Abstract Recent clinical trials have underscored the value of repeated minimal residual disease (MRD) measurements as a highly sensitive method for detecting subclinical disease and enabling dynamic risk stratification in hematologic malignancies. Despite its clinical potential, the complex and heterogeneous nature of MRD kinetics presents significant challenges for interpreting and integrating it into routine clinical decision‐making. In this study, we present a comprehensive, model‐based workflow for the longitudinal analysis of MRD trajectories designed to improve relapse risk prediction. We applied this newly developed workflow to a cohort of patients with mantle cell lymphoma (MCL). MRD measurements were collected from both bone marrow (BM) and peripheral blood (PB) over time, stored in the Fondazione Italiana Linfomi MCL0208 clinical trial. Using our functional MRD workflow, we defined four MRD dynamics that collapsed into two clinically relevant groups: favorable (rapid, sustained negativization) and unfavorable (persistent or fluctuating MRD). Patients with unfavorable profiles showed significantly shorter time to progression (TTP), with hazard ratio (HR) = 4.18 (95% CI: 2.44–7.14) in BM and HR = 5.71 (95% CI: 2.86–11.42) in PB. External validation in the European MCL Network “Younger trial” confirmed the predictive power of this stratification, with Kaplan–Meier analyses demonstrating significant prognostic discrimination. The most informative temporal windows for patient clustering vary by tissue. Early‐phase BM assessments offer greater discriminatory power, whereas late‐phase assessments are most informative in PB. These findings indicate that longitudinal MRD assessment in PB represents a clinically actionable strategy that could reduce dependence on invasive BM procedures.

BACKGROUND:Adding ibrutinib to standard, first-line immunochemotherapy improves failure-free survival in adult patients aged 18-65 years with mantle cell lymphoma, according to the first results from the TRIANGLE trial. With prolonged follow-up, we investigated whether the addition of autologous stem-cell transplantation (ASCT) to an ibrutinib-containing regimen improves failure-free survival, and evaluated effects on overall survival. METHODS:We conducted a three-arm, randomised, open-label, phase 3 superiority trial (TRIANGLE) in 165 secondary or tertiary clinical centres, with experience in mantle cell lymphoma treatment and the capability to perform ASCT or an association with such a centre, in 13 European countries and Israel. Patients aged 18-65 years with untreated, stage II-IV mantle cell lymphoma and suitable for ASCT were randomly assigned (1:1:1) to control group A or experimental groups A + I or I. Randomisation was done using computer-generated random numbers and stratified by study groups and Mantle Cell Lymphoma International Prognostic Index risk groups. Treatment in group A consisted of six alternating, 21-day cycles of R-CHOP (intravenous rituximab 375 mg/m2 on day 0 or 1, cyclophosphamide 750 mg/m2 on day 1, doxorubicin 50 mg/m2 on day 1, vincristine 1·4 mg/m2 on day 1 [up to a maximum of 2 mg], and oral prednisone 100 mg on days 1-5) and R-DHAP or R-DHAOx (intravenous rituximab 375 mg/m2 on day 0 or 1, intravenous or oral dexamethasone 40 mg on days 1-4, high-dose intravenous cytarabine 2 × 2 g/m2 for 3 h every 12 h on day 2, plus either intravenous cisplatin 100 mg/m2 over 24 h on day 1 [R-DHAP] or intravenous oxaliplatin 130 mg/m2 on day 1 [R-DHAOx]), followed by ASCT. In group A + I, oral ibrutinib (560 mg daily) was added on days 1-19 of R-CHOP cycles and as 2-year maintenance after ASCT. In group I, ibrutinib was given the same way, but ASCT was omitted. Rituximab maintenance was allowed in all treatment groups according to national guidelines. Three pairwise, one-sided, log-rank tests for the primary outcome (failure-free survival) were statistically monitored. The primary analysis was by intention to treat and included all randomly assigned patients, ignoring protocol deviations. Safety was assessed in randomly assigned patients who started any trial treatment component of the respective treatment phase. The trial is registered with ClinicalTrials.gov (NCT02858258) and is complete. FINDINGS:Between July 29, 2016, and Dec 28, 2020, 870 patients (662 [76%] were male, 208 [24%] were female) were randomly assigned to group A (n=288), group A + I (n=292), or group I (n=290). After median follow-up of 54·9 months (95% CI 54·4-56·0), group A + I did not show superiority over group I, with 4-year failure-free survival of 82% (95% CI 78-87) versus 81% (76-86; hazard ratio [HR] 0·86 [one-sided 98·33% CI 0·00-1·27]; one-sided p=0·21). Group A + I remained superior to group A (82% [78-87] vs 70% [65-76]; HR 0·63 [one-sided 98·33% CI 0·00-0·89]; one-sided p=0·0026) and, as before, group A did not show superiority over group I (70% [65-76] vs 81% [76-86]; HR 1·45 [one-sided 98·33% CI 0·00-2·02]; one-sided p=0·99). 