Anti-CD19 chimeric antigen receptor T-cell therapy for relapsed or refractory Philadelphia chromosome-positive B cell acute lymphoblastic leukemia: a multicenter retrospective study

Abstract

Background

Although chemotherapy-free regimens have improved initial remission rates in patients with newly diagnosed Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph + B-ALL), outcomes for relapsed or refractory (R/R) Ph + B-ALL remain poor. This study reports the efficacy and safety of CD19 chimeric antigen receptor (CAR) T-cell therapy in R/R Ph + B-ALL.

Methods

This study retrospectively analyzed 93 patients with R/R Ph + B-ALL who received CD19 CAR T-cell therapy across China between August 2015 and March 2024. We evaluated the overall response rates, long-term efficacy, safety, and prognostic factors associated with CD19 CAR T-cell therapy.

Results

Complete remission (CR) or CR with incomplete hematologic recovery rate was 87.1% (81/93), with 96.3% (78/81) of responders achieving minimal residual disease negativity by flow cytometry. Molecular response rate was 78.5% (73/93), including 63.4% achieving complete molecular remission and 15.1% major molecular remission. Twenty patients underwent consolidative allogeneic hematopoietic stem cell transplantation (allo-HSCT). After a median follow-up of 25.4 months (range, 0.1–68.5), the median overall survival (OS) was 20.8 months, and the median leukemia-free survival (LFS) was 8.1 months. Better Eastern Cooperative Oncology Group performance status and absence of adverse genetic features were associated with improved OS and LFS. In contrast, consolidative allo-HSCT following CAR T-cell therapy was not independently associated with improved OS or LFS. Grade ≥ 3 cytokine release syndrome and neurotoxicity occurred in 14.0% and 2.2% of patients, respectively. The most common and predictable adverse events were hematologic, primarily cytopenias, which were manageable with supportive care.

Conclusions

CD19 CAR T-cell therapy can achieve high response rates and long-term clinical benefits for patients with R/R Ph + B-ALL, with a manageable safety profile.

Statement of prior presentation

The preliminary study was presented as a poster presentation (Publication Number: 4201) at the 66th American Society of Hematology Annual Meeting, San Diego, CA, on December 7–10, 2024.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12916-026-04755-6.

Background

Philadelphia chromosome-positive (Ph +) B cell acute lymphoblastic leukemia (B-ALL) accounts for approximately 20–25% of adult B-precursor ALL, and its incidence rises with age [1, 2]. Historically, Ph + B-ALL carries a worse prognosis than Ph- disease, with higher relapse rates and poorer long-term survival [3]. Over the past 20 years, frontline therapy has shifted from intensive chemotherapy combinations to tyrosine kinase inhibitors (TKIs)-based lower-intensity regimens, including chemotherapy-free approaches, raising 3-year survival from 48 to 94% in patients with newly diagnosed Ph + B-ALL [1, 4, 5]. Nevertheless, relapse remains frequent in patients with suboptimal molecular responses or ABL1 kinase mutations that confer TKI resistance, and their long-term survival is exceptionally poor [6–8], posing continued challenges in relapse management [1].

For heavily pretreated patients with relapsed or refractory (R/R) Ph + B-ALL, the CD3/CD19-targeted bispecific T-cell engager blinatumomab and CD22-targeted antibody–drug conjugate inotuzumab ozogamicin have shown greater molecular response rates than that of standard intensive chemotherapy, and have been well-tolerated in patients with resistance to first- or second-generation TKIs [9, 10]. However, the efficacy of blinatumomab in patients exhibiting a high disease burden or extramedullary disease (EMD) remains limited [11]. The long-term outcomes of both agents are unsatisfactory when used as monotherapies [12, 13]. These limitations highlight the need to explore more potent and clinically feasible treatments for patients with R/R Ph + B-ALL.

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has become a highly promising strategy for treating B-cell malignancies, demonstrating encouraging efficacy in R/R B-ALL across both pediatric and adult populations, including those with Ph + disease. Reported overall response rates range from 60 to 90%, with median overall survival (OS) between 10 and 20 months [11, 14–17]. In addition to its efficacy in treating bone marrow relapse, CAR T-cell therapy has shown promising outcomes in central nervous system leukemia (CNSL) and other EMD, and has been effective in managing relapse post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) [7, 18].

However, published researches upon CAR T-cell therapy for patients with R/R Ph + ALL have been largely limited to single-center reports, typically involving small cohorts or short-term follow-up durations [19]. To date, no large multicenter studies have been reported. Investigating the long-term efficacy of CAR T-cell therapy in R/R Ph + ALL is essential for identifying clinical factors predictive of long-term survival and informing personalized treatment strategies [12]. Therefore, in this multicenter study, we evaluated the safety and efficacy of CD19 CAR T-cell therapy in patients with R/R Ph + B-ALL, with a focus on long-term outcomes and their correlations with clinical and disease characteristics.

