Analysis of the therapeutic effect of allogeneic hematopoietic stem cell transplantation in the treatment of pediatric acute lymphoblastic leukemia with E2A::HLF fusion gene: a case series

Introduction

Acute lymphoblastic leukemia (ALL) is a malignancy that affects white blood cells (i.e., B and T lymphocytes), resulting in the uncontrolled proliferation of immature cells and leading to the replacement of healthy cells in the bone marrow and other lymphoid organs (1). The fusion gene E2A::HLF (TCF3::HLF) is formed by t(17;19) (q21-22;p13), which is present in <1% of B-cell ALL patients, mainly in older children and adolescents (2). It is often associated with aberrant expression of CD33, hypercalcemia and acquired coagulation abnormalities (3,4). Allogeneic hematopoietic stem cell transplantation (allo-HSCT) serves as a cornerstone therapy for hematological malignancies and bone marrow disorders. However, leukemia cells harboring the E2A::HLF fusion gene exhibit resistance to intensified chemotherapy and are correlated with a dismal prognosis, even after allo-HSCT (5). We retrospectively analyzed the clinical outcomes of four pediatric ALL patients with the E2A::HLF fusion gene treated with allo-HSCT at our center, aiming to refine therapeutic strategies and improve survival for this high-risk subgroup. We present this article in accordance with the AME Case Series reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-631/rc).

Case presentation

Basic information

This was a retrospective observational study. Children suffering from ALL with the E2A::HLF fusion gene who underwent HSCT were enrolled in this study. None of the patients had human leukocyte antigen (HLA)-matched unrelated donors in the China Bone Marrow Bank. Data on the source of hematopoietic stem cells, conditioning regimen, adverse effects, and prognosis were retrospectively reviewed. The time of the last follow-up was defined as the number of days between the date of transplantation and the last clinic visit. All procedures performed in this case series were in accordance with the ethical standards of the Institutional Review Board (IRB) of Beijing Children’s Hospital, Capital Medical University (No. [2025]-E-155-R), and with the Helsinki Declaration and its subsequent amendments. Written informed consent was obtained from the patients’ parents or guardians for publication of this case series and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Four pediatric patients (male: 2, female: 2) were enrolled in this study, with a median age at diagnosis of 6.85 years (range, 1.58–11.33 years). Three patients presented with bone pain as the initial symptom, whereas one presented with pallor and fatigue (Table 1). All the patients harbored the E2A::HLF fusion gene. Bone marrow cytogenetic analysis in Patient 1 revealed a complex karyotype: 46,XY,t(17;19)(q22;p13)[14]/45,idem, der(8;9) (q10;q10)[6]. Initial laboratory findings included leukocytosis in Patient 1, leukopenia in Patient 4, and anemia/thrombocytopenia in Patients 1 and 4. Coagulation abnormalities were observed in Patients 1, 3, and 4, with hypercalcemia noted in Patients 2 and 3.

Laboratory-related information

Reverse transcription (RT)-polymerase chain reaction (PCR) for E2A::HLF

Total RNA was isolated from bone marrow cells with Trizol. RT was performed with 2 mg of total RNA, random hexamers and Superscript reverse transcriptase under conditions recommended by the manufacturer. PCR was performed using the following primers that were homologous to sequences in E2A exon 12 and exon 13, and HLF exon 4: E2A exon 12 (e12), 5'-gacatgcacacgctgctgcc-3'; E2A exon 13 (e13), 5'-gcctcatgcacaacca cgcg-3'; HLF exon 4 (e4), 5'-cccggatggcgatctggttc-3'. As a control, PCR for ABL1 was performed under the same conditions using the following primers: 5'-gtatcatctgactttgagcc-3'.

Flow cytometry (FCM) for minimal residual disease (MRD)

MRD was assessed by 8-color flow cytometry (FC) with a sensitivity of 10^-4 on bone marrow aspirate samples. The following monoclonal antibodies were used to track leukemia-associated aberrant immunophenotypes: cyCD3, mCD3, CD2, CD5, CD7, CD9, CD10, CD13, CD19, CD20, CD22, CD24, CD33, CD34, CD38, CD45, CD56, CD58, CD79a, CD99, CD117, CD123, and cTDT, acquired on a fluorescence-activated cell sorter.

Information about chimeric antigen receptor T-cell (CAR-T)

T cells were purified from the leukapheresis material of patients by CD4 isolation kit and CD8 isolation kit according to the instructions. The CAR-T structure of CD19/CD22 is a tandem single-chain variable fragment (scFv) structure followed by CD8 hinge and transmembrane domain, CD137 costimulatory domain, and the signaling portion of CD3ζ. More information about CAR-T-cell production, efficacy and safety can be seen in previous articles (6,7).

Treatment (Figure 1)

Figure 1 Treatment flowchart for ALL with E2A::HLF fusion gene positivity. ALL acute lymphoblastic leukemia; CAR-T, chimeric antigen receptor T-cell; DLI, donor lymphocyte infusion; HSCT, hematopoietic stem cell transplantation; P, patient.

