R-MINE-containing regimen in relapsed and refractory diffuse large B-cell lymphoma: a single-arm, multicenter phase II study

Abstract

Background

Improving the prognosis of patients with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL) remains an unmet need. This multicenter, phase II study (NCT05784987) aimed to assess the efficacy and safety of a mitoxantrone hydrochloride liposome (Lipo-MIT)-based rituximab with mitoxantrone/ifosfamide/mesna/etoposide (R-MINE)-containing regimen, in which conventional mitoxantrone was replaced with Lipo-MIT, with or without a targeted agent.

Methods

Patients older than 18 years with histologically confirmed R/R DLBCL were enrolled. All enrolled patients received an R-MINE-containing regimen in 21-day cycles for up to four cycles. The addition of the targeted agent (X) was individualized according to prior treatment resistance and molecular subtype. The primary endpoint was objective response rate (ORR).

Results

From March 27, 2023, to July 10, 2025, 60 patients were enrolled. Of the 55 efficacy-evaluable patients, the ORR was 76.4% (95% confidence interval [CI]: 63.0%–86.8%), and the complete response rate was 52.7% (95% CI: 38.8%–66.3%). With the median follow-up of 13.9 months, the median PFS was 17.5 months (95% CI: 10.8–24.2), and the estimated PFS rate at 1 year was 58.5%. The median OS was not reached, with an estimated 1-year OS rate of 84.0%. Late relapse after the last-line therapy was identified as the only independent predictor for ORR, with an ORR of 86.4%. Grade ≥ 3 treatment-related adverse events (TRAEs) occurred in 46.7% of patients, with neutropenia (33.3%) being the most common. No serious TRAEs or TRAE-related deaths occurred.

Conclusions

The Lipo-MIT-based R-MINE-containing regimen showed encouraging efficacy and a manageable safety profile in R/R DLBCL, warranting further validation.

Clinical Trial Registration

This trial is registered with ClinicalTrials.gov (NCT05784987). URL https//clinicaltrials.gov/study/NCT05784987.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12916-026-05051-z.

Background

The majority of patients diagnosed with diffuse large B-cell lymphoma (DLBCL) can be cured with first-line immunochemotherapy; however, approximately one-third develop relapsed or refractory (R/R) disease, which is generally associated with poor subsequent outcomes [1]. Despite the emergence of novel therapeutic approaches like chimeric antigen receptor T-cell therapy (CAR-T) and monoclonal or bispecific antibodies combined with chemotherapy, their clinical utility is restricted by accessibility and applicability [1–5]. In this setting, immunochemotherapy regimens, including rituximab (R) with or without ifosfamide/carboplatin/etoposide (ICE), gemcitabine/oxaliplatin (GemOx), gemcitabine/dexamethasone/cisplatin (GDP), dexamethasone / high-dose cytarabine/cisplatin (DHAP), and mitoxantrone/ifosfamide/mesna/etoposide (MINE), still represent the commonly used salvage approaches [6]. However, the overall response rates (ORR: 40.7%–67.0%) and complete response rates (CRR: 13.8%–31.0%) with these approaches remain suboptimal [7–12], highlighting the urgent need for further optimization of salvage immunochemotherapy strategies.

Liposomal mitoxantrone hydrochloride (Lipo-MIT), a next-generation anthracycline, utilizes liposomal technology to enhance tumor targeting via the enhanced permeability and retention effect, enabling sustained intratumoral accumulation and prolonged circulation half-life of approximately 90 h, thereby enhancing antitumor efficacy and reversing efflux-mediated resistance [13–15]. Meanwhile, Lipo-MIT reduces toxicities compared with conventional mitoxantrone, offering an improved safety profile [16, 17]. In a phase II trial of R/R peripheral T-cell lymphoma, Lipo-MIT demonstrated favorable efficacy and safety, leading to its approval by the National Medical Products Administration of China for this indication [18]. Additionally, multiple studies in DLBCL have also confirmed its clinical activity [19–21]. Collectively, replacing conventional mitoxantrone with Lipo-MIT in the R-MINE regimen may represent a promising therapeutic strategy to improve outcomes for R/R DLBCL.