Methods

Study design and patients

Overall, 98 patients diagnosed with R/R Ph + B-ALL between August 2015 and March 2024 were consecutively enrolled from seven centers across China. The data cutoff for this study was 15 May 2024. The diagnostic criteria for Ph + B-ALL were based on the World Health Organization classification. Definitions of R/R disease status followed the National Comprehensive Cancer Network (NCCN) Guidelines (Version 2.2024) for ALL. Adverse genetic features were defined according to the NCCN cytogenetic and molecular prognostic risk stratification for B-ALL, encompassing cytogenetic and molecular alterations associated with poor-risk disease. Five patients were excluded before CAR T-cell infusion due to manufacturing failure (n = 1), clinical deterioration (n = 2), death (n = 1), or adverse events following conditioning chemotherapy (n = 1). In total, 93 patients received CD19 CAR T-cell infusion and were included in both the efficacy and safety analysis cohorts (Fig. 1). Lymphodepleting chemotherapy consisting of fludarabine and cyclophosphamide was administered prior to CAR T-cell infusion. The median CAR T-cell dose administered on day 0 was 1.6 × 10⁶/kg (range, 0.2–12.7 × 10⁶/kg). The CAR constructs incorporated either a CD28 (n = 48) or a 4-1BB (n = 45) costimulatory domain, and all constructs shared a CD3ζ signaling domain. CAR transgenes were delivered using a lentiviral vector system. After CAR T-cell infusion, all patients received TKI therapy as post–CAR T maintenance. The timing of TKI initiation after CAR T-cell infusion was determined by the treating physicians based on clinical recovery and hematologic status. Baseline characteristics, treatment details, CAR T-cell-associated toxicities, and clinical outcomes were systematically collected. The research was approved by the ethics committees of all participating centers and conducted in accordance with the Declaration of Helsinki.

Fig. 1.

Study profile. Ninety-eight patients with relapsed or refractory (R/R) Philadelphia chromosome-positive (Ph +) B cell acute lymphoblastic leukemia (B-ALL) were screened. Ninety-four patients received conditioning chemotherapy, with four subsequently withdrawing. Ultimately, ninety-three patients received CD19 CAR T-cell therapy and were included in the efficacy and safety analysis cohort; one patient withdrew after conditioning

Response evaluations

Bone marrow and EMD status were assessed in every patient before therapy. Therapeutic responses were assessed according to the NCCN Guidelines, Version 2.2024, for ALL. Formal response assessment was performed at day 30 after infusion. All infused patients (n = 93) were included in an intention-to-treat (ITT) analysis for response. Within 30 days after CAR T-cell infusion, two patients died on day 4 and day 19, respectively, due to severe cytokine release syndrome (CRS) complicated by multiple organ dysfunction syndrome (MODS) and sepsis. Both patients were classified as non-responders and included in the safety analysis.

Overall response rate was defined as the proportion of patients achieving complete remission (CR) or complete remission with incomplete hematologic recovery (CRi) within 30 days after infusion. Minimal residual disease (MRD) was assessed by multiparameter flow cytometry at participating centers using institutional standard protocols. MRD negativity was defined as < 1 × 10⁻⁴ (0.01%), which was consistently applied across centers. Complete molecular remission (CMR) and major molecular remission (MMR) were defined as a BCR::ABL1/ABL transcript ratio of ≤ 0.01% and ≤ 0.1%, respectively, measured by reverse transcription-polymerase chain reaction (RT-PCR). Patients with CNSL were identified as having either CNS-2 or CNS-3 status prior to infusion. CNS-2 was defined as < 5 white blood cells (WBCs)/μL in the cerebrospinal fluid (CSF) with detectable lymphoblasts, whereas CNS-3 was defined as ≥ 5 WBCs/μL in the CSF with detectable lymphoblasts. CNS remission was defined as the achievement of CNS-1 status (no detectable lymphoblasts in CSF, irrespective of WBC count) in patients with baseline CNS-2 or CNS-3 status.

Transplantation decision-making

Consolidative allo-HSCT after CAR T-cell therapy was considered in selected patients, with individual decisions made at the discretion of treating physicians. Shared principles were applied across centers to guide eligibility, generally including achievement of MRD-negative CR and medical fitness for transplantation. Disease characteristics suggestive of increased relapse risk—such as extensive prior therapy, adverse genetic features, or TKI resistance (including T315I mutation)—were also considered. Markers of immune reconstitution, including B-cell recovery after CAR T-cell therapy, were incorporated into the overall clinical assessment, together with donor availability and patient preference. Key elements of transplant practice that may materially influence outcomes—including disease status at transplantation and timing of transplantation—were considered in transplantation planning and followed national or institutional standards.

Safety evaluations

The severity of CRS and immune effector cell-associated neurotoxicity syndrome (ICANS) was graded according to the American Society for Transplantation and Cellular Therapy Consensus Criteria [20]. Severe CRS was defined as grade ≥ 3, and severe ICANS was defined as grade ≥ 3 or the occurrence of seizure at any grade. Other adverse events were classified using the Common Terminology Criteria for Adverse Events, Version 5.0. B-cell aplasia (BCA) was defined as CD19-positive B cells accounting for < 3% of lymphocytes in peripheral blood, censored at the time of allo-HSCT.

Assessments of genetic abnormalities and serum cytokines

Chromosomal and genomic abnormalities were assessed by chromosome karyotyping, fluorescence in situ hybridization, and next-generation sequencing. RT-PCR was employed to detect the BCR::ABL1/ABL transcript ratio, including the BCR::ABL1 isoforms. Serum cytokine levels were monitored serially with enzyme-linked immunosorbent assay.