4-year overall survival was 88% (95% CI 84-92) in group A + I versus 81% (76-85) in group A (HR 0·59 [95% CI 0·38-0·92], p=0·0036) and 90% (87-94) in group I versus 81% (76-85) in group A (0·57 [0·36-0·90], p=0·0019). During maintenance or follow-up, the most common grade 3-5 adverse events were haematological disorders, reported in 127 (54%) of 234 patients in group A + I versus 74 (28%) of 269 in group I and 56 (23%) of 240 patients in group A, and infections, reported in 80 (34%) of 234 patients in group A + I versus 71 (26%) of 269 in group I and 37 (15%) of 240 patients in group A. Infections and infestations were the most common fatal adverse events during maintenance or follow-up, occurring in four (2%) of 234 patients in group A + I and five (2%) of 269 patients in group I. INTERPRETATION:After a prolonged follow-up of 55 months, both ibrutinib-containing groups showed relevant improvements not only in failure-free survival-a modified form of progression-free survival-but also in overall survival. In contrast, the addition of ASCT to an ibrutinib-containing regimen had no supplementary benefit but increased toxicity. Induction treatment with ibrutinib and R-CHOP plus R-DHAP (or R-DHAOx), followed by 2 years of maintenance treatment with ibrutinib, should be considered as a new standard of care for younger patients with mantle cell lymphoma. FUNDING:Janssen.

Brexucabtagene-autoleucel (brexu-cel) is an anti-CD19 chimeric antigen receptor T-cell (CAR-T) product approved for relapsed/refractory Mantle Cell Lymphoma (MCL) after two prior treatment lines, including Bruton tyrosine kinase inhibitors (BTKi). Patients with high-risk (hr) disease—defined by high-intermediate or high-risk MIPI-c, p53 overexpression, or TP53 alterations—have a poor prognosis, underscoring the need for improved first-line strategies. The European Mantle Cell Lymphoma Network therefore designed a phase II trial to investigate the incorporation of brexu-cel into first-line therapy for hr MCL. CARMAN is a randomized controlled, international, multicenter, open-label phase II trial evaluating efficacy, safety, and tolerability of an abbreviated induction followed by first-line brexu-cel and 6 months Ibrutinib maintenance (Arm A) as compared to standard of care induction and maintenance (Arm B). In Arm A, induction consists of two cycles of ibrutinib plus rituximab (I + R) followed by two cycles of R-CHOP plus ibrutinib (I). R-CHOP + I may be omitted in patients achieving complete or partial remission after two cycles of I + R, who then receive one additional I + R cycle before brexu-cel infusion and I maintenance. Arm B comprises a TRIANGLE-like regimen based on age, fitness, and investigator choice (alternating R-CHOP plus ibrutinib/R-DHAP or IR-bendamustine), followed by IR maintenance. Overall, 150 patients from five European countries are randomized 1:1. The primary endpoint is failure-free survival from randomization, with failure event defined as the earliest of stable disease at the end of induction (Arm B, or Arm A if brexu-cel is not infused) or within 12 weeks from CAR-T-cell infusion (Arm A), disease progression after induction, or death from any cause. Secondary endpoints include efficacy (overall and complete response rates and PET-negative CR rate 6 months from randomization as assessed according to Lugano criteria, molecular remission rate as measured by MRD), safety and tolerability (adverse events graded according to CTCAE), and patient-reported quality of life as measured by the EORTC-QLQ-C30 and EORTC-QLQ-NHL-HG29 questionnaires. When complete, CARMAN will provide important information on efficacy and safety in first-line CAR-T cell therapy in hr MCL and has the potential to prepare a practice changing confirmatory trial for these difficult-to-treat patients. Recruitment is ongoing. EU clinical trial number: 2022-502405-15-00.

