Orelabrutinib, fludarabine, cyclophosphamide, and obinutuzumab (OFCG) for first-line treatment of chronic lymphocytic leukemia: a multicenter phase II trial (cwCLL-001 trial)

Abstract

Background

This multicenter phase II trial aimed to evaluate the orelabrutinib, fludarabine, cyclophosphamide, and obinutuzumab (OFCG) regimen as first-line therapy for patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma without restriction by TP53 aberrations [del(17p) and/or TP53 mutation] and IGHV status.

Methods

Eligible patients received OFCG (orelabrutinib, orally 150 mg/day; fludarabine, intravenously 25 mg/m²/day; cyclophosphamide, intravenously 250 mg/m²/day; obinutuzumab, intravenously 1000 mg), with treatment duration and modifications based on prespecified minimal residual disease (MRD) criteria. The primary endpoint was the rate of undetectable MRD in bone marrow (BM-uMRD) after 6 cycles by flow cytometry (10⁻⁴ sensitivity). The key secondary endpoints included the rate of BM-uMRD4 at the end of cycles 3, 12, MRD in peripheral blood (PB-uMRD4) at the end of cycles 6, and complete response rate (CRR); exploratory endpoints included the rate of BM-uMRD6 (10⁻⁶ sensitivity) at the end of cycles 3, 6, and 12.

Results

Among 25 enrolled patients, 23 completed 3 cycles of OFCG therapy; of these, 13 patients achieved BM-uMRD4. After 6 cycles, 76.0 % (19/25) achieved BM-uMRD4, and 80 % (20/25) of patients achieved PB-uMRD4. CRR with BM-uMRD4 was achieved by 20.0 % (5/25), 52.0 % (13/25), and 64.0 % (16/25) of patients after 3, 6, and 12 cycles, respectively. Significant differences in BM-uMRD6 at C7D0 (odds ratio [OR], 0.03 [95 % confidence interval [CI]: 0, 0.32]; P < 0.01) and C13D0 (OR, 0.16 [95 % CI: 0.03, 0.94]; P < 0.05) were observed between IGHV-mutated (n = 10) and unmutated (n = 15) patients. Notably, 20 patients discontinued all the drugs at the data cutoff date. No treatment-related deaths occurred, and the most frequent grade 3–4 adverse effects (AEs) were neutropenia (20/25, 80.0 %), thrombocytopenia (15/25, 60.0 %), and infection (7/25, 28.0 %).

Conclusion

The study meet the primary endpoint, showing the potential efficacy and acceptable safety profile of the OFCG regimen as a first-line treatment for CLL. Trial registration: NCT05322733.

1. Introduction

Chronic lymphocytic leukemia (CLL) and its tissue counterpart, small lymphocytic lymphoma (SLL), are incurable B-cell malignancies with a heterogeneous clinical course.^1,2 CLL is the most common subtype of leukemia in Western countries. Although CLL is less common in China, its incidence has gradually increased in recent years.³ For the past several decades, chemoimmunotherapy with fludarabine, cyclophosphamide, and anti-CD20 antibody rituximab (FCR) has been established as the standard-of-care first-line therapy for young patients with CLL/SLL, with a 5-year progression-free survival (PFS) of approximately 50 %.² However, TP53 abnormalities and unmutated immunoglobulin heavy-chain gene (IGHV) have been identified as independent predictors for adverse clinical outcomes in patients treated with FCR, indicating that not all patients benefit from this regimen.⁴ Moreover, the toxic effects related to this regimen have also limited its clinical application.⁴ Therefore, further optimization of established treatments, alongside the introduction of new agents, is warranted.

Targeted therapy with Bruton's tyrosine kinase (BTK) inhibitors has changed the treatment landscape of CLL/SLL over the past decade.5, 6, 7 To enhance therapeutic outcomes, the BTK inhibitor-based combination therapies have been designed for CLL.8, 9, 10 Notably, ibrutinib combined with fludarabine, cyclophosphamide, and rituximab or obinutuzumab (iFCR or iFCG) as first-line therapy has shown promising efficacy for patients with CLL/SLL, with high rates of minimal residual disease (MRD) negativity reported.11, 12, 13, 14 These combinations also provide a fix-duration treatment option for treatment naive patients with CLL, particularly those who are young.

Orelabrutinib is a second-generation, highly selective BTK inhibitor.¹⁵ Preliminary data in relapsed/refractory (R/R) CLL/SLL suggest encouraging clinical activity, with an objective response rate (ORR) of approximately 93 % and a complete remission rate of 21.3 %, alongside acceptable tolerability.^16,17 In contrast to ibrutinib, orelabrutinib could preserve natural killer-cell-mediated antibody-dependent cellular cytotoxicity (ADCC) induced by rituximab and produce synergistic anti-tumor responses.¹⁸ Obinutuzumab, a type II anti-CD20 antibody with enhanced direct cell killing and ADCC, has shown substantial activity in CLL/SLL, and the additional benefit of obinutuzumab-based chemoimmunotherapy over rituximab-based chemoimmunotherapy was observed in the pivotal phase III CLL11 trial.19, 20, 21

Based on these evidences, we hypothesize that the combination of orelabrutinib with obinutuzumab-based chemoimmunotherapy could yield encouraging clinical outcomes. Therefore, a multicenter, investigator-initiated phase II trial was conducted to evaluate the orelabrutinib, fludarabine, cyclophosphamide, and obinutuzumab (OFCG) regimen as the first-line therapy for patients with CLL/SLL.