In parallel, genetic subtype-guided personalized treatment strategies, specifically the combination of standard immunochemotherapy with novel targeted agents (R-chemo + X), have emerged as a key focus in research on DLBCL in recent years [22–24]. Based on a simplified algorithm for genetic subtyping (termed ‘LymphPlex’), DLBCL can be classified into seven distinct genetic subtypes: MCD-like (characterized by MYD88/CD79B mutations), BN2-like (NOTCH2/BCL6 alterations), TP53^Mut (TP53 mutations), EZB-like (BCL2/EZH2 aberrations), ST2-like (JAK-STAT activation), N1-like (NOTCH1 mutations), and not otherwise specified (NOS) [25]. These subtypes exhibited marked heterogeneity in clinicopathologic features, treatment responses, and survival outcomes, underscoring the necessity of genotype-guided individualized therapeutic strategies [25, 26]. The GUIDANCE-06 trial preliminarily validated the feasibility of R-chemo + X strategy in patients with R/R DLBCL [24].

In this context, we conducted a phase II clinical trial to prospectively evaluate the feasibility of the Lipo-MIT-based R-MINE-containing regimen (R-MINE ± X), where Lipo-MIT was used in place of conventional mitoxantrone, in patients with R/R DLBCL.

Methods

Study design and participants

This was an open-label, single-arm, multicenter study of the Lipo-MIT-based R-MINE-containing regimen in patients with R/R DLBCL. The trial was conducted at five medical centers in China to evaluate the efficacy and safety of the regimen.

Eligible patients were ≥ 18 years of age with histologically confirmed R/R DLBCL. Relapsed disease was defined as lymphoma recurring after achieving a complete response (CR) to initial chemotherapy. Additional inclusion criteria included an Eastern Cooperative Oncology Group (ECOG) performance status of 0–2; a life expectancy of at least 3 months; at least one measurable lesion according to the 2014 Lugano response criteria; and adequate bone marrow, hepatic, and renal function. Key exclusion criteria included prior cumulative anthracycline exposure > 360 mg/m² (calculated as doxorubicin-equivalent dose); previous treatment with mitoxantrone or its liposomal formulation; known hypersensitivity to any study drug or its components; uncontrolled systemic diseases or significant cardiac dysfunction; active hepatitis B or C infection or HIV infection; current or prior malignancies other than the study indication; primary or secondary central nervous system lymphoma, or a history of central nervous system involvement; the presence of significant gastrointestinal disease; and pregnant or lactating women, or women not using effective contraception. Full patient eligibility is provided in the Appendix (Additional file 1: Supplementary methods 1).

The protocol and all amendments were approved by the Ethics Committee of The First Affiliated Hospital with Nanjing Medical University, Jiangsu Province Hospital (approval no. 2022-SR-682), and the trial was done per Good Clinical Practice guidelines and in accordance with the principles of the Declaration of Helsinki. All patients provided written informed consent. This trial is registered with ClinicalTrials.gov (NCT05784987).

Procedure

Detailed induction treatment schedules were provided in Fig. 1A. All enrolled patients received one cycle of the R-MINE regimen in 21-day cycles, comprising rituximab 375 mg/m² on day 0; ifosfamide 1.33 g/m² on days 1–3 with equidose mesna uroprotection; Lipo-MIT 12–20 mg/m² on day 1 (dose determined at the investigator’s discretion; and etoposide 65 mg/m² on days 1–3. The subsequent treatment strategy, including whether to add agent X, was determined based on prior clinical evidence [22, 24, 27, 28], with consideration of individual resistance patterns and molecular subtype. The rationale for the selection of specific targeted agents is provided in Additional file 1: Supplementary methods 2 [22, 24, 27–36]. The specific regimens in the combination therapy with agent X were as follows: patients with the MCD/BN2 subtype received orelabrutinib (150 mg once daily on days 1–21); those with the EZB subtype received histone deacetylase inhibitor (HDAC) inhibitor chidamide (20 mg once daily on days 1, 4, 8, and 11); those with TP53 mutations received programmed cell death protein 1 (PD-1) inhibitor penpulimab (200 mg on day 0). For patients with other subtypes, the addition of lenalidomide (25 mg once daily on days 1–10) was optional. A maximum of four cycles of induction therapy was administered. Dose adjustments of chemotherapy agents were permitted to manage toxicities. No dose modifications were recommended for the targeted agent. Participants were withdrawn from the study in cases of informed consent withdrawal, death, loss to follow-up, study termination, or other circumstances.

Fig. 1.

(A)Treatment schedules. (B) Trial profile. Abbreviation: Lipo-MIT, liposomal mitoxantrone hydrochloride; ASCT, autologous stem cell transplantation; allo-HSCT, allogeneic hematopoietic stem cell transplantation; CAR-T, chimeric antigen receptor T-cell therapy

Tumor response was assessed by investigators at baseline, mid-treatment, and at the end of treatment using positron emission tomography-computed tomography (PET-CT) or contrast-enhanced CT, according to the 2014 Lugano response criteria.

