Zanubrutinib-rituximab followed by shortened chemoimmunotherapy as frontline treatment for mantle cell lymphoma (CHESS): a phase II trial

Domain: biology
Published: 2026-03-28
Editorial outcome: major_revision

Abstract
Combination of Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib and rituximab has shown promising efficacy in mantle cell lymphoma (MCL). This phase II trial evaluated the second-generation BTK inhibitor zanubrutinib plus rituximab as induction, followed by shortened chemoimmunotherapy as frontline treatment for MCL (NCT04624958). Eligible patients had histologically confirmed stage II–IV disease requiring immediate therapy and no prior MCL-related systemic treatment. Patients received zanubrutinib-rituximab for up to 12 cycles (part A); those achieving complete response (CR) or experiencing disease progression proceeded to four cycles of R-DHSAOx (rituximab, dexamethasone, cytarabine and oxaliplatin) (part B). Patients with CR after part B received zanubrutinib maintenance for one year. The primary endpoint was the CR rate at part A completion. Forty-two patients were enrolled. The CR rate at part A completion was 88% (95% confidence interval [CI], 74-96), and 86% (95% CI, 72-95) at part B completion. Hematologic toxicities predominated: the most common grade 3-4 adverse events were neutropenia (7%) in part A, and thrombocytopenia (77%) and neutropenia (49%) in part B. In conclusion, zanubrutinib-rituximab induction followed by shortened R-DHAOx is efficacious with manageable safety as frontline therapy for MCL. This strategy allows for a reduction in chemotherapy cycles and warrants validation in randomized controlled trials.

Introduction
Mantle cell lymphoma (MCL), a subtype of mature B-cell lymphoma, is characterized by translocation of t(11;14)(q13;q32) and cyclin D1 overexpression.1 The standard of care aggressive therapy for MCL is cytarabine-based chemoimmunotherapy regimens, such as the LyMa regimen (rituximab, dexamethasone, cytarabine, and platinum [R-DHAP] × 4 cycles followed by rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisone [R-CHOP] × 4 cycles in non-complete response [CR] patients),2 the NORDIC regimen (dose-intensified R-CHOP alternating with rituximab and high-dose cytarabine × 6 cycles),3 R-CHOP + ibrutinib alternating with R-DHAP × 6 cycles,4 rituximab and bendamustine (BR) × 3 cycles followed by rituximab and high-dose cytarabine × 3 cycles,5 with optional autologous stem cell transplantation (ASCT) and maintenance therapy. For patients not suitable for aggressive induction therapies and ASCT, several less aggressive regimens without high-dose cytarabine, such as bortezomib, rituximab, cyclophosphamide, doxorubicin, and prednisone (VR-CAP) regimen,6 BR regimen,7 BR + acalabrutinib regimen,8 are recommended. Although cytarabine plays a significant role in the frontline treatment of MCL, the aforementioned cytarabine-based regimens are associated with significant myelosuppression and an increased risk of secondary malignancies.4,5,9,10

Treatment of MCL has been revolutionized by Bruton’s tyrosine kinase (BTK) inhibitors during the past decade.11-15 Several trials have shown promising efficacy of combining BTK inhibitors and rituximab as frontline treatment in MCL patients with older age or indolent disease.16-20 In the WINDOW-1 trial, the BTK inhibitor ibrutinib and rituximab as induction therapy followed by shortened R-HCVAD (rituximab, hyper-fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone) alternating with R-methotrexate-cytarabine regimen in previously untreated MCL patients resulted in 87% complete response (CR) rate after induction and 89% CR rate after R-HCVAD regimen.21

Compared with ibrutinib, the second-generation BTK inhibitor zanubrutinib has higher selectivity for BTK and thus fewer off-target toxicities.22-24 In comparison to ibrutinib, zanubrutinib demonstrated less cardiovascular toxicity and lower rate of treatment discontinuation with comparable efficacy in phase 3 trials.25,26 In a phase 2 trial for relapsed or refractory MCL, zanubrutinib achieved 78% CR rate and 33-month median progression-free survival (PFS).13 Considering the favorable efficacy and safety profile, using zanubrutinib as a frontline treatment may represent a promising approach.

We conducted this phase II trial (CHESS) to evaluate the safety and efficacy of zanubrutinib plus rituximab as induction therapy, followed by a shortened course of chemoimmunotherapy in previously untreated MCL patients. R-DHAOx regimen (rituximab, dexamethasone, cytarabine, and oxaliplatin) was selected based on its proven efficacy in clinical practice and several key factors: 1) improved outcomes of adding high-dose cytarabine to chemoimmunotherapy;27 2) a high CSR rate (77%) in the LyMa trial with the R-DHA-platinum regimen,2 which is recommended by the National Comprehensive Cancer Network (NCCN) guideline; 3) a tendency for longer disease control duration observed with oxaliplatin in subgroup analyses of the LyMa trial compared to cisplatin and carboplatin;28 and 4) Given that the potential overlapping toxicity of thrombocytopenia in high-dose cytarabine and carboplatin, as well as lower nephrotoxicity and ease of administration with oxaliplatin than cisplatin,29,30 we chose oxaliplatin as the platinum used in our regimen. In this work, we show that zanubrutinib-rituximab induction followed by shortened chemoimmunotherapy yield promising efficacy and an acceptable safety profile as frontline treatment for MCL. Based on transcriptome features, we identified three molecular subtypes that correlate with treatment response and survival outcomes.

Results
Patients
Between December 1, 2020, and December 13, 2023, 42 patients were enrolled. Baseline characteristics are summarized in Table 1. The median age was 57 years (interquartile range [IQR], 51-64), and 35 patients (83%) were male. Based on tumor tissue samples, Ki-67 was ≥ 30% in 29 (69%) patients; histological subtype was blastoid/pleomorphic in 5 (12%) patients. The risk based on the simplified Mantle Cell Lymphoma International Prognostic Index (sMIPI) was low, intermediate, and high in 24 (57%), 15 (36%), and 3 (7%) patients, respectively. Minimal residual disease (MRD) in bone marrow was detected in 36 (86%) patients. In the 35 patients with known MRD status in peripheral blood, the bone marrow and peripheral blood results were concordant except in one patient (positive in bone marrow but negative in peripheral blood) (Supplementary Table 1). In the six patients without bone marrow involvement at baseline, none had detectable MRD in peripheral blood, and they underwent MRD detection only in peripheral blood in subsequent time points.

The study profile is shown in Figure 1. All 42 enrolled patients received part A induction treatment; 39 proceeded to part B chemoimmunotherapy. All 29 patients who completed the 4-cycle part B chemoimmunotherapy, as well as five patients who discontinued part B due to toxicities, received maintenance therapy with zanubrutinib. As of November 20, 2024, 23 patients had finished the study treatment, and 11 were still receiving maintenance therapy.

Response
The median treatment cycle in part A was 4 (IQR, 4-4), and all 42 patients were evaluable for response. For the evaluation of the primary endpoint, 37 (88%; 95% CI, 74-96) patients achieved CR at part A completion. The remaining five patients (12% [95% CI, 4-26]) experienced disease progression (Table 2 and Supplementary Figure 1). CR was confirmed by 18F-fluorodeoxyglucose positron-emission tomography (PET) in all patients, plus bone marrow biopsy in patients with bone marrow involvement at baseline. Detailed information on the patients with progressive disease (PD) is provided in Supplementary Table 2. The best overall response rate (ORR) and CR rate was 98% (95% CI, 87-100) and 88% (95% CI, 74-96), respectively. The median time to CR was four months (IQR, 4-4). MRD was negative at part A completion in 33 patients (Supplementary Table 1), and the MRD-negative CR rate was 74% (31/42; 95% CI, 58-86). The dynamic change of MRD and its relationship with treatment response are presented in Supplementary Figure 2. In post-hoc subgroup analyses, the CR rate at part A completion was 40% (2/5) in patients with blastoid/pleomorphic disease and 95% (35/37) in those with classic/small cell disease; 33% (1/3) in patients with TP53 mutation and 94% (32/34) in those with wild type TP53. There were no obvious differences in other subgroups, including age, sex, Eastern Cooperative Oncology Group (ECOG) performance status, stage, bone marrow involvement, lactate dehydrogenase, Ki-67 and sMIPI risk (Supplementary Table 2).

Thirty-nine patients proceeded to part B treatment with R-DHAOx regimen. At part B completion, among the 39 patients, the CR rate was 92% (36/39; 95% CI, 79-98), and disease progressed in the remaining three patients (8%; 3/39; 95% CI, 2-21; all had PD in part A) (Table 2). Thirty-six of the 37 patients with CR at part A completion remained in CR and had undetectable MRD at part B completion (Figure S2 and Table S1); one patient was lost to follow-up after part A.

For the overall response following part B completion, 36 patients achieved CR (86% [95% CI, 72-95]), three patients had PD (7% [95% CI, 2-20], and three patients were not evaluable due to not receiving part B treatment. In post-hoc subgroup analyses, the CR rate at part B completion was 40% (2/5) in patients with blastoid/pleomorphic disease and 92% (34/37) in those with classic/small cell disease; 33% (1/3) in patients with TP53 mutation and 91% (31/34) in those with wild-type TP53 (Table S2). There were no significant differences in other subgroups.

Survival outcomes
With a median follow-up of 23 months (IQR, 14-29), the median PFS and overall survival (OS) were not reached. The analysis of the median PFS and OS were planned but the data were immature. The 2-year PFS and OS rate was 85% (95% CI, 75-97) and 90% (95% CI, 75-100), respectively (Figure 2A, B). The median duration of response (DoR) was 17 months (IQR, 8-23). Five patients experienced disease progression and three patients died due to lymphoma progression, all were non-CR patients in part A. Detailed clinical information and salvage therapies of the five PD patients in the current trial are summarized in Supplementary Table 3 and Supplementary Table 4. None of the 37 patients with CR at the completion part A had disease progression; one died of COVID-19 pneumonia. Patients with blastoid/pleomorphic disease had inferior survival outcomes than those with classic/small cell disease (Supplementary Figure 3A and Supplementary Figure 4A). Patients with TP53 mutations had inferior PFS, but no OS difference was observed. Neither PFS nor OS differed between the patients with different age (< 60 vs ≥ 60 years), stage (II vs III-IV), Ki-67 (< 30% vs ≥ 30%) or sMIPI risk (low vs intermediate vs high risk) (Supplementary Figure 3 and Supplementary Figure 4).

Adverse events
In part A, the most common treatment-related AEs (TRAEs) were neutropenia (11 [26%] of 42 patients), fatigue (11 [26%]), lymphocytopenia (11 [26%]), and rash (9 [21%]) (Table 3). Hemorrhage (all grade 1 or 2) occurred in four (10%) patients. The most common grade 3-4 TRAEs were neutropenia (3 [7%]) and lymphocytopenia (3 [7%]). No patient needed zanubrutinib dose reduction in part A. Zanubrutinib dose suspension was required in five (12%) of 42 patients (three patients for neutropenia, one for infection, and one for elevated liver enzymes). None of the patients needed recombinant human granulocyte colony-stimulating factor (rhG-CSF) supportive treatment during part A. There were no treatment-related deaths.

In part B, grade 3-4 events were predominantly hematological. The most common grade 3-4 TRAE was thrombocytopenia (30 [77%] in 39 patients), followed by leukocytopenia (23 [59%]), and neutropenia (19 [49%]) (Table 3). The most common non-hematological TRAEs were elevated liver enzymes (20 [51%]) and fatigue (20 [51%]), but most were grade 1-2. Dose reduction of chemotherapeutic agents was required in 32 (82%) of 39 patients due to toxicities (27 patients for thrombocytopenia, four for neutropenia, and one for elevated liver enzymes). Of these, 25 patients had their dose of cytarabine reduced by one dose level to 75% of the original dose, and seven patients required a reduction of two dose levels to 50%.

Thrombopoietin receptor agonists (TPO-RAs) or recombinant human thrombopoietin (rhTPO) were given in 9 patients as primary prevention for thrombocytopenia, and in 13 patients as secondary prevention. Five patients still experienced severe thrombocytopenia despite supportive care with TPO-RAs or rhTPO and dose reduction, and therefore discontinued treatment. rhG-CSF was given in 26 patients as primary prevention for neutropenia, and in all the 19 patients who had experienced grade 3-4 neutropenia as secondary prevention. None of the patients experienced further occurrence of severe neutropenia after receiving rhG-CSF supportive therapy and dose reduction. Chemotherapy dose suspension was required in 20 (51%) of 39 patients due to toxicities (18 patients for thrombocytopenia, one for neutropenia, and one for elevated liver enzymes). Five patients discontinued chemotherapy permanently due to toxicities (all due to grade 4 thrombocytopenia). There were no treatment-related deaths.

TRAEs in the zanubrutinib maintenance phase are summarized in Table 3. One patient experienced grade 3 neutropenia and recovered after supportive treatment with rhG-CSF, after which the zanubrutinib dose was reduced to 80 mg twice daily. No other dose reductions were required during the maintenance phase. Zanubrutinib dose suspension was required in six (18%) of 34 patients (two patients for neutropenia, two patients for fatigue, and one patient each for thrombocytopenia and infections). No patient required treatment discontinuation.

This trial was conducted during the COVID-19 pandemic. One patient died from COVID-19 pneumonia and respiratory failure during part B. A total of 30 patients tested positive for SARS-CoV-2 at least once, and 17 patients required treatment suspension due to SARS-CoV-2 infection.

Transcriptional and genomic analyses
Unsupervised hierarchical clustering of differentially expressed genes (DEGs) based on bulk RNA sequencing in 37 available tumor tissue samples at baseline revealed three MCL molecular subtypes (M1-M3) (Figure 3A). M1 (n=4) was characterized by activation of cell-cycle pathways, including G2M pathways, E2F targets, MYC targets and DNA replication (Figure 3B and Figure S5A). M2 (n=26) was characterized by activation and regulation of T cells (T cell activation, T cell antigen processing and presentation, and T cell co-stimulation) and DNA repairing (Figure 3B). M3 (n=7) was characterized by upregulation of inflammatory pathways (e.g., JAK-STAT3 signaling and TNF-α signaling) (Figure 3B and Supplementary Figure 5A). Activation of specific pathways (e.g., hypoxia, angiogenesis, extracellular matrix organization, and cell matrix adhesion) and the increased infiltration of stromal cells and immunosuppressive cells (cancer-associated fibroblasts and myeloid-derived suppressor cells) (Supplementary Figure 5B) indicated a more complex tumor microenvironment in M3. The genomic profiles including mutated genes and tumor mutation burden in each molecular subtype are presented in Supplementary Figure 6.

None of the four patients of the M1 subtype achieved CR in part A; all the four patients experienced PD within 9 months and died within 24 months (Figure 3C, D). In contrast, all patients of the M2 subtype obtained early-CR. In the seven patients of the M3 subtype, two achieved early-CR and the remaining five achieved late-CR (Figure 3C). The distribution of histological subtypes, sMIPI risk, and Ki-67 in different molecular subtypes are presented in Supplementary Figure 5C. In the 24 patients of the M2 subtype with positive MRD at baseline, MRD became undetectable in all 24 patients at the completion of part A. In contrast, the MRD status remained unchanged from baseline to part A completion in all patients of the M1 and M3 subtype (Figure 3E).