2. Materials and methods

2.1. Study design

The cwCLL-001 trial was a multicenter, open-label, phase II trial of OFCG in previously untreated patients with CLL/SLL without restriction by del(17p)/TP53 aberrations and/or IGHV mutation status (Fig. 1). Patients were eligible if they were aged 18–65 years and had confirmed treatment-naive CD20-positive CLL or SLL. All patients were required to meet at least one indication for treatment per the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) criteria. Patients were also required to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, a life expectancy of at least 6 months, and adequate organ and bone marrow function. Key exclusion criteria included a Cumulative Illness Rating Scale [CIRS] score of >6, creatinine clearance < 70 mL/min, uncontrolled or significant cardiovascular disease, central nervous system involvement, any history or suspicion of Richter transformation, or allergy to the study drug or any auxiliary materials. Full eligibility and exclusion criteria are provided in Supplementary Materials.

Fig. 1.

Trial profile. AE, adverse effect; ALT/AST, alanine aminotransferase/aspartate aminotransferase; BM-uMRD4, undetectable minimal residual disease in bone marrow; CLL/SLL, chronic lymphocytic leukemia/ small lymphocytic lymphoma; FC, fludarabine and cyclophosphamide; OFCG, orelabrutinib, fludarabine, cyclophosphamide, and obinutuzumab combination regimen; OG, orelabrutinib and obinutuzumab combination regimen.

This trial was conducted according to the principles of Good Clinical Practice guidelines and the Declaration of Helsinki. The institutional review boards or ethics committees of participating centers approved the study protocol (approval number of the coordinating center: 2021-SR-188). Each participating patient provided written informed consent. This study was registered with clinicaltrials.gov (Identifier, NCT05322733).

2.2. Procedure

OFCG therapy was administered in 28-day cycles. Orelabrutinib (orally 150 mg/day) was administered until confirmed undetectable MRD at a sensitivity of 10⁻⁴ (uMRD4) by flow cytometry (FCM) in BM (BM-uMRD4) at the end of cycle 12 or 24, or until disease progression or intolerable toxicity. Fludarabine (intravenously 25 mg/m²/day) and cyclophosphamide (intravenously 250 mg/m²/day) were administered on days 1–3 of each cycle for a maximum of 6 cycles. Obinutuzumab was administered (intravenously 1000 mg) on days 1, 8, and 15 of cycle 1, and on day 1 of cycle 2 onwards for a maximum of 12 cycles.

Patients received OFCG for the first three cycles. Treatment following the first three cycles of OFCG treatment was determined by prespecified MRD criteria. Patients who achieved BM-uMRD4 by FCM after three cycles (C4D0) of OFCG therapy would receive 3 cycles of orelabrutinib (orally 150 mg/day, cycle 4–6) plus obinutuzumab (intravenously 1000 mg) therapy, and other patients would receive an additional 3 cycles of OFCG therapy. After six cycles of therapy (C7D0), patients who achieved BM-uMRD4 would receive 6 cycles of orelabrutinib (orally 150 mg/day, cycle 7–12) monotherapy, and other patients would receive 6 cycles of orelabrutinib (orally 150 mg/day, cycle 4–6) plus obinutuzumab (intravenously 1000 mg) therapy. After 12 cycles of therapy (C13D0), patients who achieved BM-uMRD4 would discontinue all therapy, and others would receive 12 cycles of orelabrutinib (orally 150 mg/day, cycle 13–24) monotherapy. After 24 cycles of therapy (C25D0), patients who achieved BM-uMRD4 would discontinue all therapy, and other patients would continue treatment at the investigator’s discretion. Modification of the therapy regimen was allowed according to the safety and tolerability of patients. Detailed criteria for dose suspension and treatment discontinuation are described in Supplementary Table 1–4.

BM and peripheral blood (PB) samples were collected to assess MRD status using 8 multi-color FCM with a sensitivity of 10⁻⁴ and next-generation sequencing (NGS) with a sensitivity of 10^-6.²² Full details on BM and PB sample collection and targeted NGS are available in Supplementary Materials and Supplementary Table 5. Treatment responses were performed according to the IWCLL 2018 criteria and Lugano 2014 criteria, using enhanced computed tomography (CT) at baseline, every 3 cycles until cycle 24, and followed by every 6 cycles thereafter until disease progression. Safety evaluations including monitoring for adverse events (AEs) according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0 (NCI-CTCAE v5.0; non-hematological AEs) or IWCLL Toxicity Grading Scale (hematological AEs). Peripheral blood samples were collected to evaluate the serum IgG/IgA concentration and absolute CD4/CD8⁺ T lymphocyte count at the assessment time point. Fluorescence in situ hybridization (FISH) was performed on standard cytogenetic preparations from peripheral blood.²³ TP53 mutations sequencing and analysis were performed according to European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) guidelines.²⁴ IGHV mutation status and complex karyotype (CK) were assessed using standard methods based on cellular DNA and metaphase cytogenetics, respectively.²⁵

2.3. Endpoints

To simplify the reporting, we used the following categories for MRD description: MRD0 for MRD ≥ 10⁻¹; MRD1 for MRD ≥ 10⁻² and < 10⁻¹; MRD2 for MRD ≥ 10⁻³ and < 10⁻²; MRD3 for MRD ≥ 10⁻⁴ and < 10⁻³; MRD4 for MRD ≥ 10⁻⁵ and < 10⁻⁴ by NGS; MRD5 for MRD ≥ 10⁻⁶ and < 10⁻⁵ by NGS; and uMRD6 for undetectable MRD < 10⁻⁶ by NGS. In this study, uMRD4 specifically refers to undetectable MRD < 10⁻⁴ by FCM.

The primary endpoint was the rate of BM-uMRD4 after 6 cycles by FCM. Secondary endpoints were the rate of BM-uMRD4 by FCM at the end of cycles 3, 12, and 24, the rate of PB-uMRD4 by FCM at the end of cycles 3, 6, 12, and 24, ORR, complete response rate (CRR), 2- and 3-year PFS rates, duration of response (DoR), 3-year overall survival (OS) rate, and safety. ORR was defined as the proportion of patients with the best objective response of the complete response (CR), CR with incomplete bone marrow recovery (CRi), partial response (PR), and PR with lymphocytosis. CRR was referred to as the proportion of patients with CR and CRi. DoR was calculated as the time from first objective response to progression, or death, whichever occurred first. PFS was defined as the time from treatment initiation to disease progression or death from any cause, whichever occurred first. OS was calculated as the time from treatment initiation to death of any cause.