The TRIANGLE trial established an ibrutinib-containing therapy without autologous stem-cell transplantation (ASCT) as the new standard for younger, treatment-naïve patients with mantle cell lymphoma (MCL). However, the benefit of rituximab maintenance (RM) within this novel standard is unclear. We investigated whether RM improves progression-free survival (PFS) and overall survival (OS) with acceptable toxicity when added to the experimental arms of TRIANGLE. This secondary analysis of TRIANGLE included patients randomly assigned to ibrutinib-containing therapy without (I) or with (A + I) ASCT who responded to induction/ASCT. RM was given per national and center practice. PFS and OS of patients with and without RM were compared with inverse probability of treatment weighted Kaplan-Meier curves and log-rank tests. Among responders after induction/ASCT (I: 274; A + I: 237), RM was given to 61% (I) and 64% (A + I). RM prolonged PFS in ibrutinib-containing treatment arms (I: log-rank test: P = .003, 4-year PFS probability RM v no RM, 85% v 73%; A + I: P < .001, 90% v 75%). There were trends toward prolonged OS in RM groups. RM groups were at higher risk of grade 3 to 5 infectious toxicity (I: 34% v 11%; A + I: 41% v 18%). Our findings support adding RM to BTK inhibitor treatments in younger, untreated patients with MCL to achieve prolonged remission.

The clinical course of Mantle Cell Lymphoma (MCL) varies between individual patients. Early detection of risk is crucial to assign MCL patients to novel treatment strategies. Most of the established biomarkers of outcome require specifically trained pathologists or molecular analysis. Here we introduce MAIPI (MCL Artificial Intelligence Prognostic Index), a deep learning algorithm trained only on Hematoxylin and Eosin (H&E) images of diagnostic biopsies of n = 428 MCL patients from clinical trials to assess prognosis. The capability of MAIPI to predict disease outcome was validated in an independent cohort of n = 140 patients treated with immunochemotherapy with and without ibrutinib. MAIPI selects areas of interest by itself and provides prognostic information independent of the MCL International Prognostic Index (MIPI) and Ki67 and without the need of molecular testing or expert pathologists evaluation.

Introduction: Immunochemotherapy remains the cornerstone of treatment for Mantle Cell Lymphoma (MCL). However, 25% of patients experience early progression, with survival rates of less than two years. Current prognostic tools, such as the MCL International Prognostic Index (MIPI), and poor prognostic histological and genetic features are insufficient for stratifying patients into individualized therapeutic strategies. This study aimed to identify biomarkers for high-risk MCL patients using an integrated analysis of clinical and biological factors. Methods: We analyzed data from 299 patients enrolled in the LyMa phase 3 trial, with a focus on high-risk patients, defined by refractoriness to immunochemotherapy or relapse within 12 months post-autologous stem cell transplantation. We used optical genome mapping (OGM) on frozen samples, alongside whole-exome sequencing (WES), RNA sequencing, and DNA methylation arrays analyses on FFPE tumor biopsies to identify genetic, transcriptomic and epigenetic alterations. Machine learning models, including random forest analysis and Partial Least-Squares Discriminant Analysis (PLS-DA), were employed to predict high-risk MCL status. Results: Among the 299 patients, 31 (10.4%) were identified as high-risk (HR) with a median overall survival of 8.5 months after relapse. HR patients exhibited significantly higher levels of LDH, higher-risk MIPI scores (45% vs. 16%, p<0.001), Ki-67 >30% (71% vs. 31%, p<0.001) and blastoid/pleomorphic histology (32% vs. 9%, p<0.001). In multivariate analysis, only high-risk MIPI score, and Ki-67 >30% were associated with HR MCL. These factors were insufficient to specifically capture HR patients, as one-third of long-term responders would have been misidentified as high-risk. The high-risk (HR) subgroup displayed a greater burden of complex