Methods
Trial design and patients
This trial protocol was approved by the ethics committees of participating centers (ethics committee of Sun Yat-sen University Cancer Center, ethics committee of Huizhou Municipal Central Hospital, and ethics committee of the Second Hospital of Dalian Medical University). The trial was conducted according to the Declaration of Helsinki and all relevant regulations regarding the use of human study participants. All patients provided written informed consent. The consent to publish clinical information potentially identifying individuals was obtained. This trial is registered with ClinicalTrials.gov identifier: NCT04624958 (https://clinicaltrials.gov/study/NCT04624958; first posted on November 12, 2020).

This investigator-initiated, single-arm, phase II trial (CHESS) was conducted in three centers in China. This was an open-label trial. Participants, investigators, attending physicians and outcome assessors were not blinded. Previously untreated patients aged 18-75 years with histologically confirmed, Ann Arbor stage II-IV MCL, requiring immediate anti-lymphoma treatment were eligible. Additional inclusion criteria included: ECOG performance status score of 0 to 2, at least one measurable lesion, and adequate organ and bone marrow function. Patients were excluded if they had central nervous system involvement, hemophagocytic lymphohistiocytosis, history of active bleeding or hemorrhagic diathesis, required treatment with anticoagulants or strong CYP3A inhibitors. The full list of inclusion and exclusion criteria is presented in trial protocol in the supplementary notes.

Study treatment
The treatment consisted of three phases. A treatment schema describing the study treatment process is presented in Supplementary Figure 7. In part A (induction phase), zanubrutinib (160 mg, twice a day, orally) and rituximab (375 mg/m2, intravenously on day 1) were administered in 28-day cycles until CR, disease progression or unacceptable toxicity, with a maximum of 12 cycles. In part B (chemoimmunotherapy phase), R-DHAOx (rituximab 375 mg/m2 intravenously on day 1; dexamethasone 20 mg intravenously on days 1-4; cytarabine 2 g/m2 intravenously on days 2-3 [1 g/m2 for patients > 65 years of age]; and oxaliplatin 130 mg/m2 intravenously on day 1) was administered in 21-day cycles until disease progression or unacceptable toxicity, with a maximum of four cycles. In maintenance phase, zanubrutinib (160 mg orally, twice a day) was given until disease progression or unacceptable toxicity for up to one year. Unacceptable toxicity was defined as meeting any of the criteria for dose discontinuation of zanubrutinib or chemotherapeutic drugs as described in protocol during treatment, or if continued treatment is deemed inappropriate by the investigators.

Patients achieving CR or PD in part A proceeded to part B; patients with PR or stable disease (SD) in part A received 12 cycles of zanubrutinib-rituximab treatment prior to proceeding to part B. Patients with CR at part B completion proceeded to maintenance phase; young patients (≤ 65 years) with detectable MRD were eligible for ASCT before maintenance. Patients with PR, SD or PD at part B completion were taken off the trial.

Assessments and endpoints
Responses were evaluated by the investigators every two cycles in part A and part B, and every three cycles during maintenance therapy using PET or computerized tomography (CT) according to the Lugano 2014 criteria.32 A bone marrow biopsy is required to confirm CR in patients with bone marrow involvement at baseline. Safety assessment was performed upon all patient visits. Adverse events (AEs) were graded according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events (version 5.0).

MRD in bone marrow and/or peripheral blood was measured using flow cytometry with a sensitivity of 10-4 at baseline and upon the completion of part A and part B. Bone marrow aspiration was performed at completion of part A and part B in patients with bone marrow involvement at baseline but not routinely in patients without bone marrow involvement at baseline. The collection of peripheral blood for MRD evaluation was performed at baseline and upon the completion of part A and part B. Detectable MRD in either bone marrow or peripheral blood was considered MRD-positive.

The primary endpoint was the CR rate at the completion of part A in the intention-to-treat population, defined as proportion of patients with CR. Secondary endpoints included overall response (proportion of patients with CR plus PR), MRD, PFS, OS, DoR, time to CR, and adverse events for part A, part B and maintenance phase. PFS was defined as the time from treatment initiation to disease progression or death, whichever occurs earlier, and was censored at the last follow-up date in alive patients without disease progression. OS was defined as the time from treatment initiation to patient death or the last follow-up. DoR was defined as the time from first response to disease progression or death, whichever occurs earlier. Time to CR was defined as the time from treatment initiation to the first documentation of CR.

RNA-seq and transcriptomic analyses
Total RNA from formalin-fixed paraffin-embedded (FFPE) tumor samples obtained at baseline were extracted using the RNeasy Mini Kit (Qiagen). RNA purity was assessed using Nanodrop 2000 for A260/280 and A260/A230 ratios (Thermo Fisher Scientific). All RNA samples were quantified by Qubit 3.0 using the RNA BR Assay Kit (Life Technologies). RNA integrity was assessed using Bioanalyzer 2100 (Agilent). RIN value (RNA integrity number) higher than 6.5 was required. RNA sequencing libraries were prepared using KAPA Stranded RNA-Seq Kit with RiboErase (KAPA Biosystems). Final library was quantified using KAPA Library Quantification Kit (KAPA Bio- systems), and sequenced on the Illumina HiSeq4000 platform using PE150 sequencing chemistry (Illumina, USA) to an average of 50 M reads per sample.

FASTQ file quality control was performed using Trimmomatic,39 where N bases and low quality (score < 15) bases were removed. Reads aligning to rRNA and tRNA sequences were removed. Cleaned reads were aligned to the human reference genome (hg19) using STAR v 2.5.2b.40 Transcripts were quantified using RSEM,41 which uses an expectation maximization algorithm to optimally assign reads that maps to multiple transcripts.

Based on treatment response at the completion of part A, we categorized the patients into three groups: early-CR (achieving CR within four cycles of zanubrutinib-rituximab regimen), late-CR (achieving CR after more than four cycles of zanubrutinib-rituximab regimen), and non-CR. Differentially expressed genes (DEGs) between non-CR, late-CR and early-CR tumors were identified using edgeR package (v4.3.16) with its default parameters. DEGs were defined as genes with |log (fold change)| > 0.5 and P-value < 0.01. RNA-seq data were deconvolved with the ESTIMATE algorithm (v1.0.13) in R software and TIDE algorithm (v1.3) in python 3.12.7 software for immune infiltration analyses.

Samples were classified into three subtypes based on the normalized FPKM values of DEGs by applying an unsupervised hierarchical clustering method. When performing unsupervised hierarchical clustering, we calculated the distance matrix using the Euclidean distance metric (dist function with method = "euclidean") and applied the hclust function with the Ward's minimum variance method (method = "ward.D"), which minimizes the total within-cluster variance. The results were visualized using a dendrogram to represent the hierarchical structure of the clusters. To specify a desired number of clusters, we used the cutree function to cut the dendrogram. To visualize subtype characteristics, a heat map was generated using ComplexHeatmap R package (v 2.21.1) based on normalized FPKM values of DEGs.

Gene set enrichment analysis (GSEA) of Hallmark and Reactome pathways, and Gene Ontology terms were performed using clusterProfiler R package (v4.13.3) based on pre-ranked differentially expressed gene panels of each subtype. Single-sample GSVA scores of GOBP, Hallmark and Reactome gene sets in the MSigDB database (v2024.1.Hs) were calculated using GSVA R package (v1.53.23) and the representative significantly enriched pathways in the three subtypes were plotted using the pheatmap R package (v1.0.12).

DNA sequencing and genomic analyses
DNA from FFPE tissue and matched germline samples were extracted using a DNeasy Blood & Tissue Kit (Qiagen). Purified DNAs were quality checked using Nanodrop 2000 for A260/280 and A260/A230 ratios (Thermo Fisher Scientific). All DNA samples were quantified by Qubit 3.0 using the dsDNA HS Assay Kit (Life Technologies). Sequencing libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems). Target enrichment was performed using the xGen Exome Research Panel and Hybridization and Wash Reagents Kit (Integrated DNA Technology, USA) according to the manufacturer’s protocol. Libraries were subjected to PCR amplification with KAPA HiFi Hot-Start ReadyMix (KAPA Biosystems). The purified library was quantified using the KAPA Library Quantification Kit (KAPA Biosystems), and its fragment size distribution was analyzed using a Bioanalyzer 2100 (Agilent). Sequencing was performed on Illumina HiSeq4000 platform using PE150 sequencing chemistry (Illumina, USA).

FASTQ file quality control was performed using Trimmomatic,39 where N bases and low quality (score < 20) bases were removed. Pair-end reads were aligned to the human reference genome (hg37) using Burrows-Wheeler Aligner (BWA) with default parameters, followed by PCR deduplication with Picard V2.9.4 (Broad Institute, MA, USA). Local realignment around indels and base quality score recalibration was performed with the Genome Analysis Toolkit (GATK 3.4.0). Somatic single-nucleotide variants (SNVs) were identified using VarDict and MuTect2 for matched tumor-normal samples, and Haplotyper2 for tumor-only samples. To eliminate sequencing artifacts and common germline events in tumor-only samples, both SNVs and small insertions/deletions (indels) were filtered using an in-house common variant database that includes 2000 whole-exome sequencing (WES) normal samples. Final list of mutations was annotated using vcf2maf (available on GitHub). Resulting mutation list was filtered through an internal list. Somatic mutation calls were further filtered using the following criteria: (i) minimum 4 reads supporting the variant; (ii) ≥ 2% variant allele frequency (VAF); (iii) not present in public databases (Exome Variant Server, 1,000 genomes project and Exome Aggregation Consortium) at population frequency > 1%; (iv) in protein-coding regions. Oncoprint, constructed by R (3.5.3), was used to view the overall mutation landscape of patients. Tumor mutation burden (TMB) was counted by summing all base substitutions and indels in the coding region of targeted genes, including synonymous alterations to reduce sampling noise and excluding known driver mutations as they are over-represented in the panel.42