Exploratory endpoints included the rate of BM-uMRD6 by NGS at the end of cycles 3, 6, 12, and 24, and the rate of PB-uMRD6 by NGS at the end of cycles 3 and 6.

2.4. Statistical analysis

According to previous studies,^26,27 the rate of BM-uMRD4 after 6 cycles of FCR therapy was approximately 43–44 %. Thus, the historical control of the rate of BM-uMRD4 was set at 45 % (P0, null hypothesis). Assuming that the OFCG therapy in this trial would improve the rate of BM-uMRD4 to 75 % (P1) after 6 cycles, a total sample size of 25 patients would be sufficient with a one-sided alpha of 0.025 and a power of 80 %, as well as a 20 % dropout rate. The sample size calculation was performed using PASS software version 15.0 (NCSS, Kaysville, UT, USA).

Efficacy analysis was performed in the full analysis set (FAS) following the intention-to-treat (ITT) principle, which was defined as all patients who received at least one dose of study treatment. Safety was analyzed in the safety analysis set (SAS), which was defined as the patients who received at least one dose of study treatment and had safety records. Descriptive statistics were used to summarize patients’ characteristics, responses (the rate of BM-uMRD4, ORR, and CRR), and safety data. The Clopper-Pearson method was used to calculate the 95 % confidence intervals (CIs) of responses. PFS and OS were analyzed using the Kaplan-Meier method with corresponding 2-sided 95 % CIs. In addition, the Spearman correlation analysis was used to assess the consistency between PB and BM data measured by FCM or NGS using the absolute MRD values. Besides, univariate and multivariate analyses with the logistic proportional hazards model were carried out to identify the independent factors. Statistical analyses were conducted with SPSS version 25.0 software, R version 4.3.3, and GraphPad Prism version 10.1.2.

3. Results

3.1. Patient characteristics

Between May 30, 2022, and June 30, 2023, 30 patients were screened for eligibility, and 5 were excluded due to failure to meet the inclusion criteria (n = 3) and withdrawal of consent (n = 2). Ultimately, a total of 25 patients were enrolled and received study treatment (Fig. 1). The baseline demographic and clinical characteristics of the enrolled patients are summarized in Table 1. The median age was 48 years (interquartile range, 37–50). Among the cohort, 72.0 % (18/25) of patients were in Rai stage III or IV disease, 24.0 % (6/25) of patients had Bulky disease (≥ 10 cm), and 60.0 % (15/25) of patients were stratified into high or very high CLL International Prognostic Index score.²⁸ Unmutated IGHV, del(17p), and del(11q) were present in 60.0 % (15/25), 8.0 % (2/25), and 24.0 % (6/25) of patients, respectively. In addition, CK was present in 8 (33.3 %) of 24 patients.

Table 1.

Baseline characteristics.

Characteristics	Number of patients (n = 25)
Age, median (interquartile range), years	48 (37–50)
Male, No. ( %)	15 (60.0)
ECOG-performance score, No. ( %)
0	19 (76.0)
1	6 (24.0)
Rai stage, No. ( %)
0	0 (0.0)
Ⅰ-Ⅱ	7 (28.0)
Ⅲ-Ⅳ	18 (72.0)
Binet stage, No. ( %)
A	1 (4.0)
B	8 (32.0)
C	16 (64.0)
Baseline labs, median (interquartile range)
Absolute neutrocyte count, ×109/L	2.7 (1.8–5.7)
Absolute lymphocyte count, ×109/L	68.1 (18.4–149.7)
Hemoglobin, g/dL	101.0 (78.0–121.5)
Platelets, ×109/L	111.0 (85.0–143.0)
β2-microglobulin, mg/L	4.2 (3.3–5.1)
Lactate dehydrogenase, U/L	194.0 (175.5–256.5)
B symptoms, No. ( %)	7.0 (28.0)
Bulky lymphadenopathy, No. ( %), cm
≥ 5	14 (56.0)
≥ 10	6 (24.0)
IGHV status, No. ( %)
Unmutated	15 (60.0)
Mutated	10 (40.0)
Baseline hierarchical FISH, No. ( %)
Del(17p)	2 (8.0)
Del(11q)	6 (24.0)
Trisomy 12	1 (4.0)
Normal	7 (28.0)
Del(13q)	12 (48.0)
Complex karyotype, No. ( %)*	8 (33.3)
Baseline gene mutation, No. ( %)
TP53	3 (12.0)
ATM*	6 (25.0)
NOTCH1*	7 (29.2)
SF3B1*	3 (12.5)
MYD88*	1 (4.2)
FAT1*	2 (8.3)
KMT2D*	5 (20.8)
EGR2*	6 (25.0)
CLL-IPI	6 (25.0)
Low	6 (24.0)
Intermediate	4 (16.0)
High	12 (48.0)
Very High	3 (12.0)

^⁎A total of 24 patients had these baseline characteristics data.

Abbreviations: CLL-IPI, Chronic Lymphocytic Leukemia International Prognostic Index; ECOG, Eastern Cooperative Oncology Group. FISH, fluorescence in situ hybridization; IGHV, immunoglobulin heavy-chain gene.

3.2. Efficacy

At the data cut-off (December 31, 2024), two patients voluntarily discontinued the study after receiving 2 cycles of OFCG (Fig. 1). Among the 23 patients who completed 3 cycles of OFCG, 1 discontinued treatment after 4 cycles due to recurrent grade 4 thrombocytopenia, another withdrew from the study after 5 cycles and subsequently died from COVID-19, and a third discontinued treatment after 6 cycles of therapy due to persistent COVID-19 infection (Fig. 1). In total, 23 patients completed the 3 cycles of OFCG plus 3 cycles of OG therapy, among whom 7 patients completed the 6 cycles of OFCG therapy.