Statistical analysis

Patient demographics and treatment outcomes were described using descriptive statistics. Comparisons of categorical variables were conducted using the chi-square test or Fisher’s exact test, and 95% confidence intervals (CIs) were calculated via the Clopper–Pearson exact method. Continuous variables were compared using the Mann–Whitney U test. Spearman’s rank correlation analysis was employed to examine the relationships between ICANS occurrence and CRS severity. Fisher’s exact test was used to evaluate the association between CNS involvement and ICANS occurrence. Overall survival (OS) was defined from CAR T-cell infusion to all-cause mortality or last follow-up, and leukemia-free survival (LFS) from achievement of CR/CRi, corresponding to the day-30 landmark, to first relapse, death, or last follow-up. Probabilities of OS and LFS were estimated using the Kaplan–Meier method, with subgroup comparisons by the log-rank test. For OS analyses, baseline covariates were measured from CAR T-cell infusion, whereas response-related variables (such as CR/CRi status, MRD, and molecular remission) were assessed from the day-30 landmark.

Time-dependent Cox proportional hazards regression was used to evaluate factors associated with OS and LFS among patients achieving CR/CRi (n = 81), with day 30 after CAR T-cell infusion as the landmark. For multivariable analyses, baseline covariates assessed at the time of CAR T-cell infusion with P < 0.2 in univariate analyses—including Eastern Cooperative Oncology Group (ECOG) performance status, adverse genetic features, and T315I mutation—were considered for inclusion. Molecular remission, assessed at the day-30 landmark, was treated as a fixed covariate. Consolidative allo-HSCT was modeled as a time-dependent covariate to account for transplantation timing and to minimize immortal time bias. Non-relapse mortality (NRM) was defined as death without prior relapse after CAR T-cell infusion. The cumulative incidence of NRM and relapse (CIR) was estimated in the entire CAR T-cell cohort (n = 93, intention-to-treat population) using competing-risk methods, with relapse and NRM considered as competing events, respectively. Gray’s test was used for group comparisons of cumulative incidence. Fine–Gray subdistribution hazard models were applied to evaluate the association between baseline variables and competing risks. Subdistribution hazard ratios (sdHR) with 95% confidence intervals (CI) were reported. All statistical analyses were performed using SPSS Statistics, version 27, and R software, version 4.4.1. P-value of < 0.05 was considered statistically significant.

Results

Baseline characteristics

The clinical and demographic characteristics at baseline are outlined in Table 1. The median age at CAR T-cell infusion was 41 years (range, 6–76), with 12 patients (12.9%) aged ≥ 60 years. Nineteen patients (20.4%) had an ECOG performance status ≥ 3. The BCR::ABL1 p190 isoform was identified in 63 patients (67.7%), the p210 isoform in 28 (30.1%), and both isoforms in 2 (2.2%). ABL1 kinase mutations were detected in 57 patients (61.3%), including 43 (46.2%) with the T315I mutation. Adverse genetic features were observed in 27 patients (29.0%), including IKZF1 alterations in 7 (7.5%), and complex karyotypes in 19 (20.4%). All patients had previously received multiple lines of chemotherapy or immunotherapy, but continued to experience relapse or remained in refractory status. The median number of prior treatment lines was five (range, 2–20). Twenty-six patients (28.0%) had been exposed to third-generation TKIs, 5 (5.4%) had received blinatumomab, and 19 (20.4%) had relapsed after transplant. At CAR T-cell infusion, 48 patients (51.6%) had bone marrow blasts ≥ 20%, and 14 (15.1%) exhibited EMD. Of those with EMD, nine patients (9.7%) had CNSL, including five with CNS-3 status and four with CNS-2. Other EMD sites included the liver, spleen, uterus, gingiva, and testes. All patients in the cohort received TKI therapy following CAR T-cell infusion. Patients with a documented T315I mutation were treated with ponatinib, whereas the majority of patients without T315I mutation received dasatinib. The median time from CAR T-cell infusion to initiation of TKI therapy was 67 days (range, 12–158).

Table 1.

Characteristics of the 93 patients with R/R Ph + B-ALL at baseline

Characteristics	Value
Age at CAR T-cells infusion, years, median (range)	41(6–76)
Sex, No. (%)
Male	49(52.7)
Female	44(47.3)
Prior treatment lines, median (range)	5 (2–20)
Prior TKI exposure, No. (%)
Imatinib	47(50.5)
Dasatinib	57(61.3)
Nilotinib	4(4.3)
Flumatinib	5(5.4)
Ponatinib	20(21.5)
Olverembatinib	7(7.5)
Prior blinatumomab exposure, No. (%)
Yes	5(5.4)
No	88(94.6)
Prior allo-HSCT, No. (%)
Yes	19(20.4)
No	74(79.6)
Relapsed or refractory groups, No. (%)
Primary refractory	11(11.8)
Relapsed	82(88.2)
BCR::ABL1 isoform, No. (%)
p190	63(67.7)
p210	28(30.1)
p190 + p210	2(2.2)
T315I mutation, No. (%)
Yes	43(46.2)
No	50(53.8)
Adverse genetic featuresa, No. (%)	27(29.0)
TP53 mutation	1(1.1)
IgH rearranged	1(1.1)
JAK-STAT mutation	2(2.2)
FLT3 rearranged	2(2.2)
Alterations of IKZF1	7(7.5)
Complex karyotype	19(20.4)
BM blasts before lymphodepletion, No. (%)
< 20%	45(48.4)
≥ 20%	48(51.6)
Extramedullary diseasesb, No. (%)
Yes	14(15.1)
No	79(84.9)
ECOG performance status, No. (%)
0–2	74(79.6)
3–4	19(20.4)

^aAdverse genetic features were defined according to the NCCN (Version 2.2024) cytogenetic and molecular prognostic risk stratification for B-ALL, encompassing cytogenetic and molecular alterations associated with poor-risk disease

^bExtramedullary diseases refer to the central nervous system leukemia (CNSL) and other non-CNS extramedullary disease sites

Response rates

Among all 93 infused patients (ITT population), 81 patients (87.1%; 95% CI, 78.5–93.2) achieved CR or CRi. Of these, 66 patients (81.5%) achieved CR and 15 (18.5%) achieved CRi. Two patients who died before response assessment were classified as non-responders in the ITT analysis. Of the patients who achieved CR or CRi, 78 (96.3%) attained MRD negativity by flow cytometry. Furthermore, 73 patients (78.5%; 95% CI, 68.8–86.3) achieved molecular remission, including 59 (63.4%) with CMR and 14 (15.1%) with MMR. Among the 91 evaluable patients, the CR/CRi rate was 89.0% (95% CI, 80.7–94.6).