Statistical analysis
Sample size was estimated according to a single-stage phase 2 design based on the following assumptions: 1) CR rate of 70% for historical control2 and 90% with the regimen in this trial; 2) 90% power and two-sided 5% type I error; 3) a dropout rate of 10%. The calculation yielded 42 patients. Response assessment was performed in the full analysis set (ITT population) and per protocol set. Safety assessment was performed in the safety analysis sets, which was divided into three datasets and each contained patients who received the study treatment at least once in part A, part B, or maintenance phase. Descriptive statistics were used to present baseline characteristics, response rates, and rate of AEs. The 95% confidence interval (CI) of rates was calculated using the Clopper-Pearson method. Survival outcomes and 95% CIs were estimated using the Kaplan-Meier method. The 2-year PFS and OS were calculated. The log-rank test was used to evaluate the difference in PFS and OS between subgroups. Post-hoc analyses included the subgroup analyses of CR rate at part A completion, CR rate at part B completion, PFS, and OS. Statistical analyses were performed using SPSS software (version 25.0), R software (version 3.6.1) and python (version 3.12.7). Two-sided P of < 0.05 was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "please see attached", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "Although the combination described is of interest in MCL and window studies have now been performed with ibrutinib,\nacalabrutinib and zanubrutinib, this manuscript lacks depth in the discussion of the study results in the context of the other\ntrials, including those of BTKi +R bendamustine, R-BTKi in low volume and good risk disease, and even Zanubrutinib in\nMCL, as well as any data from these other trials, there are other aspects that need significant improvement as detailed\nbelow.\nAbstract\nThe abstract lacks any background which justifies the use of this therapy in this disease. Please add.\nPlease describe further the patient population. The description of ‘untreated MCL’ is insufficient.\nPlease define any acronyms used in the abstract eg “MCL”\nPlease explain further why 4 cycles of ‘R-DHAOX’ therapy is considered shortened therapy after the le Gouill LYMA study\nused 4 cycles and only RCHOP14 in those in <PR.\nThe treatment paradigm is not completely clear in the abstract or the methods-do all patients get RDHAOX or only those in\n‘cr’ or ‘pd’ included what if they reached the 12 cycles with SD or PR?-I know there were not any of these but clarification\nwould be helpful. Why do only those in CR get maintenance?\nPlease include the confidence interval for the CR rate.\nIntroduction.\nPlease provide more details about the regimens not just the name of the trials. The ‘Triangle regimen’ doesn’t exist, it is a\nrandomised trial with different regimens and no clear new standard, one arm assessed therapy with a BTKi, not\nimmunotherapy. Please amend.\nFurthermore, these regimens and autograft are not suitable for all patients with MCL, so please clarify this and discuss.\nIt would be useful to provide information about the outcomes from the chemotherapy backbone the authors have chosen for\ntheir study (RDHAOx), as this was not the standard therapy for the LyMA trial,\nThere is limited information on the data from Zanubrutinib in MCL in this introduction. Please refer to and discuss the\nrelevant information to provide jusfication of the use in frontline.\nMethods:\nplease include a methods section after the introduction, rather than at the end, with the standard eligibility, enrolment\nprocedures, treatment details, assessments, statistics and methods of evaluation, the entire protocol can be included,\nhowever the poignant information needs to be in the methods-the detailed sections on laboratory methods are less important\nthan the aforementioned requirements for a clinical trial. Why was an age of 75 chosen if patients are fit, and not being\ntransplanted.\nPlease define what ‘unacceptable toxicity’ is in terms of cessation of Part A.\nPlease clarify why patients in PR were taken off the study.\nPlease reference the statistical assumptions used in this trial.\nResults:\n1. It is unclear if the patient population is one that is transplant eligible or not, and although the upper age limit was 75, few\npatients over 65 appear to have been enrolled, and whether it included those with non-measurable disease.\n2. Why do the authors think there is such a high proportion in this study who are male? (83%), even with a male\npredominance, this seems high.\n3. Please state how many patients underwent PET, given the methods state PET or CT was used. Was CR confirmed with a\nbone marrow biopsy as well as PET? And did such a high number really convert from PR to CR in 8 weeks having only\nachieved a PR at cycle 2? It seems a little surprising.\n4. Please clarify if all of the PD continued to 12months of R-zanu, or whether they progressed earlier and received RDHAOx.\n5. Line 104 appears to be an error-the vs should be between the blastoid/classic categories.\n6. Please include dose disruptions as well as dose reductions in the R-zanu group. If there was not dose reduction and Zanu\nis continuous, how was the grade 3+ neutropenia managed in the 7% reported who had it. What was the management of\ninfection such as covid? Was there provision in the protocol for cessation? This study ran during the covid pandemic when\nvaccination was not available, all other BTKi studies had significant issues with COVID yet it is not mentioned here.\n7. Dose reductions of 82% for the chemo is extremely high for this regimen. Please comment on reasons, and details of the\ndose reductions that occurred. Please include description of the GCSF use.\n8. It would be useful to have the TP53 mutation status documented in the results of the manuscript – it is currently quite\ndifficult to find this in a supplementary figure. Was there any difference in response to treatment or outcomes for TP53\nmutated vs wild type patients?\n9. The frequency of TP53 mutation was lower than in other contemporary cohorts of MCL patients who have had WES\nperformed (typically 20-30%). This may have influenced the results.\n10. Why were those requiring anticoagulants excluded from the trial? (point 4 under Exclusion Criteria in the protocol). In\nclinical practice patients on anticoagulation therapy would be eligible to receive a BTKi\n11. For MRD assessment, the authors state ‘bone marrow +/- peripheral blood’ was used. Please elaborate on how this was\ndecided or if it was at random. Not all patients had bone marrow involvement at baseline – did these patients have\ndetectable disease in peripheral blood and were these patients subsequently evaluated using peripheral blood?\n12. In figure S5, the most common somatic mutation is OBSCN. Pathogenic mutations in the obscurin gene have not been\nfound in any lymphomas in online clinical databases, and are most often VUS or benign variants. Could the authors please\ncomment on their finding of OBSCN mutations in 29.7% of the cohort? Was there strong evidence for pathogenicity?\n13. Although the results here are promising, the median follow up time is 23 months and perhaps too short to conclude that\nthere was a high rate of 'sustained' CR.\nDiscussion\nThe reference to the Lugano criteria should be in a methods section, not the discussion.\nThe authors have not discussed studies of BTKi in combination with rituximab, nor any of the R-bendamustine studies which\nare relevant here due to the enrolment of patients up to age 75.\nThe limitations of the study need to be expanded, claims being made about differences in the molecular subgroups in the\nabsence of adequate power for any multivariable analysis need to be softened and noted in the limitations.\nWhy is 4 cycles of RDHAOX considered shortened in the conclusion, when it is given to patients mostly in CR?\nGeneral: there are numerous grammatical errors throughout the manuscript that are obvious to a native English speaker.\nAlthough these do not always detract from the meaning of the phrase, they are noticeable and need to be rectified.", "reviewer_4": "The multicenter, phase II trial (CHESS) evaluated zanubrutinib plus rituximab induction, followed by shortened\nchemoimmunotherapy in untreated MCL patients. The primary endpoint was the CR rate achieved after induction. Overall\nthe report is clear and comprehensive and focussed on the primary and secondary endpoints. There is some interesting\nexploratory analysis included which adds to the impact of the work.\nI have a number of fairly minor points that I believe should be addressed to improve the clarity of presentation.\nPg2. In the abstract please report estimates (CR rate, PFS and OS rate) with appropriate confidence intervals as per journal\nguidelines.\nPg2. The conclusion to the abstract should reflect the findings of a single arm phase II study. It overstates results to say the\ntreatment is \"effective and safe\" without indicating he further evaluation required in phase III. For example, in the discussion\nthe authors state: \"Further randomized controlled trials should be conducted to evaluate the efficacy and safety of the\nproposed treatment regimen in this trial.\" This should be included in any concluding statement .\nPg5. Please add more detail on treatment delivered including dose modification required and delays between cycles. If they\ndid not occur, please state as such --- I note that comment in the Discussion on page 8. For example, the protocol states:\n\"The exposure time and dose of experimental drug therapy were summarized. List the number of patients for whom the dose\nwas adjusted/suspended, along with the reasons for the dose adjustment/suspension.\" Please report this information\nappropriately, as required.\nPg 10. The conclusion should reflect the aims and have a mention of safety also. Please add.\nPg 10. Describe who was blinded or not. For example, participants and clinician not blinded. Was\noutcome assessor\nblinded?\nPg 11/14. Please define the ITT, PAS and safety population more clearly in statistical analysis section and ensure this\nrefects what is stated in the protocol and how analysing was undertaken.\nPg 14. Please clarify if the 5% type-one error is one-sided or two-sided. Fuurthemore, if it is one-sided please clarify the\nimpact this has on the confidence intervals you should report for the primary endpoint.", "reviewer_1_attached": "This clinical trial evaluated the efficacy of zanubrutinib-rituximab induction followed by a shortened\nR-DHAOx chemoimmunotherapy and zanubrutinib maintenance regimen in previously untreated\nmantle cell lymphoma patients. The study demonstrated a high complete response rate, durable\nremission, and manageable toxicities. Additionally, the authors identified three transcriptome based\nmolecular subtypes that correlated with treatment response and survival outcomes. I would recommend\nthe publication in Nature Communications with the following major revisions.\nQ1: The Abstract section would benefit from being streamlined to highlight the key points of this study\nand to emphasize the advantages of this regimen as a frontline treatment for MCL patients.\nQ2: In the Introduction section, please clarify the rationale for selecting R-DHAOx— typically used as\na salvage therapy or as first-line induction for younger lymphoma patients—as the shortened\nchemoimmunotherapy component in this regimen.\nQ3: In addition to the Study Profile, a treatment schema is needed to concisely and clearly illustrate the\nclinical trial design. For example, for patients who experienced disease progression during Part A,\nwould they still receive up to 12 cycles of the zanubrutinib-rituximab regimen or be allowed to enter\nPart B in advance? What salvage therapies were administered to patients who progressed during Part\nB or discontinued chemotherapy due to toxicities?\nQ4: Patients’ genomic information, including TP53 status and complex karyotype, should be included\nin Table 1 (Baseline Characteristics).\nQ5: Post-hoc subgroup analyses following the completion of Part B should also be presented.\nQ6: Subgroup analyses of progression-free and overall survival based on established prognostic\nmarkers in mantle cell lymphoma patients, such as karyotype and TP53 aberrations, should be included.\nQ7: In the Discussion section, the authors provide recommendations based on transcriptomic subtypes.\nPlease summarize which MCL patient populations are likely to benefit from zanubrutinib-rituximab\ninduction followed by shortened R DHAOx chemoimmunotherapy and zanubrutinib maintenance, such\nas age (e.g., above or below 65 years), performance status (fit vs. unfit), etc.\nQ8: The text is clear but would benefit from professional language refinement to further enhance\nreadability."}, "author_response": "Point-by-point responses\nReviewer #1\nThis clinical trial evaluated the efficacy of zanubrutinib-rituximab induction followed by a shortened\nR-DHAOx chemoimmunotherapy and zanubrutinib maintenance regimen in previously untreated\nmantle cell lymphoma patients. The study demonstrated a high complete response rate, durable\nremission, and manageable toxicities. Additionally, the authors identified three transcriptome based\nmolecular subtypes that correlated with treatment response and survival outcomes. I would recommend\nthe publication in Nature Communications with the following major revisions.\nQ1: The Abstract section would benefit from being streamlined to highlight the key points of this study\nand to emphasize the advantages of this regimen as a frontline treatment for MCL patients.\nAnswer: Thank you for the valuable comment. We made the following revisions to the Abstract (Line 27-\n51): (1) The abstract has been streamlined to highlight the clinical findings of this study. (2) The description\nregarding transcriptomic analyses has been removed. (3) The conclusion was expanded to emphasize the\nadvantages of the regimen and potential future directions. (4) Several other modifications to meet the word\ncount limit of 200 words required by the journal guideline.\nLine 27-51:\nAbstract\nThis multicenter, phase II trial evaluated zanubrutinib plus rituximab induction, followed by shortened\nchemoimmunotherapy as frontline treatment for mantle cell lymphoma (MCL). Forty-two patients with\nhistologically confirmed, stage II-IV MCL requiring immediate anti-lymphoma treatment were enrolled.\nPatients received zanubrutinib-rituximab regimen (part A) for up to 12 cycles; subjects achieving\ncomplete response (CR) or disease progression proceeded to four cycles of R-DHAOx (rituximab,\ndexamethasone, cytarabine and oxaliplatin) (part B). Patients with CR at part B completion received\nzanubrutinib maintenance for one year. The primary endpoint was the CR rate at part A completion,\nwhich was 88% (95% confidence interval [CI], 74-96). The CR rate at part B completion was 86% (95%\nCI, 72-95). With the median follow-up of 23 months, the 2-year progression-free survival and overall\nsurvival was 85% (95% CI, 75-97) and 90% (95% CI, 75-100), respectively. Toxicities were moderate in\npart A; hematological adverse events were common in part B. In conclusion, zanubrutinib-rituximab\ninduction followed by shortened R-DHAOx is efficacious and safe as a frontline therapy for MCL. This\nstrategy allows for a reduction in chemotherapy cycles, and should be validated in future randomized\ncontrolled trials. Clinical trial registration: NCT04624958.\nQ2: In the Introduction section, please clarify the rationale for selecting R-DHAOx— typically used as\na salvage therapy or as first-line induction for younger lymphoma patients—as the shortened\nchemoimmunotherapy component in this regimen.\nAnswer: We are grateful for your comment, and expanded the Introduction section to explain the rationale\nfor selecting R-DHAOx: (1) In the MCL Younger trial1, adding high-dose cytarabine to\nimmunochemotherapy increased the CR rate (55% for R-CHOP/R-DHAP vs 39% for R-CHOP). (2) R-\nDHA-platinum regimen is one of the preferred regimens in the NCCN guideline with a high CR rate\n(77%).2 (3) In the subgroup analyses of LyMa trial,3 there was a tendency for longer disease control\nduration with oxaliplatin than cisplatin and carboplatin. (4) Overlapping toxicity of thrombocytopenia in\nhigh-dose cytarabine and carboplatin, as well as lower nephrotoxicity and ease of administration of\noxaliplatin vs cisplatin.4,5\nWe have added relevant content in the Introduction (Line 91-101).\nLine 91-101: To expand the potential use, we chose R-DHAOx (rituximab, dexamethasone, cytarabine,\nand oxaliplatin), another commonly used cytarabine-based regimen in this trial. The R-DHAOx regimen\nwas selected for the following reasons: 1) The improved outcomes of adding high-dose cytarabine to\nchemoimmunotherapy.27 2) R-DHA-platinum regimen is one of the preferred regimens in the National\nComprehensive Cancer Network (NCCN) guideline with a high CR rate (77%) in the LyMa trial.2 3) In\nthe subgroup analyses of LyMa trial, there was a tendency for longer disease control duration than\ncisplatin and carboplatin.28 4) Given that the potential overlapping toxicity of thrombocytopenia in high-\ndose cytarabine and carboplatin, as well as lower nephrotoxicity and ease of administration with\noxaliplatin than cisplatin,29,30 we chose oxaliplatin as the platinum used in our regimen.