Of the 25 patients in the FAS, at the end of cycle 3, 6, and 12, the proportions of patients achieving PB-uMRD4 were 60.0 % (15/25; 95 % CI: 38.7, 78.9), 80.0 % (20/25; 95 % CI: 59.3, 93.2), and 84.0 % (21/25; 95 % CI: 63.9, 95.5), respectively. In parallel, 52.0 % (13/25; 95 % CI: 31.3, 72.2), 76.0 % (19/25; 95 % CI: 54.9, 90.6), and 80.0 % (20/25; 95 % CI: 59.3, 93.2) of patients had BM-uMRD4 assessed by FCM (Table 2 and Fig. 2A). Correspondingly, the CRR increased over time from 28.0 % (7/25; 95 % CI: 12.1, 49.4) after 3 cycles, to 52.0 % (13/25; 95 % CI: 31.3, 72.2) after 6 cycles, and to 68.0 % (17/25; 95 % CI: 46.5, 85.1) after 12 cycles. Similarly, the proportion of patients achieving both CRR and BM-uMRD4 increased, with 20.0 % (5/25; 95 % CI: 6.8, 40.7), 52.0 % (13/25; 95 % CI: 31.3, 72.2), and 64.0 % (16/25; 95 % CI: 42.5, 82.0) after 3, 6, and 12 cycles, respectively. The assessment at the end of 24 cycles was not performed, as nearly half of the patients had not yet reached the 24-cycle evaluation point.

Table 2.

Summary of MRD and response in the intention-to-treat population and efficacy evaluable population.

No. (%, interquartile range)	Intention-to-treat population (n = 25)			Efficacy evaluable population
	After cycle 3	After cycle 6	After cycle 12	After cycle 3 (n = 23)	After cycle 6 (n = 22)	After cycle 12 (n = 22)	After cycle 24 (n = 13)†
BM-uMRD4	13 (52.0, 31.3–72.2)	19 (76.0, 54.9–90.6)	20 (80.0, 59.3–93.2)	13 (56.5, 34.5–76.8)	19 (86.4, 65.1–97.1)	20 (90.9, 70.8–98.9)	12 (92.3, 64.0–99.8)
PB-uMRD4	15 (60.0, 38.7–78.9)	20 (80.0, 59.3–93.2)	21 (84.0, 63.9–95.5)	16 (69.6, 47.1–86.8)	20 (95.2, 76.2–99.9) #	21 (95.5, 77.2–99.9)	12 (92.3, 64.0–99.8)
ORR	22 (88.0, 68.8–97.5)	22 (88.0, 68.8–97.5)	22 (88.0, 68.8–97.5)	22 (100.0, 84.6–100.0) *	22 (100.0, 84.6–100.0)	22 (100.0, 84.6–100.0)	13 (100.0, 75.3–100.0)
CRRa	7 (28.0, 12.1–49.4)	13 (52.0, 31.3–72.2)	17 (68.0, 46.5–85.1)	7 (31.8, 13.9–54.9) *	13 (59.1, 36.4–79.3)	17 (77.3, 54.6–92.2)	12 (92.3, 64.0–99.8)
Both BM-uMRD and CRRa	5 (20.0, 6.8–40.7)	13 (52.0, 31.3–72.2)	16 (64.0, 42.5–82.0)	5 (22.7, 7.8–45.4) *	13 (59.1, 36.4–79.3)	16 (72.7, 49.8–89.3)	11 (84.6, 54.6–98.1)

^⁎Evaluable in 22 patients, due to the response assessment not available for one patient.

^#Evaluable in 21 patients, due to the MRD data being unavailable for one patient.

^†Only 13 patients completed the 24-cycle follow-up.

^aCRR was referred to as the proportion of patients with complete remission/complete remission with incomplete marrow recovery.

Abbreviations: BM, bone marrow; CRR, complete response rate; MRD, measurable residual disease; ORR, objective response rate; PB, peripheral blood; uMRD, undetectable MRD.

Fig. 2.

Response assessment. (A) Response assessment and MRD in 25 patients. (B) MRD by NGS (sensitivity of 10⁻⁶) in 25 patients with IGHV mutated and with IGHV unmutated, including TP53 aberration. (C) Response assessment and MRD4 in 10 patients with IGHV mutated regardless of TP53 aberration. (D) Response assessment and MRD4 in 15 patients with IGHV unmutated regardless of TP53 aberration. BM, bone marrow; C, cycle; CR/CRi, complete remission/complete remission with incomplete marrow recovery; D, day; IGHV, immunoglobulin heavy-chain gene; MRD, measurable residual disease; NGS, next-generation sequencing; PB, peripheral blood; uMRD, undetectable MRD.

Additionally, 23 patients who completed the 3 cycles of therapy comprised the efficacy evaluable population. The primary endpoint of BM-uMRD4 after 6 cycles of treatment was 86.4 % (19/22; 95 % CI: 65.1, 97.1), with 95.2 % (20/21; 95 % CI: 76.2, 99.9) of PB-uMRD4, and the CRR and CRR with BM-uMRD4 were both 59.1 % (13/22; 95 % CI: 36.4, 79.3). The MRD and response at the end of 6, 12, and 24 cycles of treatment in the assessment population are provided in Table 2 and Supplementary Fig. 1. The concordance between PB and BM FCM-MRD was assessed using Spearman correlation analysis, which revealed a strong positive correlation (r = 0.72, P < 0.001; Supplementary Fig. 2A). The additional details regarding the response and duration of the treatment are presented in Fig. 3.