All nine patients with CNSL achieved remission, and two of the five patients with non-CNS EMD attained CR following CAR T-cell therapy. Subgroup analyses of hematologic and molecular responses revealed no significant difference based on age, sex, number of prior therapies, prior exposure to blinatumomab or transplantation, BCR::ABL1 isoform type, genetic feature risk stratification, and disease burden status (Fig. 2).

Fig. 2.

Subgroup analysis of hematologic and molecular response. The forest plots depicted the subgroup analysis of hematologic and molecular response rates in patients with R/R Ph + B-ALL categorized by baseline characteristics. Chi-square tests and Fisher’s exact tests were performed to compare rates across subgroups, and 95% confidence intervals (CIs) were calculated by the Clopper–Pearson exact method

Long-term efficacy

The median follow-up duration for the entire cohort (n = 93) was 25.4 months (95% CI, 16.2–34.6). For all patients, the median OS was 20.8 months (95% CI, 14.0–27.5), with 6- and 12-month OS rates of 85.1% and 66.3%, respectively (Fig. 3A). Among the 81 patients who achieved CR or CRi, the median LFS was 8.1 months (95% CI, 5.5–10.7), with corresponding 6- and 12-month LFS rates of 61.8% and 42.5%, respectively (Fig. 3B). Using a day-30 landmark analysis, patients achieving CR/CRi exhibited a median OS of 24.5 months (95% CI, 17–NA), significantly longer than non-responders (13.3 months; P = 0.014; Additional file 1: Fig. S1A). Among these responders, 29 maintained sustained remissions over a median follow-up of 25.4 months from CAR T-cell infusion (range, 1–68.5), including 10 with durable responses exceeding 3 years (Fig. 4).

Fig. 3.

OS and LFS depicted by the Kaplan–Meier curve. OS analysis (A) and LFS analysis (B). OS, overall survival; LFS, leukemia-free survival

Fig. 4.

Swimmer plot demonstrating clinical outcomes for patients achieving CR/CRi at day 30 post-infusion (n = 81)

During the long-term follow-up, among the 50 patients (53.8%) with disease relapse, 31 patients (62.0%) were CD19-positive relapse. Of the 11 patients (12%) who died because of NRM, 8 died because of infection, one because of respiratory failure, one because of heart failure, and one because of acute kidney injury. The estimated NRM was 4.4% (95% CI, 0.2 to 8.7) at 3 months and 8.1% (95% CI, 2.3 to 14) at 12 months post-infusion, as shown in Additional file 1: Fig. S2. Twenty patients (24.7%) underwent consolidative allo-HSCT at a median of 3 months (range, 2–15) after CAR T-cell infusion, all in MRD-negative CR at the time of transplantation. One of these patients died of progressive disease 20 months after allo-HSCT, and 6 patients died because of NRM (of which four were because of severe infection), and the other 13 were alive at a median post-transplant follow-up of 15.7 months (range, 2.0–46.4). The cumulative incidences of non-relapse mortality (NRM) and relapse (CIR) by allo-HSCT status are shown in Additional file 1: Fig. S3. NRM was slightly higher in the allo-HSCT group than in the non-transplanted group, but the difference was not statistically significant (Gray’s test, P = 0.185). Similarly, CIR was comparable between the two groups (Gray’s test, P = 0.167).

Univariate and multivariate analysis of long-term survival

At the day-30 landmark, patients who attained MRD negativity by flow cytometry had significantly longer median OS than those with positive MRD or no response (24.5 vs 7.0 months; P < 0.001; Additional file 1: Fig. S1B). Similarly, patients achieving CMR/MMR at CR had longer median OS (24.5 vs 8.4 months; P = 0.015; Additional file 1: Fig. S1C) and a trend toward longer LFS (8.9 vs 3.1 months; P = 0.073; Additional file 1: Fig. S1D) compared with those who did not achieve molecular remission.

Kaplan–Meier analyses from the time of CAR T-cell infusion showed that patients with an ECOG performance status ≤ 2 had longer OS (NA vs 13.8 months; P = 0.019; Additional file 1: Fig. S4A). LFS was also longer in patients with ECOG ≤ 2 (14.1 vs 5.6 months; P = 0.005; Additional file 1: Fig. S4B). Additionally, patients without adverse genetic features (9.6 vs 6.1 months; P = 0.036; Additional file 1: Fig. S4C) or T315I mutation (17.3 vs 5.3 months; P = 0.017; Additional file 1: Fig. S4D) had longer LFS compared with those harboring these features. In contrast, prior therapies, disease burden, genetic stratification, and T315I mutation status were not significantly associated with OS (Additional file 1: Fig. S5A), nor were prior therapies and disease burden significantly associated with LFS (Additional file 1: Fig. S5B). Age-stratified analyses of OS and LFS further showed no significant differences among the four age groups (< 18, 18–39, 40–59, ≥ 60 years; Additional file 1: Fig. S6).