Safety evaluations were conducted after study initiation and included physical examination, ECOG performance status, laboratory tests, and electrocardiography. All adverse events (AEs) occurring during the study were monitored and recorded. AE severity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), version 5.0. Cardiotoxicity was monitored at baseline, before each treatment cycle (within 3 days prior), and during follow-up. Baseline assessments included 12-lead electrocardiogram (ECG), echocardiography, serum cardiac biomarkers (CK-MB, myoglobin, troponin), and vital signs. Echocardiographic evaluations were conducted every two cycles, while ECG and vital signs were repeated at each cycle and follow-up visit. Cardiac biomarkers were measured in patients who developed symptoms such as chest pain or palpitations.

After the end of therapy, patients were followed every 3 months during the first year and every 6 months during the second year.

Next-generation sequencing and molecular subtyping

Tumor tissue samples were obtained from all patients at baseline for targeted next-generation sequencing (NGS). The detailed sequencing procedures have been described previously in our prior study [37]. Briefly, genomic DNA was extracted using a commercially available DNA extraction kit according to the manufacturer’s instructions. Libraries were constructed using the KAPA Hyper DNA Library Preparation Kit, followed by hybrid-capture enrichment with panels covering 475 leukemia- and lymphoma-related genes. Sequencing was performed on the Illumina HiSeq 4000 platform.

Patients were classified according to the characteristic gene alterations of each subtype as defined by the LymphPlex algorithm [25]. Specifically, MCD-like cases were defined by MYD88 and/or CD79B mutations; BN2-like by NOTCH2 mutations and/or BCL6 alterations; EZB-like by BCL2 and/or EZH2 aberrations; ST2-like by genetic alterations involving the JAK–STAT signaling pathway; N1-like by NOTCH1 mutations; and TP53^Mut by TP53 mutations. Cases that did not meet the criteria for any of the above categories were classified as NOS. Owing to the limited number of N1-like cases in this study, N1-like and NOS cases were combined into a single “Others” category for subsequent analyses.

Endpoints

The primary endpoint was ORR, defined as the proportion of patients who had CR and partial response (PR) as per the 2014 Lugano response criteria. Secondary endpoints included CRR, duration of response (DoR), 1-year progression-free survival (PFS) rate, 1-year overall survival (OS) rate, and safety. CRR was defined as the proportion of patients who had a complete response. DoR was defined as the time from the first documented overall response to disease progression or death from any cause, whichever occurred first. PFS was defined as the time from treatment initiation to disease progression or death from any cause. OS was defined as the time from treatment initiation to all-cause death.

Statistical analysis

The study was designed as a single-arm trial with the ORR as the primary endpoint. Historical data suggested an ORR of about 50% [38]. We hypothesized that the investigational treatment would increase the ORR to 70%. Assuming a two-sided α of 0.05 and 80% power, a sample size of 47 patients was required. Considering a dropout rate of 20%, the final target enrolment was set at 59 patients. Sample size estimation was performed using PASS software (version 15.0, NCSS, Kaysville, UT, USA).

The primary efficacy analysis was performed in the full analysis set (FAS), which included all patients who received at least one administration of study treatment and had at least one post-baseline follow-up. As a supportive analysis, efficacy was also evaluated in the efficacy-evaluable set, which comprised all patients who received at least one administration of study treatment and had at least one post-baseline tumor assessment. Safety was evaluated in the safety analysis set (SS), comprising all patients who received at least one dose of the study drug and had available post-treatment safety data.

Statistical analyses were conducted using SPSS software version 27.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics were used to summarize the baseline characteristics and safety data. Continuous variables were reported as median (range), and categorical variables were specified as frequency (percentage). The ORR and CRR were compared using Fisher’s exact test. In univariable analyses, Fisher’s exact test was used to assess the effect of clinical variables on ORR; variables with P < 0.1 were then included in a multivariable logistic regression model to identify independent predictors of ORR. Median follow-up was estimated using the reverse Kaplan-Meier method. PFS, DoR, and OS were estimated with the Kaplan-Meier method, and differences between groups were compared with the log-rank test. A two-sided P < 0.05 was considered statistically significant, and 95% CIs were reported where applicable. For PFS, patients without an event at the time of data cutoff were censored at the date of their last disease assessment. For OS, patients who were alive at the data cutoff were censored at the date of last follow-up.