Fig. 3.

Swimmer plot of treatment duration and response. *, P01, P04, P10, P12, P18, P21, P24 received 6 cycles of OFCG. P11 received 4 cycles of OFCG and discontinued FC due to persistent COVID-19 infection. The scheduled time for therapy was put off due to COVID-19 infection and increased ALT/AST (AE3). P20 discontinued G after 3 cycles of OFCG and 1 cycle of OG due to recurrent thrombocytopenia (AE4). AE, adverse effect; BM, bone marrow; CK, complex karyotype; CR/CRi, complete remission / complete remission with incomplete marrow recovery; FC, fludarabine and cyclophosphamide; G, obinutuzumab; IGHV, immunoglobulin heavy-chain gene; mIGHV, mutant IGHV; MRD, measurable residual disease; NA, not available; O, orelabrutinib; OFCG, orelabrutinib, fludarabine, cyclophosphamide, and obinutuzumab combination regimen; OG, orelabrutinib and obinutuzumab combination regimen; PB, peripheral blood; PR, partial remission; uIGHV, unmutated IGHV.

As assessed by NGS, BM-MRD4, BM-MRD5, and BM-uMRD6 were achieved by 1, 1, and 7 patients after 3 cycles, respectively (Supplementary Fig. 3). These levels were attained by 1, 2, and 12 patients after 6 cycles; and by 4, 2, and 11 patients after 12 cycles (Supplementary Fig. 3). Among the 13 patients who completed 24 cycles of follow-up, BM-MRD4, BM-MRD5, and BM-uMRD6 were achieved by 3, 2, and 5 patients. The results of PB-MRD assessed by NGS after 3, 6, and 12 cycles of therapy are presented in Supplementary Fig. 3. The concordance between PB and BM NGS-MRD also showed a strong positive correlation (r = 0.75, P < 0.01; Supplementary Fig. 2B). Additionally, a positive correlation was observed between PB-MRD results obtained by NGS and FCM (r = 0.53, P < 0.01; Supplementary Fig. 2C), as well as between BM-MRD results from the two methods (r = 0.54, P < 0.01; Supplementary Fig. 2D).

At the data cut-off on December 31, 2024, the median follow-up time was 26.8 months (95 % CI: 24.9, 28.7). The median PFS was not reached (NR), with a 1-year PFS rate of 95.7 % (95 % CI: 87.7, 100.0) and a 2-year PFS rate of 95.7 % (95 % CI: 87.7, 100.0) (Fig. 4A). The median OS was NR, with OS rates of 96 % (95 % CI: 89.0, 100.0) at 1 year and 96 % (95 % CI: 89.0, 100.0) at 2 years. (Fig. 4B).

Fig. 4.

Kaplan-Meier outcome estimates. (A) PFS for 25 patients. (B) OS for 25 patients. OS, overall survival; PFS, progression-free survival.

3.3. Subgroup analysis

Significant differences in the ITT population between IGHV-mutated (n = 10) and IGHV-unmutated (n = 15) patients were observed at both C4D0 and C7D0 for PB-uMRD6 (C4D0: OR, 0.11 [95 % CI: 0.02, 0.72], P = 0.03; C7D0: OR, 0.13 [95 % CI: 0.02, 0.82], P = 0.04); similar results were observed at C7D0 and C13D0 for BM-uMRD6 (C7D0: OR, 0.03 [95 % CI: 0.00, 0.32], P < 0.01; C13D0: OR, 0.16 [95 % CI: 0.03, 0.94], P < 0.05; Fig. 2B). Meanwhile, in IGHV-mutated patients, both BM-uMRD6 and PB-uMRD6 were achieved more rapidly than in unmutated patients, with significant differences evident as early as cycle 3 (Supplementary Fig. 4). At C4D0, both PB-uMRD4 and BM-uMRD4 differed significantly between the IGHV-mutated and IGHV-unmutated groups, regardless of TP53 status (Fig. 2C and D). These results are also similar in the efficacy evaluable population. Between IGHV mutated (n = 9) and IGHV unmutated (n = 14), the significant differences in PB-uMRD4 (P < 0.05) and BM-uMRD4 (P < 0.05) were observed at C4D0 (Supplementary Fig. 1 and 7).

In terms of CK status, the BM-uMRD4 at C4D0 was associated with CK status (P < 0.05; Supplementary Fig. 5 and 8). Besides, the rate of CR/CRi at C13D0 was associated with TP53 aberration (P < 0.05; Supplementary Fig. 5 and 9). Two patients didn’t attain BM-uMRD4 at C13D0, continued orelabrutinib in the second year. Patient 18 (Rai stage IV, Binet stage C) with nCK, mutated NOTCH1, and unmutated IGHV underwent 6 cycles of OFCG treatment, followed by 18 cycles of O treatment. This patient achieved BM-uMRD4 at C7D0 but experienced relapse in BM at C10D0 (Supplementary Fig. 6). Patient 24 (Rai stage III, Binet stage B) with CK, del(17p) and mutated TP53, NOTCH1 and unmutated IGHV underwent 6 cycles of OFCG treatment, followed by 6 cycles of OG treatment and 12 cycles of O treatment; the patient achieved PB-uMRD4 after 15 cycles (C16D0) during the monotherapy period (Supplementary Fig. 6). In addition, two patients (Patients 02 and 21) experienced MRD relapse by FCM, both of whom had CK and unmutated IGHV (Supplementary Fig. 6). Three patients (Patients 09, 17, and 22) experienced MRD relapse in both PB and BM by NGS at a sensitivity of 10⁻⁶ at C25D0, C25D0, and C13D0, respectively, while maintaining uMRD4 in PB and BM (Supplementary Fig. 6). Importantly, among the five patients with MRD relapse, three did not achieve BM-uMRD4 at C4D0, suggesting that early deep remission may be predictive of long-term molecular relapse. Moreover, none of these experienced disease progression as per IWCLL criteria, and no changes were made to the treatment regimen.