Multivariable time-dependent Cox regression analysis was conducted to identify independent predictors of OS and LFS among patients achieving CR/CRi at day 30 (Table 2). For OS, ECOG performance status ≤ 2 was independently associated with improved survival (HR = 0.281, 95% CI 0.130–0.608; P = 0.001), whereas the presence of adverse genetic features significantly increased the risk of death (HR = 2.900, 95% CI 1.196–7.031; P = 0.018). Molecular remission at CR, T315I mutation, and consolidative allo-HSCT, modeled as a time-dependent covariate, were not significant predictors of OS (HSCT: HR = 0.754, 95% CI 0.302–1.880; P = 0.545; molecular remission: HR = 0.707, 95% CI 0.228–2.198; P = 0.549; T315I: HR = 1.311, 95% CI 0.575–2.990; P = 0.519).

Table 2.

Multivariable time-dependent Cox regression analysis of OS and LFS from the day 30 landmark in patients achieving CR/CRi

Subgroups	HR	95% CI	P
OS
Molecular remission vs no molecular remission	0.707	0.228–2.198	0.549
T315I mutation vs no T315I mutation	1.311	0.575–2.990	0.519
Adverse genetic features vs no adverse genetic features	2.900	1.196–7.031	0.018*
ECOG 0–2 vs ≥ 3	0.281	0.130–0.608	0.001*
HSCT vs no HSCT	0.754	0.302–1.880	0.545
LFS
Molecular remission vs no molecular remission	0.544	0.225–1.318	0.178
T315I mutation vs no T315I mutation	1.611	0.859–3.021	0.137
Adverse genetic features vs no adverse genetic features	2.014	1.025–3.957	0.042*
ECOG 0–2 vs ≥ 3	0.436	0.228–0.832	0.012*
HSCT vs no HSCT	0.745	0.360–1.542	0.428

^*P < 0.05

For LFS, ECOG ≤ 2 was also independently associated with prolonged survival (HR = 0.436, 95% CI 0.228–0.832; P = 0.012), and adverse genetic features were associated with higher relapse risk (HR = 2.014, 95% CI 1.025–3.957; P = 0.042). Molecular remission, T315I mutation, and allo-HSCT were not significantly associated with LFS (HSCT: HR = 0.745, 95% CI 0.360–1.542; P = 0.428; molecular remission: HR = 0.544, 95% CI 0.225–1.318; P = 0.178; T315I: HR = 1.611, 95% CI 0.859–3.021; P = 0.137).

ECOG performance status and competing-risk outcomes

Competing-risk analyses were performed to investigate the impact of baseline ECOG performance status on relapse and NRM. Poorer ECOG performance status (≥ 3) was associated with a significantly higher incidence of relapse (sdHR = 2.95, 95% CI 1.83–4.75; P < 0.001), whereas no significant association was observed with NRM (Additional file 1: Table S1, Additional file 1: Fig. S7). These results indicate that the higher risk of adverse outcomes in patients with poorer ECOG performance status appears to be driven mainly by increased relapse incidence rather than by NRM.

B-cell aplasia following CAR T-cell therapy

B-cell aplasia was assessed as a clinically accessible surrogate of functional CAR T-cell activity. The onset of B-cell aplasia from CAR T-cell infusion in our study was 3 days (range, 1–28), and the median duration of B-cell aplasia was 101 days (range, 11–705). As shown in Additional file 1: Fig. S8, patients with prolonged B-cell aplasia (≥ 3 months) were associated with a longer LFS compared with patients with shorter B-cell aplasia (< 3 months) (P = 0.037). In contrast, although patients with B-cell aplasia ≥ 3 months tended to have improved OS, the difference did not reach statistical significance (P = 0.140).

Safety

Of the 93 patients, 67 (72.0%) experienced CRS, with 13 patients (14.0%) developing grade ≥ 3 CRS. One patient died from severe CRS complicated by MODS on day 4 post-infusion, while the remaining cases were manageable. The median time from CAR T-cell infusion to CRS onset was 2 days (range, 0–17), and the median CRS duration was 7 days (range, 1–15). Predictive factors for CRS occurrence and severity are presented in Additional file 1: Fig. S9. Patients who developed CRS had significantly higher bone marrow blast counts and MRD levels prior to therapy (both P < 0.001; Additional file 1: Fig. S9A). Compared to patients who experienced mild CRS (grade 1–2), those experiencing severe CRS (grade ≥ 3) had significantly elevated serum interleukin (IL)−6 and ferritin levels (both P < 0.05; Additional file 1: Fig. S9B).

ICANS occurred in eight patients (8.6%), all of whom also developed CRS. Two patients (2.2%) experienced grade ≥ 3 ICANS, both presenting with seizures. Among the eight patients, three had previous CNSL, all of whom had CNS-3 status. No ICANS-related deaths occurred, and all symptoms resolved with supportive care. The median time from infusion to ICANS onset was 9 days (range, 4–16), and the median ICANS duration was 4 days (range, 2–10). Spearman’s rank correlation analysis identified a moderate, yet significant correlation between CRS severity and ICANS occurrence (r = 0.306, P = 0.003). Fisher’s exact test demonstrated a significant association between CNS involvement and ICANS occurrence (P = 0.028), with an odds ratio of 7.90 (95% CI, 1.69–35.37).