Results

Patients and treatments

From March 27, 2023, to July 10, 2025, 63 patients with R/R DLBCL were screened, and 60 eligible patients received Lipo-MIT-based R-MINE-containing regimens and were included in the efficacy and safety analyses (Fig. 1B). Patient characteristics are summarized in Table 1 and Additional file 1: TableS1. The median age was 61 years (range: 24–79), and 58.3% of patients were male. Forty-six patients (76.7%) had Ann Arbor stage III/IV disease, and 46 patients (76.7%) were the non-germinal center B-cell (non-GCB)-like subtype. Nearly half of the patients (46.7%) had an international prognostic index (IPI) score of 3–5, and 23 patients (38.3%) had elevated serum lactate dehydrogenase levels. Thirteen patients (21.7%) presented with B symptoms, 26 (43.3%) were diagnosed with double-expressor lymphoma, and 10 (16.7%) had bulky disease. Additionally, 40.0% of the patients exhibited two or more extranodal sites of involvement. The median number of prior lines of therapy was 2 (range: 2–7), and 19 patients (31.7%) had received ≥ 3 prior lines of therapy. Nearly all patients (98.3%) had previously been treated with anthracycline-containing regimens, with a mean baseline cumulative anthracycline dose of 150.5 mg/m². Twenty-two patients (36.7%) experienced relapse > 12 months after the first-line treatment, and the same proportion of patients experienced relapse > 12 months after the last-line treatment. Based on the LymphPlex algorithm, all 60 patients were categorized into MCD-like (14/60, 23.3%), TP53^Mut subtype (6/60, 10.0%), BN2-like (7/60, 11.7%), EZB-like (4/60, 6.7%), ST2-like (4/60, 6.7%), and other subtypes (25/60, 41.7%; Table 1).

Table 1.

Baseline characteristics of the FAS population

Characteristic	All patients (n = 60)
Age, median (range), years	61 (24, 79)
< 60	21 (35.0)
≥ 60	39 (65.0)
Sex
Male	35 (58.3)
Female	25 (41.7)
Disease stage at diagnosis
Stage I–II	14 (23.3)
Stage III–IV	46 (76.7)
IPI score
0–2	32 (53.3)
3–5	28 (46.7)
ECOG performance status
0–1	53 (88.3)
2	7 (11.7)
Cell of origin1
GCB-like subtype	14 (23.3)
Non-GCB-like subtype	46 (76.7)
Number of prior therapy lines, median (range)	2 (2, 7)
2	41 (68.3)
3	6 (10.0)
4	11 (18.3)
≥ 5	2 (3.3)
B symptoms	13 (21.7)
Double expressor lymphoma
Yes	26 (43.3)
Unknown	17 (28.3)
Bulky disease2	10 (16.7)
Baseline LDH level
Normal	19 (31.7)
Elevated	23 (38.3)
Unknown	18 (30.0)
Extranodal sites
0–1	36 (60.0)
≥2	24 (40.0)
NGS-based molecular subtype
TP53 Mut	6 (10.0)
BN2-like	7 (11.7)
EZB-like	4 (6.7)
MCD-like	14 (23.3)
ST2-like	4 (6.7)
Others	25 (41.7)
Best response to prior therapy
CR	29 (48.3)
PR	18 (30.0)
SD	3 (5.0)
PD	9 (15.0)
Unknown	1 (1.7)
Prior anthracycline-containing regimen3	59 (98.3)
Baseline cumulative anthracycline exposure (doxorubicin-equivalent dose, mg/m2), mean (standard deviation)	150.5 (60.4)
Late relapse4 after first-line therapy	22 (36.7)
Late relapse after the last-line therapy	22 (36.7)

Data are median (range) or n (%)

¹GCB or non-GCB subtyping was determined by the Hans algorithm. ²Bulky disease was defined as the presence of one or more lesions that were 7.5 cm or larger in greatest dimension. ³Anthracycline drugs include doxorubicin, liposomal doxorubicin, epirubicin, and others. ⁴Late relapse was defined as relapse occurring > 12 months after first-line therapy or the last-line therapy

Abbreviation: FAS, full analysis set; IPI, international prognostic index; ECOG, Eastern Cooperative Oncology Group; GCB, germinal center B-cell; NGS, next-generation sequencing; LDH, lactate dehydrogenase; CR, complete response; PR, partial response; CMR, complete metabolic response; SD, stable disease; PD, disease progression