3.4. Safety

Most AEs were mild and manageable by dose interruptions or supportive care within the 12 cycles of therapy (Table 3). The most common AEs of grade 1–2 were anemia (25/25, 100.0 %), nausea/vomiting (18/25, 72.0 %), and alanine aminotransferase/aspartate aminotransferase (ALT/AST) increased (13/25, 52 %). Neutropenia (20/25, 80.0 %), thrombocytopenia (15/25, 60.0 %), and infection (7/25, 28.0 %) were the most common grade 3–4 AEs. No new safety signals were identified after 12 cycles of therapy (Supplementary Table 6). Neutropenia and tumor lysis syndrome were more common grade 3–4 AEs in patients who received 6 cycles of OFCG than 3 cycles of OFCG (Supplementary Table 7). In addition, the incidence of hematological AEs (including neutropenia, anemia, and thrombocytopenia) varied with the number of treatment cycles, as illustrated in Supplementary Fig. 10. The incidence of neutropenia, anemia, and thrombocytopenia decreased as treatment cycles were increased, with a gradual decrease in grade 3–4 neutropenia, particularly after six cycles, and thrombocytopenia. No atrial fibrillation, cardiac AEs, or treatment-related deaths occurred. During the treatment, 22 (88.0 %) patients were infected with COVID-19.

Table 3.

Adverse events within the 12 cycles in the safety analysis set (n = 25).

Adverse events	Grade 1–2	Grade 3–4
Hematological adverse events, No. ( %)
Anemia	25 (100)	0 (0)
Neutropenia	5 (20)	20 (80)
Thrombocytopenia	9 (36)	16 (64)
Febrile neutropenia	-	4 (16)
Non-hematological adverse events, No. ( %)
Nausea/vomiting	18 (72)	0 (0)
Infusion related reaction	12 (48)	5 (20)
ALT/AST increased	13 (52)	3 (12)
Malaise	9 (36)	2 (8)
Infection*	1 (4)	7 (28)
Arthralgia	6 (24)	1 (4)
Skin rashes	5 (20)	1 (4)
Diarrhea	5 (20)	1 (4)
Purpura	5 (20)	0 (0)
Pruritus	4 (16)	1 (4)
Tumor lysis syndrome	-	4 (16)
Pain	2 (8)	2 (8)
Hypertension	0 (0)	1 (4)
Shingles	1 (4)	0 (0)

^⁎A total of 22 (88.0 %) patients were infected with COVID-19.

Abbreviation: ALT/AST, alanine aminotransferase/aspartate aminotransferase.

Eight patients required dose reductions in FC therapy due to recurrent grade 3 or worse hematologic AEs, while four discontinued FC therapy due to grade 3 or worse hematologic AEs or COVID-19 infection. Orelabrutinib was discontinued in 95.8 % (23/24) of patients temporarily, primarily due to COVID-19 infection (n = 17), followed by thrombocytopenia (n = 7), neutropenia (n = 4), rash (n = 3), and ALT/AST increased (n = 1). Additionally, three patients required dose reductions of orelabrutinib due to COVID-19 infection (n = 1), thrombocytopenia (n = 1), and other reasons (n = 1). In terms of serum IgG/IgA concentration and absolute CD4/CD8⁺ T lymphocyte count, no statistical difference was observed between patients who received 3 cycles of OFCG plus 3 cycles of OG and patients who received 6 cycles of OFCG (Supplementary Fig. 11). It is worth noting that patients who received 3 cycles of OFCG plus 3 cycles of OG demonstrated an early trend of recovery in absolute CD4⁺ T lymphocyte count after 3 cycles of treatment, and serum IgA concentration showed a relatively stable trend after 6 cycles of treatment (Supplementary Fig. 11).

4. Discussion

To our knowledge, this study was the first to investigate the combination of a second-generation BTKi with FC and an anti-CD20 antibody. In this multicenter phase II trial, we investigated MRD-directed, time-limited treatment with OFCG as a first-line therapy in CLL. The primary endpoint of the trial was met, with 86.4 % (19/22) patients attaining BM-uMRD4. In addition, the safety profile was tolerable and manageable. These findings provide crucial data for the clinical management of treatment-naive patients with CLL, especially those who are young.

In this study, we analyzed OFCG as the first-line treatment in CLL, irrespective of the IGHV or TP53 status. The high rates of uMRD4 in both BM and PB were encouraging and compared favorably with FCR or FCG therapy in the first-line treatment of CLL/SLL. In the CLL10 trial, six cycles of FCR therapy resulted in uMRD4 rates of 58 % and 74 % in BM and PB, respectively.²⁹ A subgroup analysis of patients with previously untreated CLL/SLL in the GREEN study showed that the rate of uMRD4 was 64.3 % in PB and 35.7 % in BM (in the MRD-evaluable population only) after six cycles of first-line FCG therapy.³⁰ This higher rate of uMRD4 might be attributable to the addition of a BTK inhibitor to our combination regimen. As reported, orelabrutinib could preserve natural killer-cell-mediated ADCC induced by anti-CD20 antibodies and produce synergistic anti-tumor responses.⁷ Additionally, the rates of uMRD4 (PB, 64.0 %; BM, 52.0 %) after 3 cycles were also superior to those observed in the iFCR (PB, 55.9 %; BM, 41.2 %) therapy¹¹, while lower than iFCG, which included only IGHV mutated (BM, 87 %) therapy.¹² Several factors might be responsible for this difference in rates of uMRD4. One consideration was that orelabrutinib had less off-target inhibition against other tyrosine kinases¹⁵, and the obinutuzumab offered stronger direct cell killing and ADCC, which might explain the superior MRD-negative rate we observed compared to the iFCR therapy. Additionally, the iFCG therapy study was limited to patients with mutated IGHV and the absence of del (17p)/TP53 mutation.¹² In contrast, our study included a majority of patients with high-risk features, including unmutated IGHV (56 %), TP53 mutation/del (17p) (16 %), or del (11q) (24 %). This higher proportion of high-risk patients in our study could plausibly explain the comparatively lower rate of uMRD4 observed relative to the iFCG therapy results. Notably, the rate of uMRD4 in BM (90 %) for our patients with IGHV mutated was also higher than the iFCG therapy,¹² despite receiving only three cycles of OFCG. The rate of uMRD6 in BM for patients with IGHV mutated in our study was also higher than the previous study (80.0 % vs. 61 %).³¹ To achieve a more thorough evaluation of these promising results, it is imperative to conduct further validation in studies with a larger sample size.