genetic alterations, with significantly increased frequencies of TP53 alterations (OR 25.4, p < 0.001), CDKN2A deletions (OR 4.5, p = 0.015), RB1 deletions (OR 4.9, p = 0.024), MYC gains (OR 5.8, p = 0.047), and MIR17HG gains (OR 11.8, p = 0.013). To improve predictive accuracy, an integrative analysis combining well-established prognostic markers with gene alterations assessed by WES, was performed. Random forest analysis achieved a test accuracy of 91% when predicting HR MCL status, with a ROC AUC of 96%. The sensitivity was 84% and the specificity was 96%, with a misclassification rate of 14%. The most influential features included the Ki-67 index, histological subtype, TP53 alterations, MIPI score, and gains of MYC and MIR17HG. Unsupervised Uniform Manifold Approximation and Projection (UMAP) analysis of gene expression profiling on 49 FFPE samples, including 15 HR MCLs, showed that HR MCLs tended to cluster together, but the distinction was not perfect. Supervised analyses, using PLS-DA, indicated potential overfitting, suggesting that transcriptomic signals alone are insufficient for perfect discrimination. In contrast, DNA methylation analysis of 29 FFPE samples, including 12 HR MCLs, revealed a distinct epigenetic signature that robustly discriminated HR MCLs from control cases. Supervised approaches (PLS-DA) identified differentially methylated probes (DMPs, n=225) that perfectly discriminated HR MCL from controls. Importantly, this epigenetic signature was validated in an independent cohort (Barcelona cohort, n=64). To explore the genome-wide impact of DNA methylation on gene expression, we performed correlation analyses between promoter methylation and transcriptomic data across all protein-coding genes. A subset of genes showed significant correlations, with a predominant inverse relationship in HR cases, absent in controls, indicating that promoter hypermethylation may drive transcriptional deregulation in this subgroup. Notably, CHL1, a tumor suppressor, and KLHL6, associated with chemoresistance, demonstrated strong inverse correlations between methylation and expression, supporting their involvement in HR MCL pathogenesis. Conclusion: This study provides an integrated characterization of high-risk MCL, identifying a novel epigenetic signature that outperform traditional prognostic markers. Our baseline epigenetic approach may enhance patient stratification and support the development of personalized therapies. These results support the combined analysis of genetic and epigenetic features to capture MCL's full biological complexity.

Hypothetical strategy allows to define an estimand for the pure treatment effect subjected to the original randomized treatments on overall survival (OS) without impact from effective subsequent therapies. Novel statistical methods including Inverse-Probability-of-Censoring Weights analysis (IPCW) and Two-Stage estimation (TSE), have been proposed for handling initiation of subsequent treatment. However, OS can vary across different types of subsequent therapies, which is ignored by these methods. Motivated by the GADOLIN trial, we propose modified methods (m-IPCW, m-TSE) to account for two different types of subsequent therapy. RCT data were simulated with various scenarios. The occurrence of subsequent therapies was simulated to vary with individual patient's characteristics and randomized treatment. The performance of different methods was evaluated using bias and root-mean-square deviation compared to the true log hazard ratio in OS without subsequent therapies. Across simulated scenarios, both modified and standard TSE and IPCW methods demonstrated superior performance to naive censoring approach. The m-TSE outperformed its standard counterpart, as confirmed by the application in the GADOLIN trial. Our results suggested that in the presence of different effective subsequent therapies, statistical methods differentiating subsequent therapy types might yield more valid estimators for investigational treatment effect on OS under the hypothetical strategy.

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.