Of 60 patients, 31 patients (51.7%) received R-MINE alone (Additional file 1: Table S2); the remaining 29 patients (48.3%) were treated with the R-MINE + X regimen, including 8 patients (13.3%) with a BTK inhibitor, 8 patients (13.3%) with a PD-1 inhibitor, 10 patients (16.7%) with lenalidomide, and 3 patients (5.0%) with HDAC inhibitor. A total of 195 cycles of the R-MINE-containing induction regimen were administered, including 99 cycles of R-MINE alone and 96 cycles of R-MINE + X. The median number of cycles per patient was 4.0 (range: 1.0–4.0). At the date of data cutoff (October 10, 2025), 36 (60.0%) completed the induction therapy, whereas 24 (40.0%) discontinued treatment owing to disease progression (PD; n = 8), physician decision (n = 9), loss to follow-up (n = 6), or voluntary withdrawal (n = 1). Ten patients (10/60; 16.7%) received subsequent maintenance therapy (Additional file 1: Table S3), with rituximab plus sintilimab and lenalidomide monotherapy being the most commonly used regimens (2 patients each; Additional file 1: Table S4). Patients who received post-progression antitumor therapy were commonly treated with glofitamab (5/60; 8.3%) or polatuzumab vedotin combined with bendamustine and rituximab (Pola-BR; 2/60; 3.3%).

Efficacy

In the FAS population, 29 patients (48.3%) achieved CR, and 13 patients (21.7%) achieved PR as their best response, resulting in an ORR of 70.0% (95% CI: 56.8%–81.2%; Table 2). The median DoR was not reached (Additional file 1: Fig. S1). With the median follow-up of 13.9 months (range: 0.03–28.0), 25 patients experienced PD or death (Fig. 2). The median PFS was 17.5 months (95% CI: 10.8–24.2), with a 1-year PFS rate of 58.5% (95% CI: 43.9%–70.5%; Fig. 3A). During the study period, 11 deaths occurred, primarily due to PD. The median OS was not reached. The 1-year and 2-year OS rates were 84.0% (95% CI: 70.4%–91.7%) and 78.0% (95% CI: 62.3%–87.8%), respectively (Fig. 3B).

Table 2.

Best tumor response per the 2014 Lugano response criteria

Response	FAS population (n = 60)	Efficacy-evaluable population (n = 55)
CR	29 (48.3; 35.2, 61.6)	29 (52.7; 38.8, 66.3)
PR	13 (21.7)	13 (23.6)
SD	5 (8.3)	5 (9.1)
PD	8 (13.3)	8 (14.5)
Not evaluated	5 (8.3)	-
ORR	42 (70.0; 56.8, 81.2)	42 (76.4; 63.0, 86.8)

Data are n (%) or n (%; 95% CI)

Abbreviation: FAS, full analysis set; CR, complete response; PR, partial response; SD, stable disease; PD, disease progression; ORR, overall response rate

Fig. 2.

Swimmer plot

Fig. 3.

Progression-free survival (A) and overall survival (B) in the FAS population. Abbreviation: FAS, full analysis set; PFS, progression-free survival; OS, overall survival; NR, not reached; CI, confidence interval

In 55 efficacy-evaluable patients, the results (ORR: 76.4% [95% CI: 63.0%–86.8%]; CRR: 52.7% [95% CI: 38.8%–66.3%]) were similar to those observed in the FAS population (Table 2).

Exploratory subgroup analyses of the FAS population are presented in Additional file 1: Tables S5–S6 and Fig. S2. Analysis by genetic subtype showed that there were no statistically significant differences in either PFS or OS among the subtypes. Furthermore, patients with late relapse after the last-line therapy demonstrated a high ORR (86.4%) and CRR (63.6%). Multivariate analysis revealed that late relapse after the last-line therapy (P = 0.039) was the only independent predictor of ORR (Additional file 1: Table S7). The 1-year PFS rate in patients with late relapse was 87.2% compared with 43.5% in those with non-late relapse after the last-line therapy. The corresponding 1-year OS rates were 100.0% and 77.0%, respectively.

Detailed efficacy data by treatment subgroup and additional exploratory analyses are provided in Additional file 1: Table S8, and Fig. S3–S4. In the R-MINE alone group (n = 31), the ORR was 74.2%, the median PFS was 17.5 months, and the 1-year OS rate was 84.3%. In the R-MINE + X group (n = 29), the ORR was 65.5%, the median PFS was not reached, and the 1-year OS rate was 84.4%.

Safety

The median single-dose intensity of Lipo-MIT was 11.7 mg/m² (range: 5.5–19.4), and the median cumulative dose of liposomal mitoxantrone was 42.0 mg/m² (range: 6.0–57.0). One patient (1.7%; Additional file 1: Table S9) required a dose reduction due to grade 2 thrombocytopenia and grade 2 leukopenia. Dose interruptions due to treatment-related adverse events (TRAEs) occurred in 9 patients (15.0%), most commonly due to leukopenia (5, 8.3%), followed by lung infection, thrombocytopenia, and neutropenia (each 3, 5.0%), anemia (2, 3.3%), and fever, respiratory failure, rash, or COVID-19 infection (each 1, 1.7%). No patient discontinued treatment owing to TRAEs.