A notable feature of our study design was that patients followed a predefined schedule for chemotherapy administration throughout the whole treatment course. In the present study, all patients received the treatment with 3 cycles of OFCG therapy, followed by an MRD-driven strategy in which FCG was given only to patients with detectable MRD in BM, which was designed to minimize chemotherapy-related toxicity while optimizing activity. In a previous study of patients treated with iFCG therapy, 91 % of enrolled patients achieved MRD negativity, and all patients discontinued treatment after 12 cycles.¹² Similarly, in our study, despite enrolling a significant number of patients with high-risk features, it was encouraging to note that an 80.0 % BM-uMRD4 rate was achieved after completing 12 cycles of therapy. Importantly, 13 (13/22, 59.1 %) patients achieved a CR/CRi with BM-uMRD4 after the 6 cycles of therapy. These results suggested that even in the high-risk group, our regimen also demonstrated remarkable efficacy in eradicating residual disease. To comprehensively assess the endurance and stability of these remission periods, sustained long-term follow-up is essential.

In this study, we also examined the rates of uMRD between patients with IGHV-mutated and those with IGHV-unmutated. The analysis revealed a statistically significant difference in the rates of BM-uMRD4 and PB-uMRD4 between the two groups at C4D0, suggesting that the IGHV mutation status may influence the early response to treatment. Specifically, 9 patients with IGHV-mutated CLL achieved both PB-uMRD4 (90.0 %) and BM-uMRD4 (90.0 %) in our study after three cycles of OFCG, markedly higher than IGHV-unmutated patients (40.0 %, 26.7 %). By comparison, the uMRD4 rates in IGHV-mutated patients observed in our study were higher than those reported in CAPTIVATE (three cycles of ibrutinib, then 12 cycles of ibrutinib plus venetoclax),³² which reported the best PB-uMRD4 and BM-uMRD4 rates of 72 % and 60 %, respectively, and AMPLIFY, which reported a PB-uMRD4 rate of 33 % in patients treated acalabrutinib plus venetoclax at the end of treatment.³³ These results indirectly indicate that, in IGHV-mutated patients, the chemoimmunotherapy-based OFCG regimen achieved higher MRD negativity rates than non-chemotherapy BTK inhibitor plus BCL2 inhibitor combinations. Meanwhile, this finding was comparable to the iFCG therapy, which resulted in a BM-uMRD4 of 87 % after cycle 3.¹² Even at a sensitivity of 10⁻⁶, BM-uMRD6 and PB-uMRD6 rates at the end of treatment were 70.0 % and 60.0 %, respectively, demonstrating the depth of response in this subgroup. Notably, 90 % of IGHV-mutated patients in our cohort achieved BM-uMRD4 after only three cycles, allowing subsequent treatment to proceed without further chemotherapy while maintaining MRD negativity, and discontinued therapy after cycle 12. Taken together, these findings suggest that patients with IGHV-mutated CLL may be candidates for BTK inhibitor–based chemoimmunotherapy approaches, which allow for reduced chemotherapy exposure while maintaining deep and durable MRD responses. However, this difference between patients with IGHV-mutated and those with IGHV-unmutated did not persist in subsequent cycles, probably due to the fact that the rate of uMRD4 in IGHV-unmutated patients increased in subsequent cycles. Interestingly, at a sensitivity of 10⁻⁶, PB-uMRD6 was achieved earlier than BM-uMRD6 in IGHV-mutated patients, as evidenced by a significant difference in PB-uMRD6 between IGHV-mutated and IGHV-unmutated patients at C4D0. With continued treatment, PB-uMRD6 rates in IGHV-unmutated patients increased, resulting in no difference between the two groups at C13D0. In contrast, a difference in BM-uMRD6 between IGHV-mutated and IGHV-unmutated patients persisted at C13D0. These findings may reflect differences in MRD kinetics and assay sensitivity between peripheral blood and bone marrow, which warrant further investigation.

In addition, due to the limited number of patients with TP53 abnormalities, the relationship between TP53 mutations and MRD status remains unclear and requires further study in larger cohorts. Meanwhile, our study observed that the rate of BM-uMRD4 at C4D0 was associated with CK status, highlighting a potential link between early BM-MRD levels and CK status. This finding underscores the potential of CK status as a predictive marker for early BM-MRD levels, which may have implications for risk stratification, treatment adaptation, and long-term disease monitoring. Further studies are needed to confirm these observations.