\nReference:\n1. Hermine O, Hoster E, Walewski J, et al. Addition of high-dose cytarabine to immunochemotherapy before\nautologous stem-cell transplantation in patients aged 65 years or younger with mantle cell lymphoma (MCL\nYounger): a randomised, open-label, phase 3 trial of the European Mantle Cell Lymphoma Network. Lancet.\n2016;388(10044):565-575.\n2. Le Gouill S, Thieblemont C, Oberic L, et al. Rituximab after Autologous Stem-Cell Transplantation in\nMantle-Cell Lymphoma. N Engl J Med. 2017;377(13):1250-1260.\n3. Tessoulin B, Chiron D, Thieblemont C, et al. Oxaliplatin before autologous transplantation in combination\nwith high-dose cytarabine and rituximab provides longer disease control than cisplatin or carboplatin in\npatients with mantle-cell lymphoma: results from the LyMA prospective trial. Bone Marrow Transplant.\n2021;56(7):1700-1709.\n4. Kuter DJ. Managing thrombocytopenia associated with cancer chemotherapy. Oncology (Williston Park).\n2015;29(4):282-294.\n5. Pabla N, Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies. Kidney Int.\n2008;73(9):994-1007.\nQ3: In addition to the Study Profile, a treatment schema is needed to concisely and clearly illustrate the\nclinical trial design. For example, for patients who experienced disease progression during Part A,\nwould they still receive up to 12 cycles of the zanubrutinib-rituximab regimen or be allowed to enter\nPart B in advance? What salvage therapies were administered to patients who progressed during Part\nB or discontinued chemotherapy due to toxicities?\nAnswer: We appreciate your thoughtful suggestions. We added a treatment schema (as Figure S7 in the\nrevised manuscript) to describe the process of study treatment. Patients who experienced disease\nprogression during part A directly proceeded to part B, but not receiving zanubrutinib-rituximab up to 12\ncycles.\nThe salvage therapies and outcomes after disease progression have been added (as Table S3 in the\nrevised manuscript). For patients who discontinued chemotherapy due to toxicities and had CR, they\nproceeded to maintenance therapy with zanubrutinib.\nFigure S7:\nTable S3:\nQ4: Patients’ genomic information, including TP53 status and complex karyotype, should be included\nin Table 1 (Baseline Characteristics).\nAnswer: Thank you for this suggestion, and we added TP53 status in Table 1. Karyotype was not routinely\nevaluated in MCL patients at the participating centers.\nTable 1:\nQ5: Post-hoc subgroup analyses following the completion of Part B should also be presented.\nAnswer: Subgroup analyses at part B completion have been added (Line 154-160). Moreover, we added a\ntable summarizing the subgroup analyses at part A and part B completion (Table S2).\nLine 154-160: For the overall response following part B completion, 36 patients achieved CR (86%\n[95% CI, 72-95]), three patients had PD (7% [95% CI, 2-20], and three patients were not evaluable due\nto not receiving part B treatment. In post-hoc subgroup analyses, the CR rate at part B completion was\n40% (2/5) in patients with blastoid/pleomorphic disease and 92% (34/37) in those with classic/small cell\ndisease; 33% (1/3) in patients with TP53 mutation and 91% (31/34) in those with wild-type TP53 (Table\nS2). There were no significant differences in other subgroups.\nTable S2:\nQ6: Subgroup analyses of progression-free and overall survival based on established prognostic\nmarkers in mantle cell lymphoma patients, such as karyotype and TP53 aberrations, should be included.\nAnswer: We appreciate your suggestion, and added the results of PFS/OS subgroup analyses based on age,\nstage, Ki67, histological subtypes, MIPI risk and TP53 mutation status (Line 170-175, Figure S3 and\nFigure S4).\nLine 170-175: Patients with blastoid/pleomorphic disease had inferior survival outcomes than those with\nclassic/small cell disease (Figure S3A and Figure S4A). Patients with TP53 mutations had inferior PFS,\nbut no OS difference was observed. Neither PFS nor OS differed between the patients with different age\n(< 60 vs ≥ 60 years), stage (II vs III-IV), Ki-67 (< 30% vs ≥ 30%) or sMIPI risk (low vs intermediate vs\nhigh risk) (Figure S3 and Figure S4).\nFigure S3:\nFigure S4:\nQ7: In the Discussion section, the authors provide recommendations based on transcriptomic subtypes.\nPlease summarize which MCL patient populations are likely to benefit from zanubrutinib-rituximab\ninduction followed by shortened R DHAOx chemoimmunotherapy and zanubrutinib maintenance, such\nas age (e.g., above or below 65 years), performance status (fit vs. unfit), etc.\nAnswer: We appreciate your comments, and revised the Discussion section to indicate the MCL patient\npopulations that are likely to benefit from zanubrutinib-rituximab induction followed by shortened R\nDHAOx chemoimmunotherapy and zanubrutinib maintenance (Line 261-265).\nLine 261-265: Additionally, according to the post-hoc subgroup analyses, patients with classic/small cell\ndisease and wildtype TP53 may benefit more from zanubrutinib-rituximab induction and R-DHAOx\nchemotherapy. No significant differences were observed based on other established prognostic markers,\nsuch as Ki-67, Ann Arbor stage and sMIPI risk.\nQ8: The text is clear but would benefit from professional language refinement to further enhance\nreadability.\nAnswer: During the revision, we worked with a professional medical editor (Kehong Zhang, M.D., Ph.D.,\nfrom the Ivy Medical Editing; formerly an Assistant Professor at the Harvard Medical School) to improve\nlanguage readability.\nReviewer #2 (ECR):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (MCL clinical):\nAlthough the combination described is of interest in MCL and window studies have now been performed\nwith ibrutinib, acalabrutinib and zanubrutinib, this manuscript lacks depth in the discussion of the study\nresults in the context of the other trials, including those of BTKi +R bendamustine, R-BTKi in low\nvolume and good risk disease, and even Zanubrutinib in MCL, as well as any data from these other\ntrials, there are other aspects that need significant improvement as detailed below.\nAnswer: Thank you very much for your valuable feedback on our manuscript. We attempted to interpret\nthe trial results in the context of existing literature in the revised manuscript. Specifically, we added\ndiscussion or comparisons with previous trials in the Introduction and Discussion sections: (1) The use of\nzanubrutinib in MCL; (2) Discussion of other cytarabine-based chemotherapy regimens; (3) Discussion of\nBTKi + R regimen in indolent clinical forms of MCL, and BTKi + R or BTKi + BR or BR regimen in\nolder MCL patients. These modifications will be detailed in the point-by-point responses to the following\nquestions.\nAbstract:\nQ1: Post-hoc subgroup analyses following the completion of Part B should also be presented.\nAnswer: Thank you for the valuable comments. We have added the results of subgroup analyses following\npart B completion in the Results section of the main text (Line 154-160 and Table S2). The results were\nnot added to the Abstract due to the word limitation (no more than 200 words in the Abstract).\nLine 154-160: For the overall response following part B completion, 36 patients achieved CR (86%\n[95% CI, 72-95]), three patients had PD (7% [95% CI, 2-20], and three patients were not evaluable due\nto not receiving part B treatment. In post-hoc subgroup analyses, the CR rate at part B completion was\n40% (2/5) in patients with blastoid/pleomorphic disease and 92% (34/37) in those with classic/small cell\ndisease; 33% (1/3) in patients with TP53 mutation and 91% (31/34) in those with wild-type TP53 (Table\nS2). There were no significant differences in other subgroups.\nTable S2:\nQ2: Please describe further the patient population. The description of ‘untreated MCL’ is insufficient.\nAnswer: We appreciate the comment, and expanded description of the study population (Line 29-30).\nLine 29-30: Forty-two patients with histologically confirmed, stage II-IV MCL requiring immediate anti-\nlymphoma treatment were enrolled.\nQ3: Please define any acronyms used in the abstract eg “MCL”\nAnswer: All abbreviations in the abstract (e.g., MCL, CR) are now clearly defined.\nQ4: Please explain further why 4 cycles of ‘R-DHAOX’ therapy is considered shortened therapy after\nthe le Gouill LYMA study used 4 cycles and only RCHOP14 in those in <PR.\nAnswer: We appreciate your insightful comment, and wish to explain that the word \"shortened\" is used in\nthe manuscript in the context of most of the cytarabine-based chemotherapy regimens, such as the 6\nalternating cycles of R-maxiCHOP/ R+high-dose cytarabine (Nordic regimen)1, 6 alternating cycles of R-\nCHOP/R-DHAP2,3, 6-8 alternating cycles of R+hyper-CVAD/R+high dose methotrexate and cytarabine4,\n6 alternating cycles of R+bendamustine/R+high-dose cytarabine5.\nAlthough the LyMa trial6 also utilized 4 cycles of R-DHAP, the intensity of the overall treatment was\nensured by (1) a routine consolidation therapy with HDT+ASCT for all patients; (2) an additional rescue\ninduction therapy with 4 cycles of R-CHOP-14 for patients with PR and whose tumor mass had been\nreduced by less than 75%; (3) 1 cycle of CVP regimen was allowed to reduce tumor burden before R-\nDHAP according to local investigator decision.\nIn our study, we anticipated to use a chemo-free induction (zanubrutinib-rituximab) and maintenance\n(zanubrutinib) to minimize the use of cytotoxic chemotherapy drugs and HDT/ASCT. At the completion\nof part B, all the CR patients were MRD-negative, and were not consolidated with HDT+ASCT. Therefore,\ncompared to the LyMa trial, the treatment intensity in our trial was decreased by using mild induction and\nmaintenance therapies.\nThe Discussion section of the manuscript has been revised to reflects these thoughts (Line 244-252),\nbut the Abstract was not expanded to due to the word limitation.\nLine 244-252: Currently, most of the cytarabine-based chemotherapy regimens, such as the NORDIC\nregimen10, alternating R-CHOP/R-DHAP regimen4,30, and R-hyper-CVAD/R-high dose methotrexate and\ncytarabine regimen31, contain six to eight cycles of treatment. The current trial used a shortened four\ncycles of R-DHAOx regimen. Notably, the LyMa trial2 also used four cycles of R-DHAP, but the\nintensity of the overall treatment was ensured by routine consolidation therapy with HDT/ASCT,\nadditional four cycles of R-CHOP for PR patients, and one cycle of CVP regimen was allowed before\nstudy treatment. The treatment intensity was lower in the current study due to mild chemotherapy-free\ninduction and maintenance therapy to minimize the use of cytotoxic chemotherapy drugs and\nHDT/ASCT.\nReference:\n1. Eskelund CW, Kolstad A, Jerkeman M, et al. 15-year follow-up of the Second Nordic Mantle Cell\nLymphoma trial (MCL2): prolonged remissions without survival plateau. Br J Haematol.\n2016;175(3):410-418.\n2. Dreyling M, Doorduijn J, Giné E, et al. Ibrutinib combined with immunochemotherapy with or without\nautologous stem-cell transplantation versus immunochemotherapy and autologous stem-cell\ntransplantation in previously untreated patients with mantle cell lymphoma (TRIANGLE): a three-arm,\nrandomised, open-label, phase 3 superiority trial of the European Mantle Cell Lymphoma Network.\nLancet. 2024;403(10441):2293-2306.\n3. Hermine O, Hoster E, Walewski J, et al. Addition of high-dose cytarabine to immunochemotherapy before\nautologous stem-cell transplantation in patients aged 65 years or younger with mantle cell lymphoma\n(MCL Younger): a randomised, open-label, phase 3 trial of the European Mantle Cell Lymphoma\nNetwork. Lancet. 2016;388(10044):565-575.\n4. Romaguera JE. et al. High rate of durable remissions after treatment of newly diagnosed aggressive\nmantle-cell lymphoma with rituximab plus hyper-CVAD alternating with rituximab plus high-dose\nmethotrexate and cytarabine. J Clin Oncol. 2005;23(28):7013-7023.\n5. Merryman RW, Edwin N, Redd R, et al. Rituximab/bendamustine and rituximab/cytarabine induction\ntherapy for transplant-eligible mantle cell lymphoma. Blood Adv. 2020;4(5):858-867.\n6. Le Gouill S, Thieblemont C, Oberic L, et al. Rituximab after Autologous Stem-Cell Transplantation in\nMantle-Cell Lymphoma. N Engl J Med. 2017;377(13):1250-1260.\nQ5: The treatment paradigm is not completely clear in the abstract or the methods-do all patients get\nRDHAOX or only those in ‘cr’ or ‘pd’ included what if they reached the 12 cycles with SD or PR?-I\nknow there were not any of these but clarification would be helpful. Why do only those in CR get\nmaintenance?\nAnswer: We thank you for this constructive suggestion, and revised the Abstract to clearly state that if\npatients reached the 12 cycles of part A with SD or PR, they would also proceed to part B (Line 349-371).\nA treatment schema has also been added based on reviewer suggestion (as Figure S7 in the revised\nmanuscript).\nThe design that only patients achieving CR proceeded to maintenance) was based on the NCCN\nguideline: (1) For patients receiving aggressive induction therapy, only CR patients are recommended to\nreceive consolidation with ASCT and/or maintenance therapy. Patients with PR are recommended to\nreceive further chemoimmunotherapy or covalent BTKi if not exposed to covalent BTKi previously (not\napplicable to the current study population). (2) For patients receiving less-aggressive induction therapy,\nCR and PR patients could consider maintenance therapy with rituximab; and for PR patients, they could\nalso receive further chemoimmunotherapy or covalent BTKi.\nIn summary, we did not impose restrictions on the subsequent therapies for patients with PR; instead,\nthey taken off the trial to ensure receiving optimal and individualized treatment. Therefore, only CR\npatients got maintenance.\nLine 349-371: The treatment consisted of three phases. A treatment schema describing the study\ntreatment process is presented in Figure S7. In part A (induction phase), zanubrutinib (160 mg, twice a\nday, orally) and rituximab (375 mg/m2, intravenously on day 1) were administered in 28-day cycles until\nCR, disease progression or unacceptable toxicity, with a maximum of 12 cycles. In part B\n(chemoimmunotherapy phase), R-DHAOx (rituximab 375 mg/m2 intravenously on day 1;\ndexamethasone 20 mg intravenously on days 1-4; cytarabine 2 g/m2 intravenously on days 2-3 [1 g/m2\nfor patients > 65 years of age]; and oxaliplatin 130 mg/m2 intravenously on day 1) was administered in\n21-day cycles until disease progression or unacceptable toxicity, with a maximum of four cycles. In\nmaintenance phase, zanubrutinib (160 mg orally, twice a day) was given until disease progression or\nunacceptable toxicity for up to one year. Unacceptable toxicity was defined as meeting any of the criteria\nfor dose discontinuation of zanubrutinib or chemotherapeutic drugs as described in protocol during\ntreatment, or if continued treatment is deemed inappropriate by the investigators.\nPatients achieving CR or PD in part A proceeded to part B; patients with PR or stable disease (SD) in\npart A received 12 cycles of zanubrutinib-rituximab treatment prior to proceeding to part B. Patients with\nCR at part B completion proceeded to maintenance phase; young patients (≤ 65 years) with detectable\nMRD were eligible for ASCT before maintenance. Patients with PR, SD or PD at part B completion were\ntaken off the trial.\nFigure S7:\nQ6: Please include the confidence interval for the CR rate.\nAnswer: We appreciate this comment, and added 95% confidence intervals for the CR rate (Line 35-37).\nLine 35-37: The primary endpoint was the CR rate at part A completion, which was 88% (95%\nconfidence interval [CI], 74-96). The CR rate at part B completion was 86% (95% CI, 72-95).\nIntroduction:\nQ1: Please provide more details about the regimens not just the name of the trials. The ‘Triangle\nregimen’ doesn’t exist, it is a randomised trial with different regimens and no clear new standard, one\narm assessed therapy with a BTKi, not immunotherapy. Please amend.\nAnswer: Thank you for the helpful suggestions. We added more details about the representative\ncytarabine-based chemoimmunotherapy regimens (Line 55-64). “Triangle regimen” has been replaced\nwith more elaborate descriptions.\nLine 55-64: The standard of care aggressive therapy for MCL is cytarabine-based chemoimmunotherapy\nregimens, such as the LyMa regimen (rituximab, dexamethasone, cytarabine, and platinum [R-DHAP] ×\n4 cycles followed by rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisone [R-CHOP]\n× 4 cycles in non-complete response [CR] patients),2 the NORDIC regimen (dose-intensified R-CHOP\nalternating with rituximab and high-dose cytarabine × 6 cycles),3 R-CHOP + ibrutinib alternating with R-\nDHAP × 6 cycles,4 rituximab and bendamustine (BR) × 3 cycles followed by rituximab and high-dose\ncytarabine × 3 cycles,5 with optional autologous stem cell transplantation (ASCT) and maintenance\ntherapy.