Overall, CRS and ICANS were effectively managed. Thirteen patients (14.0%) received tocilizumab, 6 (6.5%) received corticosteroids, and 20 (21.5%) received both agents. Other adverse events occurring within 30 days after treatment are detailed in Table 3. Hematologic toxicities were the most frequent and predictable adverse events, with over 60% of patients experiencing grade ≥ 3 neutropenia (78.5%), lymphopenia (73.1%), thrombocytopenia (64.5%), and anemia (77.4%). Additionally, 66 patients (71.0%) developed hypogammaglobulinemia, with grade ≥ 3 observed in 36 patients (38.7%). Infections were also common, likely related to lymphodepleting chemotherapy and the subsequent cytopenia. Pneumonia was diagnosed in 36 patients (38.7%), with 35 cases classified as grade ≥ 3. One patient died from sepsis on day 19 post-infusion.

Table 3.

Adverse events within 30 days after CAR T-cell infusion

	N = 93 (%)
	Any grades	Grade ≥ 3
Adverse events
Diarrhea	16 (17.2)	1 (1.1)
Oral mucositis	5 (5.4)	0
Arrhythmia	4 (4.3)	4 (4.3)
Rash	4 (4.3)	1 (1.1)
Tumor lysis syndrome	2 (2.2)	2 (2.2)
Capillary leak syndrome	3 (3.2)	3 (3.2)
Hemophagocytic syndrome	1 (1.1)	1 (1.1)
Hematologic AEs
Neutropenia	83 (89.2)	73 (78.5)
Anemia	80 (86.0)	72 (77.4)
Thrombocytopenia	75 (80.6)	60 (64.5)
Lymphopenia	84 (90.3)	68 (73.1)
Hypogammaglobulinemia	66 (71.0)	36 (38.7)
Infection
Pneumonia	36 (38.7)	35 (37.6)
Intestinal infection	1 (1.1)	1 (1.1)
Urinary tract infection	2 (2.2)	1 (1.1)
Reproductive tract infection	1 (1.1)	1 (1.1)
Sepsis	3 (3.2)	3 (3.2)
Septic shock	2 (2.2)	2 (2.2)
Laboratory abnormalities
Myocardial enzyme/BNP elevation	12 (12.9)	5 (5.4)
ALT/AST/ALP/GGT elevation	27 (29.0)	2 (2.2)
Hyperbilirubinemia	6 (6.5)	2 (2.2)
Hyperuricemia	5 (5.4)	0
Hyperlipemia	2 (2.2)	0
Creatinine elevation	3 (3.2)	0
Electrolyte disturbance	15 (16.1)	2 (2.2)
Coagulation disorders	14 (15.1)	12 (12.9)
Multisystem AEs
Fundus hemorrhage	1 (1.1)	1 (1.1)
Acute cholecystitis	1 (1.1)	1 (1.1)
Intestinal obstruction	1 (1.1)	1 (1.1)
Gastrointestinal bleeding	1 (1.1)	1 (1.1)
DAHS	1 (1.1)	1 (1.1)
Hematuria	1 (1.1)	1 (1.1)
Hydronephrosis	1 (1.1)	1 (1.1)
AKI	1 (1.1)	1 (1.1)
Heart failure	3 (3.2)	3 (3.2)
Multiple organ and system failure	2 (2.2)	2 (2.2)

Data are shown as n (%). All adverse events were documented within the first 30 days following CAR T-cell infusion for all patients who received treatment

Abbreviations: AEs adverse events, BNP brain natriuretic peptide, ALT alanine aminotransferase, AST aspartate aminotransferase, ALP alkaline phosphatase, GGT γ-glutamyl transferase, DAHS diffuse alveolar hemorrhage syndrome, AKI acute kidney injury

Long-term adverse events beyond the early post-infusion period are summarized in Additional file 1: Table S2. Prolonged cytopenias persisting or newly occurring ≥ 3 months after CAR T-cell infusion were observed in a subset of patients, including prolonged neutropenia (12.9%), thrombocytopenia (10.8%), and anemia (12.9%). The incidence of severe infections (grade ≥ 3) was 15.1% beyond 30 days, 6.5% beyond 3 months, and 2.2% beyond 6 months following CAR T-cell infusion. Hypogammaglobulinemia beyond 90 days after infusion requiring intravenous immunoglobulin replacement was observed in 14 patients (15.1%). Prolonged B-cell aplasia was recognized as a common long-term on-target adverse event, and its duration was systematically assessed and is detailed in the dedicated section above.

Discussion

Despite the increased remission rates and improved long-term survival achieved with chemotherapy-free regimens and other novel therapies for newly diagnosed Ph + ALL [1, 4, 5], long-term outcomes for patients with R/R Ph + ALL remain unfavorable [7, 8]. In this multicenter retrospective study involving 93 patients with R/R Ph + B-ALL treated with CD19 CAR T-cell therapy, we evaluated the safety and efficacy of treatment over a median follow-up of 25.4 months (range, 0.1–68.5). Additionally, this study identified clinical factors associated with treatment responses and long-term outcomes, offering insights to better understand and optimize the use of CD19 CAR T-cell therapy in patients with R/R Ph + B-ALL.