Adding ibrutinib to first-line immunochemotherapy (Ibru-R-chemo) showed superiority in younger mantle cell lymphoma (MCL) patients in the TRIANGLE trial (NCT02858258). To investigate response mechanisms and kinetics across treatment arms, we genotyped 57 patients from cell-free (cf)DNA using targeted-capture sequencing and investigated measurable residual disease (MRD) in cfDNA and peripheral blood by targeted-sequencing and qPCR. Pre-treatment cfDNA and circulating tumor (ct)DNA levels predicted outcomes, and precisely genotyped all patients. Circulating tumor cell (CTC)-clearance was more frequent and rapid than ctDNA-clearance across arms. At interim staging (IS), 55% of patients were ctDNA-positive while 35% and 41% were CTC-positive by qPCR and immunoglobulin gene (IG)-NGS. At end of induction, 43% were ctDNA-positive, while 15% (qPCR) and 25% (IG-NGS) were CTC-positive. MRD by qPCR was most predictive for outcomes. Ibru-R-chemo seemed to overcome TP53mut-mediated risk (hazard ratio 1.9 vs. 10) and induce early MRD response, represented by enhanced CTC (71% vs. 57%) and ctDNA clearance (59% vs. 24%) at IS. Flow-cytometry-based immunomonitoring showed ibrutinib’s influence on inhibitory T-cell phenotypes, showing ≥25% reduction in PD1+ and PD1+ KLRG1+ CD4+-T-cells in four patients. Taken together, besides direct anti-B-cell efficacy, ibrutinib improves chemotherapy efficiency by reconstituting an effective immune system and enhancing immune cell control.

Immunochemotherapy induces long-term responses in patients with follicular lymphoma. However, the toxicity of chemotherapy remains a relevant challenge. The Bruton tyrosine kinase inhibitor ibrutinib has shown significant activity in patients with indolent B-cell lymphoma. Combining ibrutinib with obinutuzumab may, therefore, be an attractive chemotherapy-free option. We conducted a prospective, single-arm, multicenter phase II trial to evaluate the chemotherapy-free regimen of obinutuzumab plus ibrutinib in patients with previously untreated advanced-stage follicular lymphoma. Patients received six 21-day cycles of ibrutinib and obinutuzumab for induction and 12 additional 2-month cycles for maintenance. The primary endpoint was 1-year progression-free survival (PFS). The study was powered to detect an improvement of 10% over the 1-year PFS of 85%. Ninety-eight patients were enrolled in the trial. The median follow-up was 5.5 years. After induction, five patients (5%) had a complete response and 82 (85%) had a partial response. The 1-year PFS was 80%, missing the prospected improvement of a 1-year PFS of 85% (P=0.93). The median PFS was 4.5 years; median duration of response and overall survival were not reached. The most common adverse events of grade 3/4 were neutropenia, lung infection, hypertension, fatigue, rash and thrombocytopenia. The trial of a chemotherapy-free regimen of obinutuzumab and ibrutinib in follicular lymphoma patients failed to demonstrate a 10% improvement in the primary efficacy endpoint. However, the combination produced durable and deep responses and had an acceptable safety profile. Trial registration, EudraCT-Number: 2014-005164-15.