\nQ2: Furthermore, these regimens and autograft are not suitable for all patients with MCL, so please\nclarify this and discuss.\nAnswer: We revised the text to describe less aggressive regimens for patients not suitable for cytarabine\nand transplantation (Line 66-69), and the potential risks associated with cytarabine-based regimens (Line\n69-71).\nLine 66-69: For patients not suitable for aggressive induction therapies and ASCT, several less\naggressive regimens without high-dose cytarabine, such as bortezomib, rituximab, cyclophosphamide,\ndoxorubicin, and prednisone (VR-CAP) regimen,6 BR regimen,7 BR + acalabrutinib regimen,8 are\nrecommended.\nLine 69-71: Although cytarabine plays a significant role in the frontline treatment of MCL, the\naforementioned cytarabine-based regimens are associated with significant myelosuppression and an\nincreased risk of secondary malignancies.4,5,9,10\nQ3: It would be useful to provide information about the outcomes from the chemotherapy backbone the\nauthors have chosen for their study (RDHAOx), as this was not the standard therapy for the LyMA trial.\nAnswer: We expanded the Introduction to describe the outcomes from the chemotherapy backbone (R-\nDHAOx), as well as the rationale of selecting oxaliplatin (Line 93-101).\nLine 93-101: The R-DHAOx regimen was selected for the following reasons: 1) The improved outcomes\nof adding high-dose cytarabine to chemoimmunotherapy.27 2) R-DHA-platinum regimen is one of the\npreferred regimens in the National Comprehensive Cancer Network (NCCN) guideline with a high CR\nrate (77%) in the LyMa trial.2 3) In the subgroup analyses of LyMa trial, there was a tendency for longer\ndisease control duration than cisplatin and carboplatin.28 4) Given that the potential overlapping toxicity\nof thrombocytopenia in high-dose cytarabine and carboplatin, as well as lower nephrotoxicity and ease of\nadministration with oxaliplatin than cisplatin,29,30 we chose oxaliplatin as the platinum used in our\nregimen.\nQ4: There is limited information on the data from Zanubrutinib in MCL in this introduction. Please\nrefer to and discuss the relevant information to provide jusfication of the use in frontline.\nAnswer: We expanded the Introduction to provide more relevant data on zanubrutinib in MCL patients,\nas well as the rationale of using it as frontline treatment (Line 81-88).\nLine 81-88: In comparison to ibrutinib, the second-generation BTK inhibitor zanubrutinib has higher\nselectivity for BTK and thus fewer off-target toxicities.22-24 In comparison to ibrutinib, zanubrutinib\ndemonstrated less cardiovascular toxicity and lower rate of treatment discontinuation with comparable\nefficacy in phase 3 trials.25,26 In a phase 2 trial for relapsed or refractory MCL, zanubrutinib achieved\n78% CR rate and 33-month median progression-free survival (PFS).13 Considering the favorable efficacy\nand safety profile, using zanubrutinib as a frontline treatment may represent a promising approach.\nMethods:\nQ1: please include a methods section after the introduction, rather than at the end, with the standard\neligibility, enrolment procedures, treatment details, assessments, statistics and methods of evaluation,\nthe entire protocol can be included, however the poignant information needs to be in the methods-the\ndetailed sections on laboratory methods are less important than the aforementioned requirements for a\nclinical trial. Why was an age of 75 chosen if patients are fit, and not being transplanted.\nAnswer: We appreciate for the valuable suggestion, but the method section should be placed at the end of\nmanuscript based on journal policy/style. The detailed eligibility criteria (Line 337-344), enrolment\nprocedures & treatment details (Line 349-371 and Figure S7), assessments & endpoints & methods of\nevaluation (Line 373-393), statistics (Line 475-491) have been provided in the revised manuscript. Detailed\nmethods of sequencing have been reduced in the main text (Line 395-403) and moved to supplemental\nmaterials.\nUsually, previous trials of MCL defined an upper age of 65 years for patients suitable for ASCT and\nreceived aggressive chemoimmunotherapy. In our study, the selection of age 18-75 years involves the\nfollowing considerations: (1) The R-DHAOx regimen contains cytarabine and oxaliplatin. Patients aged ≥\n75 years often have reduced renal and hepatic function as well as hematopoietic reserve, leading to\npotential higher rates of severe cytopenia and liver/renal dysfunction. An upper age limit of 75 years was\nchosen to consider the balance between patient benefits and risks. (2) Transplantation was not mandatory\nin our trial, and only patients ≤ 65 years with detectable MRD at part B completion were candidate for\nASCT, so we did not set an upper age limit at 65 years.\nLine 337-344: Previously untreated patients aged 18-75 years with histologically confirmed Ann Arbor\nstage II-IV MCL requiring immediate anti-lymphoma treatment were eligible. Additional inclusion\ncriteria included: ECOG performance status score of 0 to 2, at least one measurable lesion, and adequate\norgan and bone marrow function. Patients were excluded if they had central nervous system\ninvolvement, hemophagocytic lymphohistiocytosis, history of active bleeding or hemorrhagic diathesis,\nrequired treatment with anticoagulants or strong CYP3A inhibitors. The full list of inclusion and\nexclusion criteria is presented in the supplementary materials (Protocol).\nLine 349-371: The treatment consisted of three phases. A treatment schema describing the study\ntreatment process is presented in Figure S7. In part A (induction phase), zanubrutinib (160 mg, twice a\nday, orally) and rituximab (375 mg/m2, intravenously on day 1) were administered in 28-day cycles until\nCR, disease progression or unacceptable toxicity, with a maximum of 12 cycles. In part B\n(chemoimmunotherapy phase), R-DHAOx (rituximab 375 mg/m2 intravenously on day 1;\ndexamethasone 20 mg intravenously on days 1-4; cytarabine 2 g/m2 intravenously on days 2-3 [1 g/m2\nfor patients > 65 years of age]; and oxaliplatin 130 mg/m2 intravenously on day 1) was administered in\n21-day cycles until disease progression or unacceptable toxicity, with a maximum of four cycles. In\nmaintenance phase, zanubrutinib (160 mg orally, twice a day) was given until disease progression or\nunacceptable toxicity for up to one year. Unacceptable toxicity was defined as meeting any of the criteria\nfor dose discontinuation of zanubrutinib or chemotherapeutic drugs as described in protocol during\ntreatment, or if continued treatment is deemed inappropriate by the investigators.\nPatients achieving CR or PD in part A proceeded to part B; patients with PR or stable disease (SD) in\npart A received 12 cycles of zanubrutinib-rituximab treatment prior to proceeding to part B. Patients with\nCR at part B completion proceeded to maintenance phase; young patients (≤ 65 years) with detectable\nMRD were eligible for ASCT before maintenance. Patients with PR, SD or PD at part B completion were\ntaken off the trial.\nFigure S7:\nLine 373-393: Responses were evaluated by the investigators every two cycles in part A and part B, and\nevery three cycles during maintenance therapy using PET or computerized tomography (CT) according\nto the Lugano 2014 criteria.32 A bone marrow biopsy is required to confirm CR in patients with bone\nmarrow involvement at baseline. Safety assessment was performed upon all patient visits. Adverse\nevents (AEs) were graded according to the National Cancer Institute’s Common Terminology Criteria for\nAdverse Events (version 5.0).\nMRD in bone marrow and/or peripheral blood were measured using flow cytometry with a\nsensitivity of 10-4 at baseline and upon the completion of part A and part B. Bone marrow aspiration was\nperformed at completion of part A and part B in patients with bone marrow involvement at baseline but\nnot routinely in patients without bone marrow involvement at baseline. The collection of peripheral\nblood for MRD evaluation was performed at baseline and upon the completion of part A and part B.\nDetectable MRD in either bone marrow or peripheral blood was considered MRD-positive.\nThe primary endpoint was the CR rate at the completion of part A in the intention-to-treat\npopulation, defined as proportion of patients with CR. Secondary endpoints included overall response\n(proportion of patients with CR plus PR), MRD, PFS, OS, and adverse events for part A, part B and\nmaintenance phase. PFS was defined as the time from treatment initiation to disease progression or\ndeath, whichever occurs earlier, and was censored at the last follow-up date in alive patients without\ndisease progression. OS was defined as the time from treatment initiation to patient death or the last\nfollow-up.\nLine 475-491: Sample size was estimated according to a single-stage phase 2 design based on the\nfollowing assumptions: 1) CR rate of 70% for historical control2 and 90% with the regimen in this trial;\n2) 90% power and two-sided 5% type I error; 3) a dropout rate of 10%. The calculation yielded 42\npatients.\nResponse assessment was performed in the full analysis set (ITT population) and per protocol set.\nSafety assessment was performed in the safety analysis set, which was divided into three datasets and\neach contained patients who received the study treatment at least once in part A, part B, or maintenance\nphase.\nDescriptive statistics were used to present baseline characteristics, response rates, and rate of AEs.\nThe 95% confidence interval (CI) of rates was calculated using the Clopper-Pearson method. Survival\noutcomes and 95% CIs were estimated using the Kaplan-Meier method. The 2-year PFS and OS were\ncalculated. The log-rank test was used to evaluate the difference in PFS and OS between subgroups.\nPost-hoc analyses included the subgroup analyses of CR rate at part A completion, CR rate at part B\ncompletion, PFS, and OS. Statistical analyses were performed using SPSS software (version 25.0) and R\nsoftware (version 3.6.1). Two-sided P of < 0.05 was considered statistically significant.\nLine 395-403: Total RNA and genomic DNA were extracted from formalin-fixed paraffin-embedded\n(FFPE) tumor samples obtained at baseline. Based on treatment response at the completion of part A, we\ncategorized the patients into three groups: early-CR (achieving CR within four cycles of zanubrutinib-\nrituximab regimen), late-CR (achieving CR after more than four cycles of zanubrutinib-rituximab\nregimen), and non-CR. Bulk RNA-sequencing was conducted and samples were classified into three\nsubtypes based on the differentially expressed genes among non-CR, late-CR and early-CR tumors.\nWhole-exome sequencing (WES) was conducted to analyze somatic mutations and tumor mutation\nburden. Detailed methods of RNA sequencing and WES are presented in the supplementary materials.\nQ2: Please define what ‘unacceptable toxicity’ is in terms of cessation of Part A.\nAnswer: We added the definition of “unacceptable toxicity” to the text (Line 359-361).\nLine 359-361: Unacceptable toxicity was defined as meeting any of the criteria for dose discontinuation\nof zanubrutinib or chemotherapeutic drugs as described in protocol during treatment, or if continued\ntreatment is deemed inappropriate by the investigators.\nQ3: Please clarify why patients in PR were taken off the study.\nAnswer: Thank you for raising this critical point. The design that only patients achieving CR proceeded\nto maintenance) was based on the NCCN guideline: (1) For patients receiving aggressive induction therapy,\nonly CR patients are recommended to receive consolidation with ASCT and/or maintenance therapy.\nPatients with PR are recommended to receive further chemoimmunotherapy or covalent BTKi if not\nexposed to covalent BTKi previously (not applicable to the current study population). (2) For patients\nreceiving less-aggressive induction therapy, CR and PR patients could consider maintenance therapy with\nrituximab; and for PR patients, they could also receive further chemoimmunotherapy or covalent BTKi.\nIn summary, we did not impose restrictions on the subsequent therapies for patients with PR; instead,\nthey taken off the trial to ensure receiving optimal and individualized treatment. Therefore, only CR\npatients got maintenance.\nLine 349-371: The treatment consisted of three phases. A treatment schema describing the study\ntreatment process is presented in Figure S7. In part A (induction phase), zanubrutinib (160 mg, twice a\nday, orally) and rituximab (375 mg/m2, intravenously on day 1) were administered in 28-day cycles until\nCR, disease progression or unacceptable toxicity, with a maximum of 12 cycles. In part B\n(chemoimmunotherapy phase), R-DHAOx (rituximab 375 mg/m2 intravenously on day 1;\ndexamethasone 20 mg intravenously on days 1-4; cytarabine 2 g/m2 intravenously on days 2-3 [1 g/m2\nfor patients > 65 years of age]; and oxaliplatin 130 mg/m2 intravenously on day 1) was administered in\n21-day cycles until disease progression or unacceptable toxicity, with a maximum of four cycles. In\nmaintenance phase, zanubrutinib (160 mg orally, twice a day) was given until disease progression or\nunacceptable toxicity for up to one year. Unacceptable toxicity was defined as meeting any of the criteria\nfor dose discontinuation of zanubrutinib or chemotherapeutic drugs as described in protocol during\ntreatment, or if continued treatment is deemed inappropriate by the investigators.\nPatients achieving CR or PD in part A proceeded to part B; patients with PR or stable disease (SD) in\npart A received 12 cycles of zanubrutinib-rituximab treatment prior to proceeding to part B. Patients with\nCR at part B completion proceeded to maintenance phase; young patients (≤ 65 years) with detectable\nMRD were eligible for ASCT before maintenance. Patients with PR, SD or PD at part B completion were\ntaken off the trial.\nFigure S7:\nQ4: Please reference the statistical assumptions used in this trial.\nAnswer: We appreciate your valuable comment, and added the reference for statistical assumptions (70%)\nto the text (Line 475-478), which was based on the results of the LyMA trial.1 The LyMa trial reported a\nCR rate of 41% (124/299) and an unconfirmed CR rate of 36% (107/299) after R-DHA-platinum induction.\nHowever, there are still some differences compared with the current trial: 1) In the LyMa trial,\ncarboplatin, and oxaliplatin were used in 76 and 38 patients, respectively; the remainder received cisplatin.\nIn contrast, all subjects in the current study received oxaliplatin; 2) Efficacy was evaluated using the 1999\nInternational Working Group criteria in the LyMa trial versus the Lugano 2014 criteria in the current study.\nAccordingly, unconfirmed CR was included in the CR rate in the LyMa trial but not the current study; 3)\nIn the LyMa trial, 35 patients (12%) received 1 cycle of CVP before R-DHAP, and 20 patients (7%)\nreceived R-CHOP because of insufficient response after R-DHAP.\nIn sample size estimation, we roughly took the number of patients achieving CR (n=124) or CRu\n(n=107) in the LyMA trial, minus the number of patients who received R-CHOP due to inadequate response\n(n=20), resulting in approximately 70% (211/299).\nLine 475-478: Sample size was estimated according to a single-stage phase 2 design based on the following\nassumptions: 1) CR rate of 70% for historical control2 and 90% with the regimen in this trial;\n2) 90% power and two-sided 5% type I error; 3) a dropout rate of 10%.\nReference:\n1. Le Gouill, S., et al. Rituximab after Autologous Stem-Cell Transplantation in Mantle-Cell Lymphoma.\nThe New England journal of medicine 377, 1250-1260 (2017).\nResults:\nQ1: It is unclear if the patient population is one that is transplant eligible or not, and although the upper\nage limit was 75, few patients over 65 appear to have been enrolled, and whether it included those with\nnon-measurable disease.\nAnswer: Thank you for raising these important points.\n(1) Transplant eligibility:\nIn the current trial, we did not set strict criteria for eligibility for transplantation. According to the study\ndesign, the ASCT was only used for patients aged ≤ 65 years with positive MRD at part B completion.\nEventually, no patient underwent ASCT because all the CR patients who proceeded to part B achieved\nMRD negativity, obviating the need for transplant.\n(2) Age distribution:\nThe patients in the current study were indeed younger than that in several clinical trials without age limit,1-\n3 as well as several real-world studies in MCL patients.4-6 First, First, since the age was set between 18 and\n75 years, so patients over 75 were not included, resulting in a relatively low proportion of patients aged 65\nand older. Second, during patient recruitment, there was a tendency for older patients to refuse to participate\nwhen they learned chemotherapeutic drugs are used in part B of the trial.\n(3) Measurable disease:\nFor enrollment, all patients were required to have at least one measurable lesion (Line 337-341).