In the safety population, TRAEs occurred in 56 patients (93.3%). The most common any-grade hematologic TRAEs were leukopenia and anemia (both 75.0%), and hypokalemia was the most frequent non-hematologic event (60.0%; Table 3). Grade ≥ 3 TRAEs were reported in 28 patients (46.7%), primarily neutropenia (33.3%) among hematologic toxicities and hypokalemia (11.7%) among non-hematologic toxicities. Two cardiac-related TRAEs (grade 1 high-sensitivity troponin T increased and sinus tachycardia) were reported. No serious TRAEs or TRAE-related deaths were observed. Safety data for different treatment subgroups are summarized in Additional file 1: Tables S9–S10.

Table 3.

Treatment-related adverse events in the safety population

TRAEs*	All patients (n = 60)
	Any grade	Grade ≥ 3
Hematologic toxicity
Leukopenia	45 (75.0)	17 (28.3)
Anemia	45 (75.0)	14 (23.3)
Neutropenia	39 (65.0)	20 (33.3)
Thrombocytopenia	35 (58.3)	7 (11.7)
Lymphopenia	5 (8.3)	4 (6.7)
Non-hematologic toxicity
Hypokalemia	36 (60.0)	7 (11.7)
Hyponatremia	33 (55.0)	1 (1.7)
Hypocalcemia	32 (53.3)	1 (1.7)
Blood lactate dehydrogenase increased	23 (38.3)	0
Hyperglycemia	17 (28.3)	0
Hypoalbuminemia	14 (23.3)	0
Fibrinogen decreased	14 (23.3)	0
Hypertriglyceridemia	12 (20.0)	0
Constipation	11 (18.3)	0
Aspartate aminotransferase increased	11 (18.3)	0
Fever	9 (15.0)	0
Cough	9 (15.0)	0
Alkaline phosphatase increased	7 (11.7)	1 (1.7)
Alanine aminotransferase increased	7 (11.7)	0
Productive cough	7 (11.7)	0
Lung infection	6 (10.0)	3 (5.0)
Nausea	6 (10.0)	0
Hypernatremia	6 (10.0)	0
Diarrhea	5 (8.3)	0
Cholesterol high	4 (6.7)	0
Vomiting	4 (6.7)	0
Insomnia	4 (6.7)	0
Hypoglycemia	3 (5.0)	0
Abdominal distension	3 (5.0)	0
Hyperkalemia	3 (5.0)	0
Hyperuricemia	3 (5.0)	0
Creatinine increased	3 (5.0)	0
Pain	3 (5.0)	0
Pruritus	2 (3.3)	0
COVID-19 infection	2 (3.3)	0
Blood bilirubin increased	2 (3.3)	0
Anorexia	2 (3.3)	0
Respiratory failure	1 (1.7)	1 (1.7)
Urinary tract infection	1 (1.7)	1 (1.7)
Rash acneiform	1 (1.7)	0
Sinus tachycardia	1 (1.7)	0
Pneumonitis	1 (1.7)	0
Abdominal pain	1 (1.7)	0
Hypercalcemia	1 (1.7)	0
High-sensitivity troponin T increased	1 (1.7)	0
Activated partial thromboplastin time prolonged	1 (1.7)	0
Mucositis oral	1 (1.7)	0
Rash	1 (1.7)	0
Dyspnea	1 (1.7)	0
Malaise	1 (1.7)	0
Peripheral sensory neuropathy	1 (1.7)	0
Chest tightness	1 (1.7)	0
Influenza virus infection	1 (1.7)	0

Data are n (%)

*Listing of all TRAEs observed in 60 patients. Abbreviation: TRAEs, treatment-related adverse events

Discussion

To our knowledge, this study is the first prospective evaluation of a Lipo-MIT-based R-MINE-containing regimen that replaced conventional mitoxantrone with Lipo-MIT, with a subset of patients also receiving genotype-guided targeted therapy for R/R DLBCL. With the best ORR of 70.0%, this study successfully met its prespecified primary endpoint in R/R DLBCL, reaching the benchmark considered encouraging in the statistical assumption of this trial. The objective and deep responses (ORR: 76.4%; CRR: 52.7%) were more evident in the efficacy-evaluable population. The Lipo-MIT-based R-MINE-containing regimen resulted in durable responses, with the median DoR not yet reached after a median follow-up of 13.9 months. The 1-year OS rate was 84.0%, indicating a meaningful survival benefit. Exploratory subgroup analyses further revealed consistent clinical benefit in patients with adverse prognostic factors [7, 39–42], including those with advanced-stage disease (ORR: 63.0%), non-GCB subtype (ORR: 71.7%), and double-expressor lymphoma (ORR: 76.9%). Moreover, patients experiencing late relapse showed significantly higher response and survival rates compared to those with non-late relapse. The regimen exhibited a manageable safety profile, with no new safety concerns identified. Collectively, these findings support the potential of the Lipo-MIT-based R-MINE-containing regimen as a promising treatment option in the management of R/R DLBCL.