Previous studies of CAR T-cell therapy in R/R B-ALL that included a small subset of patients with Ph + disease reported favorable CR/CRi rates ranging from 80 to 100%. However, these findings require further validation due to the limited sample size of this subgroup [14–16, 18]. In this multicenter study with a relatively large-scale cohort, we observed an overall CR/CRi of 87.1% (81/93), with 96.3% (78/81) of responders achieving MRD-negative status by flow cytometry. This response rate is comparable to the 91.1% CR/CRi reported in a single-center retrospective study of 56 patients with R/R Ph + B-ALL treated with CAR T-cell therapy [19], and notably exceeds the response rates achieved by inotuzumab ozogamicin (73% CR/CRi) and blinatumomab (36% CR/CRi) reported previously [9, 10]. Achieving CMR or MMR has been associated with improved long-term survival [21, 22]. In this study, 78.5% of patients (73/93) achieved molecular remission, with a CMR rate of 63.4% (59/93) and MMR rate of 15.1% (14/93). Our CMR rate was similar to prior CAR T-cell studies in R/R Ph + ALL [19]. It exceeded the CMR rate of 56% (10/18) observed in a Phase 1/2 trial of inotuzumab ozogamicin plus bosutinib in patients with R/R Ph + ALL or lymphoid blast phase of chronic myeloid leukemia [23]. Our data demonstrate that CD19 CAR T-cell therapy is capable of achieving great hematologic and molecular responses in patients with R/R Ph + B-ALL, even in the presence of adverse genetic features or substantial disease burden.

The median OS for the entire cohort in our study was 20.8 months, which appears notably longer than the median OS reported for patients with R/R Ph + ALL receiving blinatumomab (7.1 months) and for those treated with inotuzumab ozogamicin (7.4–8.7 months) [9, 10]. It seems that CAR T-cell therapy offers greater survival benefits for patients with R/R Ph + ALL compared with blinatumomab or inotuzumab ozogamicin, and this advantage, as suggested by previous studies, may be explained by several factors, including (1) blinatumomab relies on the immune system to exert its anti-leukemic effect; its activity is limited by the quantity and functional capacity of endogenous T cells [14], and its efficacy may be compromised in patients with R/R disease whose immune systems are often weakened by prior intensive treatments; (2) inotuzumab ozogamicin, as an antibody–drug conjugate, is susceptible to drug resistance with repeated exposure, partly due to the evasion of DNA damage-induced apoptosis through the disruption of the G1/S DNA damage checkpoint [24]; (3) in contrast, CAR T-cells can rapidly expand in vivo and generate both memory and effector lymphocytes, thereby providing long-term tumor cell eradication and immune surveillance [14, 25].

Of note, the median OS was 24.5 months in patients achieving CR/CRi in the day-30 landmark analysis, with 17 individuals surviving beyond 36 months of follow-up from infusion (of whom one experienced a late relapse at 36.6 months, another at 47.1 months, one patient died due to disease progression, and ten maintained durable responses). Consistent with prior evidence recognizing MRD as a key prognostic factor in ALL [26], patients achieving MMR/CMR had prolonged median OS (24.5 vs 8.4 months) and LFS (8.9 vs 3.1 months), though the difference in LFS did not reach statistical significance. These observations suggest the promise of increasing MRD eradication through CAR T-cell therapy to improve long-term outcomes. Moreover, the use of more sensitive MRD detection methods, such as high-throughput next-generation sequencing with a sensitivity of 10⁻⁶, has been recommended to guide treatment decisions—either to escalate or adjust therapy to prevent disease progression, or to de-escalate treatment to minimize treatment-related toxicity and adverse events [27].

While treatment outcomes following CAR T-cell therapy were encouraging, disease relapse remained a substantial clinical challenge in this cohort. Consolidative strategies after CAR T-cell therapy have therefore been actively explored, including allo-HSCT [5, 28], particularly in patients with high-risk disease features. Previous studies in R/R B-ALL have suggested a potential survival benefit of consolidative allo-HSCT following CAR T-cell therapy [29–31]. Similar observations have also been reported in selected cohorts of relapsed Ph + ALL [19, 32]. Nevertheless, the reported benefit has not been uniform, and results have varied across disease subtypes, risk groups, and study designs. Consolidative allo-HSCT was not independently associated with improved OS or LFS in our cohort of patients with R/R Ph + B-ALL. These results indicate that the association between consolidative allo-HSCT after CAR T-cell therapy and survival outcomes should be interpreted cautiously. Several factors may contribute to the discrepancy between our findings and prior reports, including the limited number of transplanted patients, patient selection bias, and the biological heterogeneity of Ph + ALL, which may influence post–CAR T-cell disease control.

In line with previous reports, allo-HSCT after CAR T-cell therapy was not associated with excess treatment-related mortality in our study [33]; however, its role as a routine consolidative strategy remains uncertain [34]. Taken together, our data do not support a definitive survival benefit of routine consolidative allo-HSCT after CAR T-cell therapy in R/R Ph + B-ALL. Instead, they underscore the need for individualized post-remission strategies and further prospective studies to define optimal patient selection. Alternative approaches, including immunotherapy- or TKI-based consolidation strategies, warrant further investigation as potential complements or alternatives to allo-HSCT in this setting [32].

Consistent with previous reports [6, 31], patients with adverse genetic features or impaired performance status had inferior outcomes. These findings suggest that CD19 CAR T-cell therapy may be more effective in Ph + B-ALL when administered earlier in the treatment course and combined with tailored post-remission strategies guided by refined risk stratification. We previously reported the initial results of a phase 2, single-arm clinical trial evaluating the combination of dasatinib and CAR T-cell therapy in patients with newly diagnosed Ph + ALL, which demonstrated 2-year overall survival and leukemia-free survival rates of 92% [35]. We anticipate that further investigation of CAR T-cell therapy as a frontline treatment in Ph + ALL will be essential to define its clinical role. In addition, Wang et al. proposed a refined risk stratification model for patients with BCR::ABL1-positive ALL that integrates IKZF1^plus genotyping with MRD assessment, providing criteria to help guide transplantation decisions for improving disease control [26]. However, given the predominance of antigen-positive relapse, strategies to enhance CAR T-cell persistence—through sequential or combinatorial regimens with targeted or immunomodulatory agents, or through next-generation CAR T-cell constructs such as armored or dual-targeted designs—merit further clinical investigation in R/R Ph + B-ALL.

