: HRs were 2.07 (P=0.04), 1.96 (P<0.01), 1.32 (P<0.01), and 1.21 (P=0.13). EN involvement was non-significant across all groups: HRs were 1.37 (P=0.34), 1.26 (P=0.05), 0.98 (P=0.74), and 1.06 (P=0.67). The IPI C-statistics was 0.676 overall and declined with age: ≤40 years (0.708), 41–60 years (0.683), 61–80 years (0.641), and >80 years (0.598). We next stratified the IPI factors into patient- (age, ECOG PS) and disease-related (clinical stage, LDH, EN involvement). In patients ≤40 years, disease-related factors outperformed patient-related (C-index 0.693 vs 0.602), as in the 41–60 group (C-index 0.677 vs 0.589). In patients aged 61–80, predictive values were comparable (0.613 vs 0.622). In >80-year-olds, both declined substantially (0.580 vs 0.568). Conclusion The IPI remains a valuable prognostic tool in LBCL, particularly among younger patients. However, its overall predictive accuracy declines with age. In patients ≤60 years old, prognosis is primarily driven by disease-related factors, suggesting a role for molecular classifiers to enhance risk stratification. In older patients, incorporation of additional patient-level factors, such as comorbidities and nutritional status could be considered. These findings underscore the need for more individualized prognostic tools for LBCL patients. Funding: NU21-03-00411, P50 CA97274, U01 CA195568, Charles University Haematology-Oncology Cooperatio Program.

A large body of evidence suggests that hypoxia drives aggressive molecular features of malignant cells irrespective of cancer type. Non-Hodgkin lymphomas (NHL) are the most common hematologic malignancies characterized by frequent involvement of diverse hypoxic microenvironments. We studied the impact of long-term deep hypoxia (1% O2) on the biology of lymphoma cells. Only 2 out of 6 tested cell lines (Ramos, and HBL2) survived ≥ 4 weeks under hypoxia. The hypoxia-adapted (HA)b Ramos and HBL2 cells had a decreased proliferation rate accompanied by significant suppression of both oxidative phosphorylation and glycolytic pathways. Transcriptome and proteome analyses revealed marked downregulation of genes and proteins of the mitochondrial respiration complexes I and IV, and mitochondrial ribosomal proteins. Despite the observed suppression of glycolysis, the proteome analysis of both HA cell lines showed upregulation of several proteins involved in the regulation of glucose utilization including the active catalytic component of prolyl-4-hydroxylase P4HA1, an important druggable oncogene. HA cell lines demonstrated increased transcription of key regulators of auto-/mitophagy, e.g., neuritin, BCL2 interacting protein 3 (BNIP3), BNIP3-like protein, and BNIP3 pseudogene. Adaptation to hypoxia was further associated with deregulation of apoptosis, namely upregulation of BCL2L1/BCL-XL, overexpression of BCL2L11/BIM, increased binding of BIM to BCL-XL, and significantly increased sensitivity of both HA cell lines to A1155463, a BCL-XL inhibitor. Finally, in both HA cell lines AKT kinase was hyperphosphorylated and the cells showed increased sensitivity to copanlisib, a pan-PI3K inhibitor. In conclusion, our data report on several shared mechanisms of lymphoma cell adaptation to long-term hypoxia including: 1. Upregulation of proteins responsible for glucose utilization, 2. Degradation of mitochondrial proteins for potential mitochondrial recycling (by mitophagy), and 3. Increased dependence on BCL-XL and PI3K-AKT signaling for survival. In translation, inhibition of glycolysis, BCL-XL, or PI3K-AKT cascade may result in targeted elimination of HA lymphoma cells.