\nLine 337-341: Previously untreated patients aged 18-75 years with histologically confirmed, Ann Arbor\nstage II-IV MCL, requiring immediate anti-lymphoma treatment were eligible. Additional inclusion\ncriteria included: ECOG performance status score of 0 to 2, at least one measurable lesion, and adequate\norgan and bone marrow function.\nReferences:\n1. Kumar A, Soumerai J, Abramson JS, et al. Zanubrutinib, obinutuzumab, and venetoclax for first-line\ntreatment of mantle cell lymphoma with a TP53 mutation. Blood. 2025;145(5):497-507.\n2. Ruan J, Martin P, Shah B, et al. Lenalidomide plus Rituximab as Initial Treatment for Mantle-Cell\nLymphoma. N Engl J Med. 2015;373(19):1835-1844.\n3. Giné E, de la Cruz F, Jiménez Ubieto A, et al. Ibrutinib in Combination With Rituximab for Indolent\nClinical Forms of Mantle Cell Lymphoma (IMCL-2015): A Multicenter, Open-Label, Single-Arm, Phase\nII Trial. J Clin Oncol. 2022;40(11):1196-1205.\n4. Yang P, Cai QQ, Zhang W, et al. Real-world treatment and outcome patterns of patients with mantle cell\nlymphoma in China: A large, multicenter retrospective analysis. Cancer Med. 2023;12(12):13204-13216.\n5. He Y, Wang C, Pan T, et al. POD24-Based prognostic signature enables personalized risk stratification in\nmantle cell lymphoma. Sci Rep. 2025;15(1):8687.\n6. Zeng D, Fang Y, Fei Y, et al. Treatment patterns and clinical outcomes of mantle cell lymphoma: A\nretrospective cohort study by CHOICE. Int J Cancer. 2023;153(5):1016-1025.\nQ2: Why do the authors think there is such a high proportion in this study who are male? (83%), even\nwith a male predominance, this seems high.\nAnswer: Thank you for raising this important question. We believe that the high proportion of male in the\ncurrent study is due to chance for two reasons: 1) male predominance in the MCL population; 2) small\nsample size (n=42) of our trial. Sex imbalance is a well-recognized feature of MCL. We reviewed the male\nproportion reported in previous trials, including the Nordic1, Triangle2, LYM-30023, LyMA4, SHINE5,\nECHO6, and European MCL Younger trials7, and the male proportion ranged from 69% to 79%.\nFurthermore, according to several recent large retrospective studies from China, the male predominance\nranged from 76%-78%.8-10\nReference:\n1. Geisler CH, Kolstad A, Laurell A, et al. Long-term progression-free survival of mantle cell lymphoma after\nintensive front-line immunochemotherapy with in vivo-purged stem cell rescue: a nonrandomized phase 2\nmulticenter study by the Nordic Lymphoma Group. Blood. 2008;112(7):2687-2693.\n2. Dreyling M, Doorduijn J, Giné E, et al. Ibrutinib combined with immunochemotherapy with or without\nautologous stem-cell transplantation versus immunochemotherapy and autologous stem-cell\ntransplantation in previously untreated patients with mantle cell lymphoma (TRIANGLE): a three-arm,\nrandomised, open-label, phase 3 superiority trial of the European Mantle Cell Lymphoma Network. Lancet.\n2024;403(10441):2293-2306.\n3. Robak T, Jin J, Pylypenko H, et al. Frontline bortezomib, rituximab, cyclophosphamide, doxorubicin, and\nprednisone (VR-CAP) versus rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-\nCHOP) in transplantation-ineligible patients with newly diagnosed mantle cell lymphoma: final overall\nsurvival results of a randomised, open-label, phase 3 study. Lancet Oncol. 2018;19(11):1449-1458.\n4. Le Gouill S, Thieblemont C, Oberic L, et al. Rituximab after Autologous Stem-Cell Transplantation in\nMantle-Cell Lymphoma. N Engl J Med. 2017;377(13):1250-1260.\n5. Wang ML, Jurczak W, Jerkeman M, et al. Ibrutinib plus Bendamustine and Rituximab in Untreated Mantle-\nCell Lymphoma. N Engl J Med. 2022;386(26):2482-2494.\n6. Wang M, Salek D, Belada D, et al. Acalabrutinib Plus Bendamustine-Rituximab in Untreated Mantle Cell\nLymphoma. J Clin Oncol. 2025;43(20):2276-2284.\n7. Hermine O, Hoster E, Walewski J, et al. Addition of high-dose cytarabine to immunochemotherapy before\nautologous stem-cell transplantation in patients aged 65 years or younger with mantle cell lymphoma (MCL\nYounger): a randomised, open-label, phase 3 trial of the European Mantle Cell Lymphoma Network.\nLancet. 2016;388(10044):565-575.\n8. Yang P, Cai QQ, Zhang W, et al. Real-world treatment and outcome patterns of patients with mantle cell\nlymphoma in China: A large, multicenter retrospective analysis. Cancer Med. 2023;12(12):13204-13216.\n9. He Y, Wang C, Pan T, et al. POD24-Based prognostic signature enables personalized risk stratification in\nmantle cell lymphoma. Sci Rep. 2025;15(1):8687.\n10. Zeng D, Fang Y, Fei Y, et al. Treatment patterns and clinical outcomes of mantle cell lymphoma: A\nretrospective cohort study by CHOICE. Int J Cancer. 2023;153(5):1016-1025.\nQ3: Please state how many patients underwent PET, given the methods state PET or CT was used. Was\nCR confirmed with a bone marrow biopsy as well as PET? And did such a high number really convert\nfrom PR to CR in 8 weeks having only achieved a PR at cycle 2? It seems a little surprising.\nAnswer: We appreciate your valuable comments, and revised the manuscript to clearly state: 1) In part A,\nwe recommended that patients to undergo a CT scan after 2 cycles and a PET scan after 4 cycles (with the\nexception of one patient who had a low tumor burden and underwent PET scan after 2 cycles, and achieved\nCR). Bone marrow biopsy was required for patients with bone marrow infiltration at baseline to confirm\nCR.\nAs a result, all CR were confirmed by PET and bone marrow biopsy (Line 130-132 and Line 376-\n377).\nAs for the condition that a high PR rate after cycle 2 and then most of the patients obtained CR after\ncycle 4, it might partly due to the use of PET after cycle 4.\nLine 130-132 (Results): CR was confirmed by 18F-fluorodeoxyglucose positron-emission tomography\n(PET) in all patients, plus bone marrow biopsy in patients with bone marrow involvement at baseline.\nLine 376-377 (Methods): A bone marrow biopsy is required to confirm CR in patients with bone marrow\ninvolvement at baseline.\nQ4: Please clarify if all of the PD continued to 12months of R-zanu, or whether they progressed earlier\nor whether they progressed earlier and received RDHAOx.\nAnswer: Thank you for this important suggestion. Patients with PD in part A directly proceeded to part B\nto receive R-DHAOx, but not continued until 12 cycles of zanubrutinib-rituximab. We revised the Methods\nsection (Line 362-364) and added a treatment schema (Figure S7) to clarify this issue.\nLine 362-364: Patients achieving CR or PD in part A proceeded to part B; patients with PR or stable\ndisease (SD) in part A received 12 cycles of zanubrutinib-rituximab treatment prior to proceeding to part\nB.\nFigure S7:\nQ5: Line 104 appears to be an error-the vs should be between the blastoid/classic categories.\nAnswer: We are sorry for this mistake. The text has been revised accordingly (Line 138-141).\nLine 138-141: In post-hoc subgroup analyses, the CR rate at part A completion was 40% (2/5) in patients\nwith blastoid/pleomorphic disease and 95% (35/37) in those with classic/small cell disease; 33% (1/3) in\npatients with TP53 mutation and 94% (32/34) in those with wild type TP53.\nQ6: Please include dose disruptions as well as dose reductions in the R-zanu group. If there was not\ndose reduction and Zanu is continuous, how was the grade 3+ neutropenia managed in the 7% reported\nwho had it. What was the management of infection such as covid? Was there provision in the protocol\nfor cessation? This study ran during the covid pandemic when vaccination was not available, all other\nBTKi studies had significant issues with COVID yet it is not mentioned here.\nAnswer: We deeply appreciated for this valuable comment, and added detailed description of dose\nsuspension/reduction during the trial period (Line 180-183, Line 188-202, and Line 203-206). According\nto the protocol, grade 3-4 hematological toxicities required a treatment suspension until recovery to ≤ grade\n1, with zanubrutinib maintained at 160mg BID during zanubrutinib-rituximab treatment. Dose reduction\nto 80mg BID was implemented only upon recurrence of the same toxicity. In our study, three patients (7%)\nexperienced grade 3-4 neutropenia for once; therefore, none needed dose reductions.\nThank you for highlighting this critical aspect of trial conduct during the pandemic. With regards to\nthe COVID-19 issue, only one patient death was attributed to COVID-19, presumably due to relatively\nstrict preventive measures in China.\n(1) The management of SARS-CoV-2 infection during pandemic: All patients underwent\nmandatory SARS-CoV-2 PCR testing prior to hospitalization between 2020 and 2023. Outpatients\nreceiving zanubrutinib at home were tested immediately if they developed fever or respiratory symptoms.\nIn patients with positive result, all anti-lymphoma treatments were suspended and patients received\nimmediate COVID-directed therapy. Anti-lymphoma treatment resumed only after complete recovery (two\nconsecutive negative PCR tests at least 24 h apart). Routine screening before hospitalization was\ndiscontinued after 2023. However, all patients developing fever (≥ 38℃), cough, dyspnea, or new hypoxia\nwould still underwent prompt PCR testing.\n(2) Provision in the protocol for cessation: Measures for SARS-CoV-2-related cessation rule was\nnot included in the trial protocol, but decided by the investigators.\n(3) SARS-CoV-2-related information in our trial: A total of 30 patients tested positive for SARS-\nCoV-2, and treatment was suspended in 17 patients. One patient died due to SARS-CoV-2. The Results\nsection has been expanded to describe these findings (Line 208-211).\nLine 180-183: No patient needed zanubrutinib dose reduction in part A. Zanubrutinib dose suspension\nwas required in five (12%) of 42 patients (three patients for neutropenia, one for infection, and one for\nelevated liver enzymes).\nLine 188-202: Dose reduction of chemotherapeutic agents was required in 32 (82%) of 39 patients due to\ntoxicities (27 patients for thrombocytopenia, four for neutropenia, and one for elevated liver enzymes).\nOf these, 25 patients had their dose of cytarabine reduced by one dose level to 75% of the original dose,\nand seven patients required a reduction of two dose levels to 50%. Five patients were still unable to\ntolerate thrombocytopenia after dose reduction and discontinued part B treatment. Thrombopoietin\nreceptor agonists (TPO-RAs) or recombinant human thrombopoietin (rhTPO) were given in 9 patients as\nprimary prevention for thrombocytopenia, and in 13 patients as secondary prevention. Recombinant\nhuman granulocyte colony-stimulating factor (rhG-CSF) was given in 26 patients as primary prevention\nfor neutropenia, and in all the 19 patients who had experienced grade 3-4 neutropenia as secondary\nprevention. Chemotherapy dose suspension was required in 20 (51%) of 39 patients due to toxicities (18\npatients for thrombocytopenia, one for neutropenia, and one for elevated liver enzymes).\nLine 203-206: One patient needed zanubrutinib dose reduction to 80 mg twice a day due to grade 3\nneutropenia. Zanubrutinib dose suspension was required in six (18%) of 34 patients (two patients for\nneutropenia, two patients for fatigue, and one patient each for thrombocytopenia and infections).\nLine 208-211: This trial was conducted during the COVID-19 pandemic. One patient died from COVID-\n19 pneumonia and respiratory failure during part B. A total of 30 patients tested positive for SARS-CoV-\n2 at least once, and 17 patients required treatment suspension due to SARS-CoV-2 infection.\nQ7: Dose reductions of 82% for the chemo is extremely high for this regimen. Please comment on\nreasons, and details of the dose reductions that occurred. Please include description of the GCSF use.\nAnswer: We appreciate your constructive comments and added a description of the G-CSF use to the text.\nWe agree that dose reduction during part B in 32 (82%) patients represents an important issue. Upon a\ncloser inspection, does reduction was due to thrombocytopenia in most cases (n=27), followed by\nneutropenia (n=4) and elevated liver enzymes (n=1).\nIn the current trial, we recommended using appropriate medications for primary prevention of\nhematological toxicities in patients in older age, had relatively poor physical or nutritional status, low\nplatelet or neutrophil counts at baseline, and significant bone marrow involvement. 26 patients received\nG-CSF as primary prevention for neutropenia; while only 9 patients received recombinant TPO or TPO\nreceptor agonists as primary prevention for thrombocytopenia partly due to the high out-of-pocket\nexpenses.\nThe detailed information of dose reduction, and medication for thrombocytopenia and neutropenia\nhave been added to the revised manuscript (Line 188-202).\nLine 188-202: Dose reduction of chemotherapeutic agents was required in 32 (82%) of 39 patients due to\ntoxicities (27 patients for thrombocytopenia, four for neutropenia, and one for elevated liver enzymes).\nOf these, 25 patients had their dose of cytarabine reduced by one dose level to 75% of the original dose,\nand seven patients required a reduction of two dose levels to 50%. Five patients were still unable to\ntolerate thrombocytopenia after dose reduction and discontinued part B treatment. Thrombopoietin\nreceptor agonists (TPO-RAs) or recombinant human thrombopoietin (rhTPO) were given in 9 patients as\nprimary prevention for thrombocytopenia, and in 13 patients as secondary prevention. Recombinant\nhuman granulocyte colony-stimulating factor (rhG-CSF) was given in 26 patients as primary prevention\nfor neutropenia, and in all the 19 patients who had experienced grade 3-4 neutropenia as secondary\nprevention. Chemotherapy dose suspension was required in 20 (51%) of 39 patients due to toxicities (18\npatients for thrombocytopenia, one for neutropenia, and one for elevated liver enzymes).\nQ8: It would be useful to have the TP53 mutation status documented in the results of the manuscript –\nit is currently quite difficult to find this in a supplementary figure. Was there any difference in response\nto treatment or outcomes for TP53 mutated vs wild type patients?\nAnswer: Thank you for this suggestion. We moved the TP53 status to Table 1. Patients with TP53\nmutations had relatively lower CR rate and inferior PFS, but no OS difference was observed. Subgroup\nanalyses of CR rate (Line 138-141, Line 156-159 and Table S2) and survival (Line 172, Figure S3F, and\nFigure S4F) have also been provided in the revised manuscript.\nTable 1:\nLine 138-141 (part A completion): In post-hoc subgroup analyses, the CR rate at part A completion was\n40% (2/5) in patients with blastoid/pleomorphic disease and 95% (35/37) in those with classic/small cell\ndisease; 33% (1/3) in patients with TP53 mutation and 94% (32/34) in those with wild type TP53.\nLine 156-159 (part B completion): In post-hoc subgroup analyses, the CR rate at part B completion was\n40% (2/5) in patients with blastoid/pleomorphic disease and 92% (34/37) in those with classic/small cell\ndisease; 33% (1/3) in patients with TP53 mutation and 91% (31/34) in those with wild-type TP53 (Table\nS2).\nTable S2:\nLine 172: Patients with TP53 mutations had inferior PFS, but no OS difference was observed.\nFigure S3F (Progression-free survival):\nFigure S4F (Overall survival):\nQ9: The frequency of TP53 mutation was lower than in other contemporary cohorts of MCL patients\nwho have had WES performed (typically 20-30%). This may have influenced the results.\nAnswer: We are grateful for your constructive comments. The rate of TP53 mutation in the current study\n(8.1%) was indeed lower than that reported in several prospective trials1-3 and genomic studies4-6 (15% to\n32% in untreated MCL tumor tissue samples), as well as in a phase 2 trial conducted in China (13%)7. We\nspeculate that such a discrepancy was due to the relative sample size.\nWe have initiated a phase 2 trial in untreated MCL patients (NCT06846489) in which genetic testing\nwill be conducted before study treatment instead of as a post-hoc testing, which may give further evidence\nof TP53 mutation rate in Chinese MCL patients.\nReference:\n1. Wang ML, Jain P, Zhao S, et al. Ibrutinib-rituximab followed by R-HCVAD as frontline treatment for\nyoung patients (≤65 years) with mantle cell lymphoma (WINDOW-1): a single-arm, phase 2 trial. Lancet\nOncol. 2022;23(3):406-415.\n2. Jain P, Zhao S, Lee HJ, et al. Ibrutinib With Rituximab in First-Line Treatment of Older Patients With\nMantle Cell Lymphoma. J Clin Oncol. 2022;40(2):202-212.\n3. Giné E, de la Cruz F, Jiménez Ubieto A, et al. Ibrutinib in Combination With Rituximab for Indolent\nClinical Forms of Mantle Cell Lymphoma (IMCL-2015): A Multicenter, Open-Label, Single-Arm, Phase\nII Trial. J Clin Oncol. 2022;40(11):1196-1205.\n4. Yi S, Yan Y, Jin M, et al. Genomic and transcriptomic profiling reveals distinct molecular subsets\nassociated with outcomes in mantle cell lymphoma. J Clin Invest. 2022;132(3):e153283.