Despite the heavily pretreated status of the patient population, treatment was administered with an acceptable and manageable toxicity profile. The incidences of severe CRS and ICANS observed in our study were 14.0 and 2.2%, respectively. Compared to previous reports, no increase in incidence was observed, suggesting that the presence of the BCR::ABL1 fusion gene does not elevate the risk of CRS or ICANS following CD19 CAR T-cell therapy [11, 14, 15]. Moreover, NRM remained relatively low throughout the follow-up period, with cumulative incidences of 4.4% (95% CI, 0.2 to 8.7), 8.1% (95% CI, 2.3 to 14), and 12.3% (95% CI, 5.0 to 19.6) at 3, 12, and 24 months, respectively, reflecting a favorable safety profile of CAR T-cell therapy. Patients who underwent CRS had a significantly higher bone marrow tumor burden prior to treatment than those who did not. Consistent with prior reports, a higher tumor burden in ALL is correlated with an increased likelihood and severity of CRS [14, 36]. Therefore, pre-infusion reduction of tumor burden should be considered a potential strategy to lower the risk of adverse events. Moreover, elevated serum cytokines, such as IL-6 and ferritin, may serve as predictors of CRS severity. Similar to prior findings, the presence of CNSL and severe CRS was associated with the occurrence of ICANS [18, 36], indicating that early intervention in CRS may help decrease the incidence of neurotoxicity.

The present multicenter study, while having several strengths, also has limitations that warrant consideration. First, the retrospective nature of this study may introduce inherent selection bias, preclude definitive causal inference, and result in incomplete availability of certain detailed data, such as CAR T-cell kinetics. Second, heterogeneity in clinical practice across participating centers, including real-world differences in CAR T-cell constructs and manufacturing processes, as well as supportive care and toxicity management, may have contributed to inter-institutional variability in clinical outcomes. Third, although the median follow-up was 25.4 months, a subset of patients had relatively limited follow-up, which may have led to underestimation of late relapses and long-term toxicities. Last, multiple subgroup analyses were performed without formal adjustment for multiple comparisons, which may increase the risk of spurious associations. Accordingly, these subgroup findings should be interpreted as exploratory and hypothesis-generating rather than definitive.

Conclusions

In summary, this multicenter retrospective study demonstrates that CD19 CAR T-cell therapy is an effective treatment option for patients with R/R Ph + B-ALL, with manageable toxicity. Survival outcomes were primarily influenced by baseline patient and disease characteristics, as patients with better performance status (ECOG ≤ 2) and without adverse genetic features experienced superior survival. In contrast, consolidative allo-HSCT after CAR T-cell therapy was not independently associated with improved overall or leukemia-free survival. Together, these findings underscore the prognostic importance of baseline risk stratification and support an individualized approach to post–CAR T-cell management. Prospective studies are warranted to further refine patient selection and optimize treatment strategies in this population.

Abbreviations

Allo-HSCT

Allogeneic hematopoietic stem cell transplantation

B-ALL

B-cell acute lymphoblastic leukemia

CAR

Chimeric antigen receptor

CIs

Confidence intervals

CMR

Complete molecular remission

CNSL

Central nervous system leukemia

CR

Complete remission

CRi

Complete remission with incomplete hematologic recovery

CRS

Cytokine release syndrome

CSF

Cerebrospinal fluid

ECOG

Eastern Cooperative Oncology Group

EMD

Extramedullary disease

ICANS

Immune effector cell-associated neurotoxicity syndrome

ITT

Intention-to-treat

LFS

Leukemia-free survival

MMR

Major molecular remission

MODS

Multiple organ dysfunction syndrome

MRD

Minimal residual disease

NCCN

National Comprehensive Cancer Network

NRM

Non-relapse mortality

OS

Overall survival

Ph + 

Philadelphia chromosome-positive

R/R

Relapsed or refractory

RT-PCR

Reverse transcription-polymerase chain reaction

sdHR

Subdistribution hazard ratios

TKI

Tyrosine kinase inhibitor

Authors’ contributions

GW, YH and HH were responsible for designing the study. JY, FS, MZ2, WS, SJ, LW, XW, ST and YL contributed to collecting the required data. JY and MZ1 conducted the statistical analysis; JY drafted the manuscript; GW, YH and HH provided guidance and revised the manuscript. All the authors have read and approved the submitted version.

Funding

The research was funded by the National Natural Science Foundation of China (82341206, 82270234, 82370223 and 82522004), and the Key Project of Science and Technology Department of Zhejiang Province (2024C03156).

Data availability

The data generated in this study are available upon request from the corresponding author.

Declarations

Ethics approval and consent to participate

This multicenter retrospective study was approved by the Ethics Committee of The First Affiliated Hospital, Zhejiang University School of Medicine, the coordinating center (Approval No. IIT20241434A), and was conducted in accordance with the Declaration of Helsinki. The ethics committees approved a waiver of informed consent owing to the minimal risk to patients and the impracticality of obtaining consent in this retrospective study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.