Background:Covalent BTK inhibitors are the backbone of treatment for patients with Waldenström's Macroglobulinemia (WM). Addition of rituximab to ibrutinib has shown a remarkable efficacy in WM in the iNNOVATE trial, including patients carrying CXCR4 mutations or MYD88 wildtype (Dimopoulos et al, NEJM 2018, Buske et al. JCO 2022). In addition, the proteasome inhibitor Bortezomib (B) has shown significant activity in WM as single agent or combined with Rituximab, Dexamethasone and Cyclophoshamide (DRC) (Buske et al., JCO 2023). The ECWM-2 trial of the European Consortium for Waldenström's Macroglobulinemia (NCT03620903) aimed at evaluating the efficacy and toxicity of Bortezomib-Ibrutinib/Rituximab (B-IR) as first line treatment in WM. Methods:In this multicenter European single-arm phase II trial, treatment naïve patients with WM requiring therapy received 6 cycles (C) (d=28) of Bortezomib (1.6 mg/ m2 s.c. d1,8,15), Rituximab (375 mg/m2 i.v (C1d1), 1400 mg absolute s.c (C2-6 d1) and Ibrutinib (420 mg p.o. daily) followed by maintenance with Rituximab (1400 mg absolute s.c; D1 every 2nd month) combined with Ibrutinib for 24 months and subsequent ibrutinib treatment until progression or non-tolerated toxicity. Primary endpoint was the 1-year progression free survival (PFS) rate (1YPFS). Secondary endpoints included response rates, PFS, overall survival (OS), and toxicity. Plasma cell-free DNA (cfDNA) and digital droplet PCR (ddPCR) was used for identifying and quantifying MYD88L265P mutational burden in patients at different time points before and during treatment. Results: 53 patients were included and started trial treatment. Median age was 63 years (range 36-84), 62% were male and 70% of patients had intermediate/high risk according to the ISSWM prognostic score. Median baseline hemoglobin was 10.1 g/dl (7.1-14.5) and median baseline IgM 33.9 g/l (3.05-102.87). Mutational status was available for 51 patients with 31 pts (60.8%) showing mutated MYD88 (MYD88MT) and CXCR4 wildtype (CXCR4WT), 18 patients (35.3%) MYD88MT/CXCR4MT and 2 pts (3.9%) MYD88 wildtype (MYD88WT). Of note, no patients showed progression after a median follow-up of 37 months. The primary endpoint 1YPFS was 93% (38/41, p<0.001 in a one-sided exact binomial test to reject 1YPFS ≤ 60%), with 3 deaths. 2YPFS was 0.88 (95% CI: 0.79-0.97). OS probabilities were identical to PFS probabilities due to the fact of lacking progressions. B-IR reduced rapidly deep responses with an overall response rate (ORR) and a major response rate (MRR) of 98% and 70%, respectively, after 3 cycles, with a median reduction of IgM serum levels by 74%. At best response 98% of all patients achieved a MRR with 100% ORR. The proportion of patients with VGPR/CR increased over time with 19% versus 28% versus 38% after 6 cycles, 12 cycles and at best response, respectively. Median time to major response was 2.8 months. Responses were largely independent of CXCR4 mutations with an MRR of 76 and 70 % in the CXCR4WT vs CXCR4MT patients, respectively, at end of induction. In total 104 cfDNA plasma samples from 32/53 patients were analyzed by ddPCR for MYD88L265P mutation: 93% of patients (30/32) showed MYD88L265P at baseline (median AF: 3.8%; range 50%-0.24%) with 41% of samples reaching MRD negativity and an almost 1 log reduction among MRD positive cases (median residual AF: 0.63%; range 5.6%-0.1%) after 3 cycles and a MRD negativity rate of 65% (median residual AF of 0.16% (range 2.34%-0.035%) at end of induction. Grade ≥3 AEs related to treatment occurred in 45% of all patients. Most common grade ≥3 AEs included COVID-19 pneumonia (13.0 %), lower respiratory tract infection (11.1 %), and anemia (7.4%). Overall, 12 pts (22.6 %) developed infections grade ≥3. Peripheral sensory neuropathy occurred in 8 patients (all grade 1 and 2). There have been 8 deaths in the course of the study in total, 5 caused by COVID-19 and three caused by respiratory tract infection. Conclusion: With a 1YPFS of 93%, a major response rate of 98%, 65% MRD negativity after 6 cycles of treatment and no observed progression of disease after a median follow-up of 37 months, B-IR shows impressive efficacy in WM. All deaths were caused by respiratory infections, for which COVID-19 was confirmed as the cause in the majority of cases, reflecting patient recruitment of this trial in the COVID-19 pandemic. These data characterize B-IR as a novel and powerful treatment option for patients with WM.