Introduction. Circulating tumor DNA (ctDNA) was identified as a powerful biomarker for treatment response evaluation and outcome prediction in malignant tumors, including diffuse large B-cell lymphoma (DLBCL). However, more studies are necessary to validate ctDNA utilization in general, and specifically in a real-world setting to facilitate standardization and transition into routine clinical practice. Therefore, we assessed pre-treatment ctDNA in 169 unselected previously untreated DLBCL patients consequently uniformly treated with R-CHOP chemoimmunotherapy at five academic hematology centers in the Czech Republic. Methods. ctDNA was analyzed by CAPP-Seq (CAncer Personalized Profiling by deep Sequencing) and a custom panel of 521 genes. DNA alterations were identified by VarScan2. ctDNA concentration was calculated from average variant allele frequencies and reported as human haploid genome equivalents (hGE) per ml of plasma. Germinal center B-cell like (GCB) and non-GCB subtyping was done by immunohistochemistry. Genetic classes were analyzed by LymphGen 2.0, COSMIC (Catalogue Of Somatic Mutations In Cancer) signatures by SigProfiler. Results. In all patients, the median age at diagnosis was 64 years (range 24-81), clinical stage III–IV in 68%, more than one extranodal involved sites in 40%, PS ECOG 2–4 in 24%, elevated LDH in 60%, International Prognostic Index (IPI) 3–5 in 50%, bulky disease over > 7.5 cm in 40%, and non-GCB DLBCL subtype in 47% of patients. Median follow-up was 2.73 years. The 2-year progression free and overall survivals (PFS and OS) were 73.6% and 84.3%, respectively. Without any threshold for input DNA quality and quantity, ctDNA was detected in 136 out of 169 analyzed patients (80%). Clinical characteristics of patients with and without detected ctDNA did not show any differences. The median plasma ctDNA concentration was 995 hGE/ml. ctDNA concentration was significantly higher in patients with clinical stage III–IV (median 1416.9 vs. 542.7 hGE/ml, p = 2.62e-5), age > 60 years (median 1110.7 vs. 691.2, p = 0.04), PS ECOG 2–4 (median 2130.9 vs. 824.2 hGE/ml, p = 2.62e-5), elevated LDH serum levels (median 1804.1 vs. 513.3 hGE/ml, p = 1.14e-9), bulky disease over > 7.5 cm (median 2234.9 vs. 624.8 hGE/ml, p = 4.58e-6), and in patients with IPI 3-5 (median 1646.7 vs. 631.7 hGE/ml, p = 8.33e-5). To find a pre-treatment ctDNA plasma concentration that would identify high risk patients (based on PFS analysis), patients from the General University Hospital (n = 72) were used as a discovery cohort and patients from other centers (n = 64) as a validation cohort (clinically well-balanced). In the discovery cohort, the threshold was determined at 3000 hGE/ml using Harrell's C-index. High ctDNA (> 3000 hGE/ml, 32% of patients) was associated with inferior PFS (2-year PFS 46% vs. 88%, 5-year PFS 38% vs. 71%, HR 3.99, p = 0.001). In the validation cohort, ctDNA concentration > 3000 hGE/ml (19% of patients) was also associated with inferior PFS (2-year PFS 50% vs. 78%, 5-year PFS 40% vs. 67%, HR 2.9, p = 0.024). It confirmed identified threshold that was consequently used for further analyses. In all patients, high ctDNA level (> 3000 hGE/ml, 26% of patients) was associated with inferior PFS (2-year PFS 48% vs. 83%, 5-year PFS 38% vs. 66%, HR 3.37, p < 0.001), and, importantly, also with inferior OS (2-year OS 74% vs. 88%, 5-year OS 54% vs. 75%, HR 2.56, p = 0.016). Moreover, ctDNA was an independent prognostic factor for PFS in multivariate analysis with IPI score (high ctDNA, HR 2.12, p = 0.029; IPI ordinal, HR 1.42, p = 0.01). High ctDNA patients had more advanced clinical stage, worse PS ECOG, higher LDH levels, higher IPI score (all with p < 0.001), and higher proportion of bulky disease (p = 0.005). The LymphGen assigned a genetic subtype to 44% of cases, reflecting the capability of the LympGen model to classify 50-60% of tumors (if copy number and translocation information is available). COSMIC mutational signatures showed differences between genetic and cell of origin subtypes, e.g., activation induced cytidine deaminase activity specifically in non-GCB DLBCL. Conclusion. Our study confirmed pre-treatment ctDNA concentration as a strong and independent risk factor associated with unfavorable DLBCL survival in a routine clinical setting, allowing parallel genetic and biological evaluation. First two authors contributed equally. Supported by NU21-03-00411, DRO-VFN00064165, LX22NPO5102, and SVV 260637.