MRD was also monitored in both PB and BM in the present study. Our findings revealed that the uMRD4 increased with the number of treatment cycles in both PB and BM. Besides, the concordance between PB and BM NGS-MRD also showed a strong positive correlation. Previous studies have confirmed that achieving MRD negativity in BM was associated with a better prognosis, making it crucial to select the appropriate tissue sample for testing.^34,35 Although the rate of PB-uMRD4 was slightly higher than that in BM at cycles 3 and 6 in our study, given the invasive, time-consuming, and expensive nature of testing for MRD in BM,³⁶ PB samples might serve as a more convenient option for MRD assessment when BM sampling is unfeasible. Additionally, the choice of detection method was equally pivotal. In this study, we used two methods to assess MRD, FCM and NGS. Consistent results were observed for both methods, both in PB and BM. As reported, early achievement of MRD negativity using a high-sensitivity NGS assay identifies patients who have a very low risk of relapse.³⁷ Similarly, in our study, we observed that NGS detected a higher relapse rate than FCM, with a 10⁻⁶ detection threshold identifying more positive patients than other thresholds. These results suggested that a 10⁻⁶ detection threshold might be more appropriate for identifying patients eligible for treatment discontinuation within this therapeutic and could serve as a criterion for discontinuing treatment. However, the short follow-up period limited our ability to establish a clear relationship between MRD relapse and disease progression, warranting further investigation.

In general, the present study showed an acceptable safety profile for OFCG in patients with CLL. The majority of AEs were regarded as grade 1–2, which was comparable to those of iFCG or iFCR.^11,12 The most common grade 1–2 AEs were anemia, nausea/vomiting, and ALT/AST increased, which were consistent with the safety profile of those previously reported for each regimen, with no new safety signals identified.^16,30 Importantly, the majority of AEs were generally mild, tolerable, and reversible. In our study, 32 % of patients required dose reductions due to grade ≥ 3 hematologic AEs, a lower proportion compared with iFCG (FC therapy dose-reduction, 43 %).¹² Moreover, no treatment-related deaths were observed, with only one patient dying due to the COVID-19 pandemic. In addition, BTK inhibitor therapy is potentially constrained by a high incidence of cardiac AEs, notably arrhythmias, which could entail major health risks, increased mortality, and escalated healthcare costs.^38,39 Interestingly, there were no cardiac AEs observed in our study, which was lower than the iFCG therapy in CLL/SLL (atrial fibrillation, 11 %).¹² The lower cardiac AE incidence observed in our study might be attributable to the effect that orelabrutinib had remarkably less off-target inhibition against ibrutinib. Additionally, patients who received 3 cycles of OFCG plus 3 cycles of OG demonstrated an early trend of recovery in absolute CD4⁺T lymphocyte count after 3 cycles of treatment. This early recovery in CD4⁺ T lymphocyte count indicated immune reconstitution, potentially enhancing infection control and tumor surveillance while informing treatment decisions. Nevertheless, the results should be interpreted prudently due to the small sample size. Taken together, these findings suggested that OFCG therapy was well tolerated in a broad population of patients with CLL.

Several limitations of our study should be considered when interpreting the results. Firstly, this was a single-arm phase II trial, with a relatively small sample size, especially in subgroup analyses, the statistical power was constrained, and the findings should be interpreted with caution. Further validation in larger studies is warranted. Secondly, in the absence of a randomized trial, it is uncertain whether only achieving an MRD negativity through our treatment strategy would translate into improved outcomes. Thirdly, survival data were not mature, and the follow-up remained relatively short for a first-line study. Fourthly, our study excluded patients with a history or suspicion of Richter transformation; however, as highlighted by Maher et al., Richter transformation remains a potential risk in CLL, warranting ongoing monitoring.⁴⁰ Future studies should include this patient population to further assess the safety and efficacy of OFCG in the context of Richter transformation risk. In addition, the relatively younger age of our patients compared to most patients with CLL might limit the generalizability of AE data; however, all adverse events were systematically recorded, centrally monitored, and graded using standardized criteria (CTCAE v5.0), with strict protocol adherence and independent verification, minimizing potential reporting bias. Finally, although chemoimmunotherapy is recommended by current treatment guidelines to be an option when pathway inhibitors are not available or not feasible, our study demonstrated that OFCG would be a MRD driven strategy to optimize the efficacy and toxicity, especially favorable in IGHV-mutated patients. These findings support OFCG as a treatment option, and future studies should identify the patient populations likely to benefit from this combination therapy.

5. Conclusions

This multicenter phase II study in previously untreated CLL showed that MRD-directed OFCG therapy had an acceptable safety profile and met its primary endpoint, with 86.4 % of patients reaching BM-uMRD4. Moreover, this time-limited treatment approach could increase the potential for OFCG to cure patients with mutated IGHV and, importantly, might extend this potential to patients without high-risk factors. Given these data, a larger randomized controlled trial is needed in the future to validate these findings.

CRediT author contribution statement

Yi Miao: Resources, Writing – original draft, Formal analysis and Funding acquisition. Ming Liu: Writing – original draft, Data curation and Formal analysis. Shuchao Qin: Resources and Data curation. Fei Li, Jin Zhang, Hongling Peng, Chunyan Ji, Luomengjia Dai, Ziyuan Zhou, Chongyang Ding, Zhen Wang, Yeqin Sha, Tonglu Qiu, Hanning Tang, Hui Jin, Lei Fan, Wei Xu: Resources. Jianyong Li: Conceptualization, Project administration, Supervision and Funding acquisition. Yi Xia: Resources, Data curation, Funding acquisition. Huayuan Zhu: Conceptualization, Writing – original draft, Formal analysis, Project administration, Supervision and Funding acquisition. All authors: Writing – review & editing.

Ethics statement

This trial was conducted according to the principles of Good Clinical Practice guidelines and the Declaration of Helsinki. The institutional review boards or ethics committees of participating centers approved the study protocol (approval number of the coordinating center: 2021-SR-188). Each participating patient provided written informed consent.

Funding

This study was supported by the National Natural Science Foundation of China (grant number: 82170166); Natural Science Foundation of Jiangsu Province (BK20251955); Suqian Science and Technology Program (grant number: KY202305); The Specialized Diseases Clinical Research Fund of Jiangsu Province Hospital (grant number: DL202406). The study was supported in part by Roche and InnoCare Pharma. The pharmaceutical had no role in the collection or analysis of the data.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.