Several salvage therapies, such as the conventional MINE regimen (ORR: 43.8%–48.0%; CRR: 21.0%–22.0%) [38, 43], (R)-GDP regimen (ORR: 46.10%–67.7%; CRR: 13.80%–31.0%) [8, 12], (R)-DHAP regimen (ORR: 42.2%–62.8%; CRR: 14.6%-27.7%) [7–9], R-ICE regimen (ORR: 63.5%; CRR: 24.4%) [7], and the (R)-GemOx regimen (ORR: 38.0%–78.0%; CRR: 25.3%–50.0%) [10, 11, 44–46], have demonstrated clinical activity in patients with R/R DLBCL, and remain widely used treatment options in this setting. However, direct comparisons of efficacy outcomes between the Lipo-MIT-based R-MINE-containing regimen evaluated in the present study and these established salvage regimens are difficult because of discrepancies in study design and baseline characteristics. Nevertheless, the findings of the present study suggest that the Lipo-MIT-based R-MINE regimen may be effective in patients with R/R DLBCL. Given the single-arm design, limited sample size, and treatment heterogeneity, these observations should be considered preliminary and hypothesis-generating and warrant further validation in larger prospective studies.

Time to relapse is recognized as an important predictor of treatment response [47]. Patients with early relapse are typically less responsive to salvage regimens and achieve lower ORR, whereas those with late relapse tend to exhibit higher response rates [7, 9]. Consistent with these observations, our exploratory analysis suggested that patients with late relapse had numerically higher response and survival rates than those with non-late relapse after treatment with the Lipo-MIT-based R-MINE-containing regimen. It should be noted that patients with late relapse had previously received R-CHOP as first-line therapy and R-Gemox with or without PD-1 inhibitor or BR in second-line or later settings, with 27.3% receiving ≥ 3 prior lines. In contrast, those with non-late relapse had undergone more complex regimens including R-Gemox plus BTKi, R-Gemox plus PD-1 inhibitor, rituximab plus lenalidomide and BTKi, or Pola-BR, with 34.2% receiving ≥ 3 prior lines. These findings suggest that patients with non-late relapse represented a more refractory population, which may account for their comparatively lower responses to the study regimen.

In the overall safety population, the Lipo-MIT-based R-MINE-containing regimen demonstrated a favorable overall safety profile with no new safety signals detected. The majority of TRAEs in this study were manageable through dose adjustments and supportive care with growth factors, rarely leading to treatment discontinuation and deaths. It is well recognized that anthracycline-based chemotherapy is associated with cardiotoxicity, a risk that has limited the clinical application of conventional mitoxantrone [48]. In this study, cardiotoxicity occurred in only two cases, and no new cardiac safety signals were identified with a median follow-up of 13.9 months, which is probably because Lipo-MIT is capable of mediating the drug distribution in tumor tissues, thus enhancing cardiac safety [13]. Long-term follow-up after completion of chemotherapy is still required for patients to continuously monitor cardiac function. Furthermore, no cases of hand-foot syndrome were observed in this study, which is consistent with prior observations in patients with R/R mature T-cell and NK-cell lymphoma [18]. Similar to approved salvage therapies for R/R DLBCL, neutropenia was the most frequent grade ≥ 3 TRAE, observed in 33.3% of patients, within the range reported for existing regimens (15.0%–32.0%) [7–9]. Although neutropenia represents a key risk factor for infection [49], timely use of granulocyte colony-stimulating factor and/or anti-infective therapy in this study resulted in a lower incidence of grade ≥ 3 infection (6.7%) than that previously reported for immunochemotherapy in the treatment of R/R DLBCL (13.0%–17.0%) [7–9]. In summary, the R-MINE-containing regimen can be administered safely, supporting its clinical feasibility in patients with R/R DLBCL.