\n5. Jain P, Nomie K, Kotlov N, et al. Immune-depleted tumor microenvironment is associated with poor\noutcomes and BTK inhibitor resistance in mantle cell lymphoma. Blood Cancer J. 2023;13(1):156.\nPublished 2023 Oct 12.\n6. Khouja M, Jiang L, Pal K, et al. Comprehensive genetic analysis by targeted sequencing identifies risk\nfactors and predicts patient outcome in Mantle Cell Lymphoma: results from the EU-MCL network trials.\nLeukemia. 2024;38(12):2675-2684.\n7. Zhang H, Su L, Liu L, et al. Orelabrutinib-lenalidomide-rituximab in patients with untreated mantle cell\nlymphoma (MCL): updated results of the multicenter, phase II POLARIS study. 2024 European\nHematology Association Meeting. 419228; P1141.\nQ10: Why were those requiring anticoagulants excluded from the trial? (point 4 under Exclusion\nCriteria in the protocol). In clinical practice patients on anticoagulation therapy would be eligible to\nreceive a BTKi\nAnswer: We agree that anticoagulation therapy does not represent a contraindication for BTKi treatment\nin clinical practice. However, hemorrhage events are frequent in patients with B-cell malignancies treated\nwith BTKi (ibrutinib as well as 2nd generation BTKis like zanubrutinib).1 Supporting evidence mainly\ncomes from a phase II trial of zanubrutinib in relapsed/refractory MCL2, where hemorrhage events\noccurred in 25.6% of patients and major hemorrhage was reported in 3 patients (3.5%), including one with\ncerebral hemorrhage and two with gastrointestinal hemorrhages. Consequently, the zanubrutinib\nprescription information explicitly warns that coadministration of zanubrutinib with antiplatelet or\nanticoagulant medications may further increase the risk of hemorrhage\n(https://www.brukinsa.com/prescribing-information.pdf). Based on these concerns, we excluded patients\nwho require anticoagulants from the current trial. Similar design has been used in most BTKi-containing\nclinical trials in MCL, eg. the SHINE trial3, the ECHO trial4, and the WINDOW-1 trial5.\nReference:\n1. von Hundelshausen P, Siess W. Bleeding by Bruton Tyrosine Kinase-Inhibitors: Dependency on Drug Type\nand Disease. Cancers (Basel). 2021;13(5):1103.\n2. Song Y, Zhou K, Zou D, et al. Zanubrutinib in relapsed/refractory mantle cell lymphoma: long-term\nefficacy and safety results from a phase 2 study. Blood. 2022;139(21):3148-3158.\n3. Wang ML, Jurczak W, Jerkeman M, et al. Ibrutinib plus Bendamustine and Rituximab in Untreated Mantle-\nCell Lymphoma. N Engl J Med. 2022;386(26):2482-2494.\n4. Wang M, Salek D, Belada D, et al. Acalabrutinib Plus Bendamustine-Rituximab in Untreated Mantle Cell\nLymphoma. J Clin Oncol. 2025;43(20):2276-2284.\n5. Wang ML, Jain P, Zhao S, et al. Ibrutinib-rituximab followed by R-HCVAD as frontline treatment for\nyoung patients (≤65 years) with mantle cell lymphoma (WINDOW-1): a single-arm, phase 2 trial. Lancet\nOncol. 2022;23(3):406-415.\nQ11: For MRD assessment, the authors state ‘bone marrow +/- peripheral blood’ was used. Please\nelaborate on how this was decided or if it was at random. Not all patients had bone marrow involvement\nat baseline – did these patients have detectable disease in peripheral blood and were these patients\nsubsequently evaluated using peripheral blood?\nAnswer: We appreciate for this suggestion. In our trial, for patients with bone marrow involvement at\nbaseline, a bone marrow aspiration was performed for MRD detection at completion of part A and part B.\nFor patients without bone marrow involvement at baseline, bone marrow aspiration was not routinely\nperformed. The collection of peripheral blood for MRD evaluation was performed at baseline and upon\nthe completion of part A and part B.\nMRD assessment deviated from the plan during the trial process in some patients refused sample\ncollection of bone marrow or peripheral blood, which in turn led to discrepancies in the number of available\nMRD results. We added the sample collection protocol/rules for MRD assessment in Methods (Line 381-\n385) and specific collection at each time point in annotation of Table S1.\nIn the six patients without bone marrow involvement at baseline, none had detectable MRD in\nperipheral blood; these patients were evaluated subsequently using peripheral blood for MRD. The Results\nsection has been revised to describe these facts (Line 118-120).\nLine 381-385: Bone marrow aspiration was performed at completion of part A and part B in patients with\nbone marrow involvement at baseline but not routinely in patients without bone marrow involvement at\nbaseline. The collection of peripheral blood for MRD evaluation was performed at baseline and upon the\ncompletion of part A and part B.\nTable S1:\nLine 118-120: In the six patients without bone marrow involvement at baseline, none had detectable\nMRD in peripheral blood, and they underwent MRD detection only in peripheral blood in subsequent\ntime points.\nQ12: In figure S5, the most common somatic mutation is OBSCN. Pathogenic mutations in the obscurin\ngene have not been found in any lymphomas in online clinical databases, and are most often VUS or\nbenign variants. Could the authors please comment on their finding of OBSCN mutations in 29.7% of\nthe cohort? Was there strong evidence for pathogenicity?\nAnswer: We appreciate for this important comment, and updated WES analysis by further including 12\nnegative control samples (bringing the total to 23 cases with negative control samples). Additionally, the\nreview of the WES results in last version of manuscript showed a large number of low-abundance, splice\nvariants, which were inconsistent with actual conditions. Consequently, we increased the threshold for\nsomatic mutation calling from 3 reads to 4 reads for all the genes. The detailed methods have been revised\nin the supplementary methods.\nIn the updated analysis, the mutation rate of OBSCN was 10.8% (Figure S6), including missense\nmutations in four patients: c.556G>A(p.A186T), c.2765C>T(p.T922M), c.2997_2998inv(p.A1000T), and\nc.20648G>C(p.R6883P). The somatic mutation of OBSCN was uncommon in MCL in most of previous\nstudies, but a recent genomic study reported 5% OBSCN mutation rate among 123 newly-diagnosed MCL\npatients.1 Then we compared the expression level of OBCSN between patients with versus without OBSCN\nmutation based on RNAseq (figure below), but found no significant differences. For response and survival\noutcomes, patients with OBSCN mutation had a CR rate of 50% (2/4), and an inferior PFS than those\nwithout mutation, but no difference in OS (figures below).\nTaken together, based on the current findings, no conclusions can be drawn regarding the clinical and\npathogenic value of OBSCN mutations in MCL.\nFigure S6:\nExpression level based on RNAseq:\nPFS and OS:\nReference:\n1. Yi S, Yan Y, Jin M, et al. Genomic and transcriptomic profiling reveals distinct molecular subsets\nassociated with outcomes in mantle cell lymphoma. J Clin Invest. 2022;132(3):e153283.\nQ13: Although the results here are promising, the median follow up time is 23 months and perhaps too\nshort to conclude that there was a high rate of 'sustained' CR.\nAnswer: Thank you for this suggestion. We refrained from claiming “sustained CR” in the revised\nmanuscript (Line 326-328).\nLine 326-328: In conclusion, zanubrutinib-rituximab induction followed by a shortened R-DHAOx\nchemoimmunotherapy and maintenance with zanubrutinib as the frontline treatment resulted in a high\nrate of sustained CR in patients with untreated MCL.\nDiscussion:\nQ1: The reference to the Lugano criteria should be in a methods section, not the discussion.\nAnswer: We appreciate for the comment, and added reference citation to the Lugano criteria to the\nMethods section (Line 373-376).\nLine 373-376: Responses were evaluated by the investigators every two cycles in part A and part B, and\nevery three cycles during maintenance therapy using PET or computerized tomography (CT) according\nto the Lugano 2014 criteria.32\nQ2: The authors have not discussed studies of BTKi in combination with rituximab, nor any of the R-\nbendamustine studies which are relevant here due to the enrolment of patients up to age 75.\nAnswer: We are grateful for this suggestion, and expanded the Discussion section to address combination\nof BTKi with rituximab, bendamustine-rituximab with or without BTKi (ibrutinib or acalabrutinib) (Line\n287-297). Also, a comprehensive comparison with the WINDOW-1 trial, which was similar in study design\nto our trial, has already been provided (Line 267-286).\nLine 287-297: A recent phase II trial reported 80% CR rate when using the ibrutinib-rituximab regimen\nin treating untreated indolent clinical forms of MCL (non-blastoid variants, Ki-67 ≤ 30%, asymptomatic\ndisease, ECOG 0-1, and largest lesion diameter ≤ 3cm).16 Five out of 42 patients in the current trial had\nuntreated indolent clinical forms of MCL using the same criteria and all achieved CR. In addition, our\ntrial included nine patients 65-75 years of age, and achieved a CR rate of 88% in this group of patients.\nIn previous trials for MCL patients of 65 years of age or older, ibrutinib-rituximab regimen resulted in a\nCR rate of 71%,17 whereas the CR rate in patients receiving BR regimen with or without BTK inhibitors\n(ibrutinib or acalabrutinib) was 65.5%-66.6% and 53.5%-57.6%.8,35 A phase II trial investigating BR plus\nlow-dose cytarabine regimen in MCL patients aged > 65 years or 60-65 years but ineligible for\nHDT/ASCT reported a CR rate of 91% at the end of treatment.36\nLine 267-286: Compared to the WINDOW-1 trial,21 patients in this trial had numerically higher\npercentage of Ki-67 ≥ 30%, higher MIPI risk, but lower rate of TP53 mutation. The duration of induction\ntreatment was also shorter in this trial (median cycles: 4 vs 7). Despite these differences, the response to\nzanubrutinib-rituximab induction in this trial (CR, 88%; ORR, 98%) was similar to that reported for\nibrutinib plus rituximab in the WINDOW-1 trial (CR, 87%; ORR, 98%). The fewer cycles needed to\nachieve CR in this trial may reflect improved efficacy; alternatively, this difference may be attributable to\nthe fact that responses were assessed using PET scans routinely after cycle 4 in this trial but only used to\nverify CR after CT screening in the WINDOW-1 trial. Temporal pattern of treatment failure also differed:\nall five PD events in this trial occurred within nine months of treatment initiation; in contrast, the PD\nevents in the WINDOW-1 trial occurred over a much longer period, even after 24 months. This\ndifference might be related to the maintenance therapy with zanubrutinib following\nchemoimmunotherapy in our trial, but an extended follow-up is warranted since the median follow-up\nwas only 23 months.\nNone of the patients in this trial required dose reduction of zanubrutinib during zanubrutinib-\nrituximab induction treatment. In contrast, one third of patients in the WINDOW-1 trial required dose\nreduction of ibrutinib due to AEs. Such a difference may reflect a more favorable safety profile with the\nzanubrutinib-rituximab regimen than ibrutinib plus rituximab. Additionally, this difference could also be\nattributable to the shorter induction cycles in our trial.\nQ3: The limitations of the study need to be expanded, claims being made about differences in the\nmolecular subgroups in the absence of adequate power for any multivariable analysis need to be\nsoftened and noted in the limitation.\nAnswer: Thank you for this suggestion. We expanded the limitation section to included likely confounding\nby molecular subgroups in the absence of adequate power for multivariable analysis (Line 323-325).\nLine 323-325: Third, in the molecular subtype analyses, the absence of multivariable analysis and\nindependent validation has resulted in a reduced reliability of the findings, necessitating further\nverification.\nQ4: Why is 4 cycles of RDHAOX considered shortened in the conclusion, when it is given to patients\nmostly in CR?\nAnswer: We appreciate your insightful comment, and wish to explain that the word \"shortened\" is used in\nthe manuscript in the context of most of the cytarabine-based chemotherapy regimens, such as the 6\nalternating cycles of R-maxiCHOP/ R+high-dose cytarabine (Nordic regimen)1, 6 alternating cycles of R-\nCHOP/R-DHAP2,3, 6-8 alternating cycles of R+hyper-CVAD/R+high dose methotrexate and cytarabine4,\n6 alternating cycles of R+bendamustine/R+high-dose cytarabine5.\nAlthough the LyMa trial6 also utilized 4 cycles of R-DHAP, the intensity of the overall treatment was\nensured by (1) a routine consolidation therapy with HDT+ASCT for all patients; (2) an additional rescue\ninduction therapy with 4 cycles of R-CHOP-14 for patients with PR and whose tumor mass had been\nreduced by less than 75%; (3) 1 cycle of CVP regimen was allowed to reduce tumor burden before R-\nDHAP according to local investigator decision.\nIn our study, we anticipated to use a chemo-free induction (zanubrutinib-rituximab) and maintenance\n(zanubrutinib) to minimize the use of cytotoxic chemotherapy drugs and HDT/ASCT. At the completion\nof part B, all the CR patients were MRD-negative, and were not consolidated with HDT/ASCT. Therefore,\ncompared to the LyMa trial, the treatment intensity in our trial was decreased by using mild induction and\nmaintenance therapies.\nThe Discussion section of the manuscript has been revised to reflects these thoughts (Line 244-252).\nLine 244-252: Currently, most of the cytarabine-based chemotherapy regimens, such as the NORDIC\nregimen10, alternating R-CHOP/R-DHAP regimen4,30, and R-hyper-CVAD/R-high dose methotrexate and\ncytarabine regimen31, contain six to eight cycles of treatment. The current trial used a shortened four cycles\nof R-DHAOx regimen. Notably, the LyMa trial2 also used four cycles of R-DHAP, but the intensity of the\noverall treatment was ensured by routine consolidation therapy with HDT/ASCT, additional four cycles of\nR-CHOP for PR patients, and one cycle of CVP regimen was allowed before study treatment. The\ntreatment intensity was lower in the current study due to mild chemotherapy-free induction and\nmaintenance therapy to minimize the use of cytotoxic chemotherapy drugs and HDT/ASCT.\nReference:\n1. Eskelund CW, Kolstad A, Jerkeman M, et al. 15-year follow-up of the Second Nordic Mantle Cell\nLymphoma trial (MCL2): prolonged remissions without survival plateau. Br J Haematol.\n2016;175(3):410-418.\n2. Dreyling M, Doorduijn J, Giné E, et al. Ibrutinib combined with immunochemotherapy with or without\nautologous stem-cell transplantation versus immunochemotherapy and autologous stem-cell\ntransplantation in previously untreated patients with mantle cell lymphoma (TRIANGLE): a three-arm,\nrandomised, open-label, phase 3 superiority trial of the European Mantle Cell Lymphoma Network.\nLancet. 2024;403(10441):2293-2306.\n3. Hermine O, Hoster E, Walewski J, et al. Addition of high-dose cytarabine to immunochemotherapy\nbefore autologous stem-cell transplantation in patients aged 65 years or younger with mantle cell\nlymphoma (MCL Younger): a randomised, open-label, phase 3 trial of the European Mantle Cell\nLymphoma Network. Lancet. 2016;388(10044):565-575.\n4. Romaguera JE. et al. High rate of durable remissions after treatment of newly diagnosed aggressive\nmantle-cell lymphoma with rituximab plus hyper-CVAD alternating with rituximab plus high-dose\nmethotrexate and cytarabine. J Clin Oncol. 2005;23(28):7013-7023.\n5. Merryman RW, Edwin N, Redd R, et al. Rituximab/bendamustine and rituximab/cytarabine induction\ntherapy for transplant-eligible mantle cell lymphoma. Blood Adv. 2020;4(5):858-867.\n6. Le Gouill S, Thieblemont C, Oberic L, et al. Rituximab after Autologous Stem-Cell Transplantation in\nMantle-Cell Lymphoma. N Engl J Med. 2017;377(13):1250-1260.\nGeneral:\nQ1: there are numerous grammatical errors throughout the manuscript that are obvious to a native\nEnglish speaker. Although these do not always detract from the meaning of the phrase, they are\nnoticeable and need to be rectified.\nAnswer: During the revision, we worked with a professional medical editor (Kehong Zhang, M.D., Ph.D.,\nfrom the Ivy Medical Editing; formerly an Assistant Professor at the Harvard Medical School) to improve\nlanguage readability.\nReviewer #4 (Biostats):\nThe multicenter, phase II trial (CHESS) evaluated zanubrutinib plus rituximab induction, followed by\nshortened chemoimmunotherapy in untreated MCL patients. The primary endpoint was the CR rate\nachieved after induction. Overall the report is clear and comprehensive and focussed on the primary\nand secondary endpoints. There is some interesting exploratory analysis included which adds to the\nimpact of the work.\nI have a number of fairly minor points that I believe should be addressed to improve the clarity of\npresentation.\nQ1: Pg2. In the abstract please report estimates (CR rate, PFS and OS rate) with appropriate confidence\nintervals as per journal guidelines.\nAnswer: We appreciate this comment, and added 95% confidence intervals (Line 35-37 and Line 38-39).\nLine 35-37: The primary endpoint was the CR rate at part A completion, which was 88% (95%\nconfidence interval [CI], 74-96). The CR rate at part B completion was 86% (95% CI, 72-95).