Background: Follicular lymphoma (FL) remains a clinical challenge due to its relapsing course and difficulty in tailoring therapies to individual risk. Frontline treatment commonly includes an anti-CD20 antibody with either bendamustine or CHOP/CVP. Bendamustine has been associated with longer progression-free survival (PFS) compared to CHOP/CVP in the GALLIUM trial. However, bendamustine was also associated with more grade 3-5 infections, prolonged T-cell reduction, fatal events, and second neoplasms(Hiddemann, 2017). Additional concerns include its immunosuppressive effects and potential to impair subsequent T-cell based therapies including CAR-T cells (Iacoboni, 2024). This highlights the need to better identify patients who benefit most and least from bendamustine. Here, we used our gene mutation data to identify predictive biomarkers of bendamustine benefit and validated our findings in a large international real-life cohort. Methods: The GALLIUM trial (NCT01332968) enrolled 1,202 patients with previously untreated, advanced-stage FL (stage III/IV or bulky stage II) and ECOG 0–2, all requiring treatment per GELF criteria. Patients were randomized to receive rituximab or obinutuzumab with CHOP, CVP, or bendamustine allocated by the treating center (Marcus, 2017). Targeted sequencing of recurrently mutated genes was available for diagnostic biopsies from 418 evaluable cases. Kaplan-Meier and Cox regression analyses were used to correlate gene mutations with clinical outcomes. The median follow-up in the GALLIUM cohort was 6.9 years; 283 patients (68%) received bendamustine, 135 (33%) received CHOP or CVP. The validation cohort consisted of 473 patients from Australia (N=208), USA (N=138), and Canada (N=127). The median follow-up in the validation cohort was 5.5 years; 181 patients (38%) received bendamustine- and 292 (62%) received CHOP- or CVP-based immunochemotherapies. Results: In this updated analysis with longer follow-up, we confirmed that the previously described clinicogenetic risk model m7-FLIPI (Pastore, 2015) predicts PFS in CHOP/CVP–treated patients, but not in those receiving bendamustine. The mutation status of two of the seven m7-FLIPI genes was associated with differing PFS outcomes in patients treated with CHOP/CVP vs bendamustine: Patients with wild-type EZH2 had significantly shorter PFS with CHOP/CVP-based therapies compared to EZH2-mutated cases (HR=0.43, p=0.020); notably, no difference in PFS was observed in the bendamustine cohort. In addition, CREBBP mutations were associated with shorter PFS after CHOP/CVP (HR=2.05, p=0.037), but not after bendamustine. None of the other m7-FLIPI gene mutations predicted PFS in bendamustine-treated patients. Based on these findings, we combined the EZH2 and CREBBP mutation status to stratify patients. Only patients with both a CREBBP mutation and wild-type EZH2 showed a clinically relevant benefit from bendamustine, with significantly longer PFS compared to CHOP/CVP-treated patients (HR=0.44, p=0.00011) and similar OS (HR=0.62, p=0.27). All other patients had similar PFS with either regimen (HR=1.05, p=0.86), but we observed a trend to longer OS with CHOP/CVP (HR=2.19, p=0.11). These results were confirmed in the validation cohort: EZH2 mutations were associated with longer PFS in patients receiving CHOP/CVP (HR=0.62, p=0.025), while CREBBP mutations were associated with shorter PFS (HR 1.42, p=0.033). Again, none of the m7-FLIPI gene mutations were predictive of PFS in bendamustine-treated patients. Stratification based on EZH2 and CREBBP mutation status validated the previous results: only patients with both a CREBBP mutation and wild-type EZH2 had a PFS benefit from bendamustine (HR 0.61, p=0.027), again with similar OS (HR=0.75, p=0.48). Likewise, all other patients experienced similar PFS with either regimen (HR=1.07, p=0.73), but again had a trend towards longer OS when treated with CHOP/CVP (HR=1.75, p=0.068). Conclusion: Our findings suggest that EZH2 and CREBBP mutation status can inform treatment selection in FL. Patients with CREBBP mutations and wild-type EZH2 benefit most from bendamustine with significantly longer PFS and similar OS; all other patients had similar PFS with either regimen but a trend towards longer OS with CHOP/CVP. These data may indicate a distinct biology in FL with CREBBP mutations and wild-type EZH2, and support the use of simple mutation testing to guide personalized therapy in newly diagnosed advanced-stage patients.