Given the marked biological heterogeneity of R/R DLBCL, a stratified treatment approach was adopted, administering R-MINE alone or in combination with targeted agents according to molecular characteristics and prior treatment history. This strategy aimed to provide personalized therapeutic options for a broad patient population, including those sensitive or resistant to targeted therapies. As a single-arm phase II study, the primary objective was to evaluate the efficacy and safety of the Lipo-MIT-based R-MINE-containing regimen rather than the relative benefit of different treatment strategies. Because treatment allocation was biologically driven rather than randomized, and because of the limited sample size and baseline imbalances between treatment subgroups, the study design precluded attributing the benefit specifically to the genetic subtype-guided treatment strategy or to Lipo-MIT. Additionally, the limited sample size may constrain statistical power and affect the generalizability of the findings, particularly in subgroup analyses. Therefore, any apparent subgroup-specific signals should be interpreted as hypothesis-generating rather than evidence of definitive therapeutic benefit. Furthermore, the OS data are currently immature, and a longer follow-up is required to obtain more reliable survival outcomes.

Conclusions

In conclusion, the findings of this phase II study indicate that the Lipo-MIT-based R-MINE-containing regimen demonstrated promising clinical efficacy and a manageable safety profile, underscoring its potential as a valuable therapeutic option for patients with R/R DLBCL.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

R/R DLBCL

relapsed or refractory diffuse large B-cell lymphoma

R-MINE

rituximab with mitoxantrone/ifosfamide/mesna/etoposide

ORR

objective response rate

CI

confidence interval

TRAEs

treatment-related adverse events

DLBCL

diffuse large B-cell lymphoma

R/R

relapsed or refractory

CAR-T

chimeric antigen receptor T-cell therapy

R

rituximab

ICE

ifosfamide/carboplatin/etoposide

GemOx

gemcitabine/oxaliplatin

GDP

gemcitabine/dexamethasone/cisplatin

DHAP

dexamethasone / high-dose cytarabine/cisplatin

MINE

mitoxantrone/ifosfamide/mesna/etoposide

CRR

complete response rates

Lipo-MIT

Liposomal mitoxantrone hydrochloride

R-MINE ± X

R-MINE-containing regimen

CR

complete response

ECOG

Eastern Cooperative Oncology Group

HDAC

histone deacetylase inhibitor

PD-1

programmed cell death protein 1

PET-CT

positron emission tomography-computed tomography

AEs

adverse events

NCI CTCAE

National Cancer Institute Common Terminology Criteria for Adverse Events

ECG

electrocardiogram

CK-MB

cardiac biomarkers

NGS

next-generation sequencing

PR

partial response

DoR

duration of response

PFS

progression-free survival

OS

overall survival

FAS

full analysis set

SS

safety analysis set

non-GCB

non-germinal center B-cell

IPI

international prognostic index

Author contributions

J.H.L.: Conceptualization, Methodology, Data curation, Formal analysis, Investigation, Software, Validation, Visualization, Writing – original draft, Writing – review & editing; X.Y.Z.: Conceptualization, Methodology, Data curation, Formal analysis, Investigation, Software, Validation, Visualization, Writing – original draft, Writing – review & editing; L.T.B.: Data curation, Resources, Writing – review & editing; W.W.Q.: Data curation, Resources, Writing – review & editing; L.P.S.: Data curation, Resources, Writing – review & editing; J.B.L.: Data curation, Resources, Writing – review & editing; J.Z.W.: Data curation, Resources, Writing – review & editing; H.Y.: Data curation, Resources, Writing – review & editing; Y.L.K.: Data curation, Resources, Writing – review & editing; H.R.S.: Data curation, Resources, Writing – review & editing; J.Y.L.: Data curation, Resources, Writing – review & editing; Y.L.: Conceptualization, Methodology, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing; L.W.: Conceptualization, Methodology, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing; W.X.: Conceptualization, Methodology, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing.All authors read and approved the final manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grant number 82200887 and 82370194), Jiangsu Science and Technology Department (grant number BK20220716 and BE2023780), China Postdoctoral Science Foundation (grant number 2022M7114034 and 2023M741463), Nanjing Municipal Returned Overseas Scholars Foundation (grant number BSHNJ2023011), Jiangsu Province Hospital Talent Specialized Foundation (grant number MXJL202107), Jiangsu Province “333” Project (grant number BRA2018085), Jiangsu Province Hospital Clinical Diagnosis and Treatment Technologies Innovation Project Foundation (grant number JBGS202405).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The protocol and all amendments were approved by the Ethics Committee of The First Affiliated Hospital with Nanjing Medical University, Jiangsu Province Hospital (approval no. 2022-SR-682), and the trial was done per Good Clinical Practice guidelines and in accordance with the principles of the Declaration of Helsinki. All patients provided written informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.