\nLine 38-39: With the median follow-up of 23 months, the 2-year progression-free survival and overall\nsurvival was 85% (95% CI, 75-97) and 90% (95% CI, 75-100), respectively.\nQ2: Pg2. The conclusion to the abstract should reflect the findings of a single arm phase II study. It\noverstates results to say the treatment is \"effective and safe\" without indicating he further evaluation\nrequired in phase III. For example, in the discussion the authors state: \"Further randomized controlled\ntrials should be conducted to evaluate the efficacy and safety of the proposed treatment regimen in this\ntrial.\" This should be included in any concluding statement .\nAnswer: We are grateful for your valuable comment, and revised the conclusion part of Abstract (Line 47-\n51) as well as the Discussion (Line 326-331).\nLine 47-51: In conclusion, zanubrutinib-rituximab induction followed by shortened R-DHAOx is\nefficacious and safe as a frontline therapy for MCL. This strategy allows for a reduction in chemotherapy\ncycles, and should be validated in future randomized controlled trials.\nLine 326-331: In conclusion, zanubrutinib-rituximab induction followed by a shortened R-DHAOx\nchemoimmunotherapy and maintenance with zanubrutinib as the frontline treatment resulted in a high\nrate of CR in patients with untreated MCL. The induction phase and maintenance phase were well-\ntolerated; however, significant myelosuppression was still observed during the chemoimmunotherapy\nphase. Future randomized controlled trials are required to validate strategies that minimize chemotherapy\nuse in first line treatment of MCL.\nQ3: Pg5. Please add more detail on treatment delivered including dose modification required and delays\nbetween cycles. If they did not occur, please state as such --- I note that comment in the Discussion on\npage 8. For example, the protocol states: \"The exposure time and dose of experimental drug therapy\nwere summarized. List the number of patients for whom the dose was adjusted/suspended, along with\nthe reasons for the dose adjustment/suspension.\" Please report this information appropriately, as\nrequired.\nAnswer: We deeply appreciated for this valuable comment, and added detailed description of dose\nsuspension/reduction during the trial period (Line 180-183, Line 188-202, and Line 203-206). According\nto the protocol, grade 3-4 hematological toxicities required a treatment suspension until recovery to ≤ grade\n1, with zanubrutinib maintained at 160mg BID during zanubrutinib-rituximab treatment. Dose reduction\nto 80mg BID was implemented only upon recurrence of the same toxicity. In our study, three patients (7%)\nexperienced grade 3-4 neutropenia once; therefore, none needed dose reductions.\nLine 180-183: No patient needed zanubrutinib dose reduction in part A. Zanubrutinib dose suspension\nwas required in five (12%) of 42 patients (three patients for neutropenia, one for infection, and one for\nelevated liver enzymes).\nLine 188-202: Dose reduction of chemotherapeutic agents was required in 32 (82%) of 39 patients due to\ntoxicities (27 patients for thrombocytopenia, four for neutropenia, and one for elevated liver enzymes).\nOf these, 25 patients had their dose of cytarabine reduced by one dose level to 75% of the original dose,\nand seven patients required a reduction of two dose levels to 50%. Five patients were still unable to\ntolerate thrombocytopenia after dose reduction and discontinued part B treatment. Thrombopoietin\nreceptor agonists (TPO-RAs) or recombinant human thrombopoietin (rhTPO) were given in 9 patients as\nprimary prevention for thrombocytopenia, and in 13 patients as secondary prevention. Recombinant\nhuman granulocyte colony-stimulating factor (rhG-CSF) was given in 26 patients as primary prevention\nfor neutropenia, and in all the 19 patients who had experienced grade 3-4 neutropenia as secondary\nprevention. Chemotherapy dose suspension was required in 20 (51%) of 39 patients due to toxicities (18\npatients for thrombocytopenia, one for neutropenia, and one for elevated liver enzymes).\nLine 203-206: One patient needed zanubrutinib dose reduction to 80 mg twice a day due to grade 3\nneutropenia. Zanubrutinib dose suspension was required in six (18%) of 34 patients (two patients for\nneutropenia, two patients for fatigue, and one patient each for thrombocytopenia and infections).\nLine 208-211: This trial was conducted during the COVID-19 pandemic. One patient died from COVID-\n19 pneumonia and respiratory failure during part B. A total of 30 patients tested positive for SARS-CoV-\n2 at least once, and 17 patients required treatment suspension due to SARS-CoV-2 infection.\nQ4: Pg 10. The conclusion should reflect the aims and have a mention of safety also. Please add.\nAnswer: We appreciate the comments and added safety to the conclusion (Line 326-331).\nLine 326-331: In conclusion, zanubrutinib-rituximab induction followed by a shortened R-DHAOx\nchemoimmunotherapy and maintenance with zanubrutinib as the frontline treatment resulted in a high\nrate of CR in patients with untreated MCL. The induction phase and maintenance phase were well-\ntolerated; however, significant myelosuppression was still observed during the chemoimmunotherapy\nphase. Future randomized controlled trials are required to validate strategies that minimize chemotherapy\nuse in first line treatment of MCL.\nQ5: Pg 10. Describe who was blinded or not. For example, participants and clinician not blinded. Was\noutcome assessor blinded?\nAnswer: This was an open-label trial. Participants, investigators, attending physicians and outcome\nassessors were not blinded. We apologize for not making it clear in the original manuscript, and revised\nthe Methods accordingly (Line 336-337).\nLine 336-337: This was an open-label trial. Participants, investigators, attending physicians and outcome\nassessors were not blinded.\nQ6:Pg 11/14. Please define the ITT, PAS and safety population more clearly in statistical analysis section\nand ensure this refects what is stated in the protocol and how analysing was undertaken.\nAnswer: We appreciate the comment, and added term definitions to the statistical analysis section (Line\n479-491 and Line 390-393).\nLine 479-491: Response assessment was performed in the full analysis set (ITT population) and per\nprotocol set. Safety assessment was performed in the safety analysis sets, which was divided into three\ndatasets and each contained patients who received the study treatment at least once in part A, part B, or\nmaintenance phase.\nDescriptive statistics were used to present baseline characteristics, response rates, and rate of AEs.\nThe 95% confidence interval (CI) of rates was calculated using the Clopper-Pearson method. Survival\noutcomes and 95% CIs were estimated using the Kaplan-Meier method. The 2-year PFS and OS were\ncalculated. The log-rank test was used to evaluate the difference in PFS and OS between subgroups.\nPost-hoc analyses included the subgroup analyses of CR rate at part A completion, CR rate at part B\ncompletion, PFS, and OS. Statistical analyses were performed using SPSS software (version 25.0) and R\nsoftware (version 3.6.1). Two-sided P of < 0.05 was considered statistically significant.\nLine 390-393: PFS was defined as the time from treatment initiation to disease progression or death,\nwhichever occurs earlier, and was censored at the last follow-up date in alive patients without disease\nprogression. OS was defined as the time from treatment initiation to patient death or the last follow-up.\nQ7:Pg 14. Please clarify if the 5% type-one error is one-sided or two-sided. Fuurthemore, if it is one-\nsided please clarify the impact this has on the confidence intervals you should report for the primary\nendpoint.\nAnswer: Thank you for your helpful suggestions. We revised the Methods section to clearly state the the\n5% type-one error was two-sided (Line 475-478).\nLine 475-478: Sample size was estimated according to a single-stage phase 2 design based on the\nfollowing assumptions: 1) CR rate of 70% for historical control2 and 90% with the regimen in this trial;\n2) 90% power and two-sided 5% type I error; 3) a dropout rate of 10%. The calculation yielded 42\npatients."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have provided a comprehensive point-by-point response and revised the manuscript accordingly. Only minor\nissues remain, and upon satisfactory revision, I would recommend the manuscript for publication in Nature Communications.\n1. In the Abstract, start with a brief rationale for clinical trial design, followed by a concise description of the methods. The\nkey results should then be presented, beginning with the number of patients enrolled and summarizing the major efficacy\nand safety outcomes. Currently, the Methods and Results are somewhat interwoven, which may confuse readers.\n2. The toxicity description in the Abstract is unclear. A more focused summary of the key safety findings would be helpful.\n3. In the Results–Adverse Events section, the authors should briefly summarize key supportive treatments and patient\nrecovery outcomes.\n4. Please note the placement of the subtitles on page 8, line 193.\n5. Did the authors compare the three MCL molecular subtypes (M1-M3) with the four clusters (C1-C4) identified by Yi S et al.\n(Yi S, Yan Y, Jin M, et al. J Clin Invest. 2022;132(3):e153283)? This analysis may help identify the most suitable patient\ngroup for this therapy in future studies.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "thank you for the detailed responses.", "reviewer_4": "My comments have been addressed adequately. Many thanks!", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Point-by-point responses\nReviewer #1\nThe authors have provided a comprehensive point-by-point response and revised the manuscript\naccordingly. Only minor issues remain, and upon satisfactory revision, I would recommend the\nmanuscript for publication in Nature Communications.\nQ1: In the Abstract, start with a brief rationale for clinical trial design, followed by a concise\ndescription of the methods. The key results should then be presented, beginning with the number of\npatients enrolled and summarizing the major efficacy and safety outcomes. Currently, the Methods\nand Results are somewhat interwoven, which may confuse readers.\nAnswer: Thank you for the valuable suggestion. The abstract has been revised accordingly (Line 27-43\nin the clean version of manuscript). The major revisions included: (1) the addition of a brief rationale\nfor the study design; (2) a separate and more detailed method description; (3) clarification on the\ndefinition of “untreated patients”; (4) expansion of the toxicity-related description.\nLine 27-43: Combination of Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib and rituximab has\nshown promising efficacy in mantle cell lymphoma (MCL). This phase II trial evaluated the second-\ngeneration BTK inhibitor zanubrutinib plus rituximab as induction, followed by shortened\nchemoimmunotherapy as frontline treatment for MCL (NCT04624958). Eligible patients had\nhistologically confirmed stage II–IV disease requiring immediate therapy and no prior MCL-related\nsystemic treatment. Patients received zanubrutinib-rituximab for up to 12 cycles (part A); those\nachieving complete response (CR) or experiencing disease progression proceeded to four cycles of R-\nDHAOx (rituximab, dexamethasone, cytarabine and oxaliplatin) (part B). Patients with CR after part B\nreceived zanubrutinib maintenance for one year. The primary endpoint was the CR rate at part A\ncompletion. Forty-two patients were enrolled. The CR rate at part A completion was 88% (95%\nconfidence interval [CI], 74-96), and 86% (95% CI, 72-95) at part B completion. Hematologic\ntoxicities predominated: the most common grade 3-4 adverse events were neutropenia (7%) in part A,\nand thrombocytopenia (77%) and neutropenia (49%) in part B. In conclusion, zanubrutinib-rituximab\ninduction followed by shortened R-DHAOx is efficacious with manageable safety as frontline therapy\nfor MCL. This strategy allows for a reduction in chemotherapy cycles and warrants validation in\nrandomized controlled trials.\nQ2: The toxicity description in the Abstract is unclear. A more focused summary of the key safety\nfindings would be helpful.\nAnswer: We appreciate for this comment. The abstract has been revised to provide a more focused and\ndetailed summary of safety (Line 38-40).\nLine 38-40: Hematologic toxicities predominated: the most common grade 3-4 adverse events were\nneutropenia (7%) and lymphocytopenia (7%) in part A, and thrombocytopenia (77%) and neutropenia\n(49%) in part B.\nQ3: In the Results–Adverse Events section, the authors should briefly summarize key supportive\ntreatments and patient recovery outcomes.\nAnswer: A summary of supportive treatment and recovery outcomes have been added in the Result\nsection (Line 171-172, Line 182-189 and Line 194-197).\nLine 171-172: None of the patients needed recombinant human granulocyte colony-stimulating factor\n(rhG-CSF) supportive treatment during part A.\nLine 182-189: Thrombopoietin receptor agonists (TPO-RAs) or recombinant human thrombopoietin\n(rhTPO) were given in 9 patients as primary prevention for thrombocytopenia, and in 13 patients as\nsecondary prevention. Five patients still experienced severe thrombocytopenia despite supportive care\nwith TPO-RAs or rhTPO and dose reduction, and therefore discontinued treatment. rhG-CSF was given\nin 26 patients as primary prevention for neutropenia, and in all the 19 patients who had experienced\ngrade 3-4 neutropenia as secondary prevention. None of the patients experienced further occurrence of\nsevere neutropenia after receiving rhG-CSF supportive therapy and dose reduction.\nLine 194-197: One patient experienced grade 3 neutropenia and recovered after supportive treatment\nwith rhG-CSF, after which the zanubrutinib dose was reduced to 80 mg twice daily. No other dose\nreductions were required during the maintenance phase.\nQ4: Please note the placement of the subtitles on page 8, line 193.\nAnswer: Thank you for the kind reminding, and placement of the subtitle has been adjusted.\nQ5: Did the authors compare the three MCL molecular subtypes (M1-M3) with the four clusters (C1-\nC4) identified by Yi S et al. (Yi S, Yan Y, Jin M, et al. J Clin Invest. 2022;132(3):e153283)? This\nanalysis may help identify the most suitable patient group for this therapy in future studies.\nAnswer: Thank you for the valuable suggestion. During the revision, we performed further comparison\nbetween the three subtypes (M1-M3) with the four subtypes (C1-C4) identified by Yi S et al.\nFirst, using RNAseq data, we compared the gene expression signatures using normalized\nenrichment score (NES) (Figure A: M1-M3; Figure B: C1-C4 from Yi S et al.). Interestingly, similar to\nthe C4 subtype from Yi S et al., patients with the M1 subtype were characterized by activation of cell\ncycle and MYC pathways, and showed poor clinical outcomes. Other subtypes showed different\ncharacteristics.\nThen we compared the genomic characteristics using WES data (Figure C). We analyzed the\nreported genomic features of C1-C4 from Yi S et al. and evaluated their occurrence in our cohort.\nHowever, the results showed that the M1-M3 subtypes did not have enough features that can be highly\nmatched with those of C1-C4.\nCollectively, the differences might be explained by that the C1-C4 subtypes from Yi S et al. were\nidentified based on WES characteristics, while our M1-M3 subtypes were identified from transcriptional\ncharacteristics. Future molecular and genomic studies are needed to further identify the patients who\nmay benefit the most from the frontline use of BTK inhibitors.\n**REVIEWER #1 ON BEHALF OF REVIEWER #3**:\nQ1: Abstract Q2: The definition of “untreated MCL” would benefit from additional clarification. The\nauthors should explicitly state that this refers to patients who had never received any disease-related\nsystemic therapy.\nAnswer: Thank you for the valuable suggestion. The clarification of “untreated MCL” has been\nprovided in the abstract (Line 30-32).\nLine 30-32: Eligible patients had histologically confirmed stage II–IV disease requiring immediate\ntherapy and no prior MCL-related systemic treatment.\nQ2: Please carefully review the entire manuscript to correct typographical and formatting errors. For\nexample, “in patients withour]t bone marrow involvement” (Line 354, Page 14).\nAnswer: Thank you for this helpful comment. We have carefully checked the entire manuscript and\ncorrected the typographical and formatting errors throughout.\nQ3: In introduction, MCL is characterized by translocation of t(11;14)(q13;q32), not\nt(11;14)(q14;q32).\nAnswer: This mistake has been corrected in the revised manuscript.\nQ4: The manuscript would benefit from improved conciseness while preserving the existing content.\nAnswer: Thank you so much for your effort during the revision. We have carefully revised the\nmanuscript to improve conciseness, particularly in the Abstract and Discussion sections."}]

Decision letter