Before HSCT

All patients received chemotherapy via the Chinese Children Leukemia Group (CCLG)-2018-ALL high-risk protocol (8). Following intensive therapy, two patients exhibited persistent negativity for MRD and E2A::HLF fusion in the bone marrow. Patient 1 maintained non-remission (NR) after 4 months of chemotherapy (MRD: 4.3×10⁻⁴; E2A::HLF transcript level: 8.2×10⁻²) and subsequently received murine-derived CD19-targeted CAR-T therapy, achieving sustained MRD and E2A::HLF negativity in the bone marrow. Patient 4 also did not achieve complete remission (CR) after 6 months of chemotherapy (MRD: 3.59×10⁻¹; E2A::HLF transcript level: 1.16×10⁻¹) and underwent autologous CD19 CAR-T-cell therapy (cell dose: 2.03×10⁶). Posttreatment assessments revealed sustained MRD and E2A::HLF negativity in the bone marrow.

HSCT treatment (Table 2)

All four patients achieved CR before HSCT and were negative for MRD and E2A::HLF fusion in the bone marrow. The median interval from diagnosis to haploidentical HSCT was 208.5 days (range, 177.0–244.0 days). The donors included the mother (Patient 2), father (Patients 3 and 4) or sister (Patient 1) of the patients, all with 5/10 HLA matching. The conditioning regimens used were as follows: fludarabine (Flu) + busulfan (Bu) + cyclophosphamide (Cy) + anti-thymocyte globulin (ATG) (Patient 1), cytarabine (Ara-C) + Bu + Cy + ATG + Semustine (Mc-CCNU) (Patients 2 and 3), and total body irradiation (TBI) + Flu + Cy + ATG + thiotepe (TT) (Patient 4). All patients received GVHD prophylaxis with cyclosporin a (CsA) + mycophenolate mofetil (MMF) + methotrexate (MTX) (Figure 2). The median infused mononuclear cell count was 9.96 (6.02–11.37) ×10⁸/kg, and the median infused CD34⁺ cell count was 9.72 (6.61–17.43) ×10⁶/kg. All four patients were engrafted successfully, with a mean time of neutrophil recovery of 12 days (range, 11–15 days) and a median time of platelet recovery of 14.5 days (range, 8–23 days).

Figure 2 Conditioning regimen. Ara-c, cytarabine; ATG, anti-thymocyte globulin; BM, bone marrow; Bu, Busulfan; CsA, cyclosporin a; Cy, cyclophosphamide; Flu, fludarabine; Me-CCNU, semustine; MMF, mycophenolate mofetil; MTX, methotrexate; P, patient; PBSC, peripheral blood stem cells; TBI, total body irradiation; TT, thiotepa.

In our study, 3 patients (Patients 1, 2 and 3) experienced acute GVHD (aGVHD), two of whom were grade I–II (skin stage 1–3) and one of whom was grade IV (skin stage 4 and gastrointestinal stage 2). All GVHD manifestations resolved following therapeutic intervention with corticosteroids, cyclosporine, and/or ruxolitinib, with no GVHD-related mortality observed. According to the diagnostic criteria of thrombotic microangiopathy (TMA) (9), 3 patients fit the criteria for TMA (Patients 1, 2 and 4). Patient 1 was diagnosed with veno-occlusive disease (VOD) on day +11 and treated with defibrotide, low-molecular-weight heparin and alprostadil. Patient 3 was diagnosed with transplant-associated TMA (TA-TMA) on day +32 and treated with defibrotide, plasma exchange, low-molecular-weight heparin and alprostadil. Patient 4 was diagnosed with VOD on day +43 and treated with defibrotide, low-molecular-weight heparin and alprostadil. Patients 1 and 4 demonstrated clinical improvement following therapeutic intervention. Patient 3 succumbed to diffuse alveolar hemorrhage syndrome and respiratory failure on post-transplantation day 104. Hemorrhagic cystitis and cytomegalovirus (CMV) infection were observed in 2 patients (Patient 1 and Patient 4).

Treatment after HSCT

Patient 1 received prophylactic donor lymphocyte infusion (Pro-DLI) starting at +250 d after HSCT to prevent relapse. Six cumulative DLI doses (total 5×10⁷/kg) were administered. Subsequent monitoring revealed sustained CR with negative MRD [flow cytometry and next-generation sequencing (NGS)] and E2A::HLF fusion in the bone marrow.

Relapse was detected on day +182 after HSCT in Patient 2 (MRD: 1.63×10⁻²; E2A::HLF fusion qualitative 3+). Donor-derived CD19 CAR-T-cell therapy (4.8×10⁷ cells, transduction efficiency 67.49%) was administered on day +215, and the patient achieved molecular and MRD-negative remission (CR2) within one week. Prophylactic CD22 CAR-T cells (1.1×10⁸ cells, transduction efficiency 44.83%) were administered on day +326. Transient E2A::HLF fusion positivity (qualitative 2+) with negative MRD was observed on day +341 (15 days post-second CAR-T-cell therapy), followed by full molecular remission on day +354. Pro-DLI (5 cumulative doses, total 4.5×10⁷/kg) was initiated on day +368. The second relapse occurred on day +625 (bone marrow MRD: 1.57×10⁻²; E2A::HLF qualitative 1+; cerebrospinal fluid tumor cells detected). Chemotherapy (vincristine, pegaspargase, MTX, dexamethasone) and triple intrathecal therapy were administered. Persistent MRD positivity (8.35×10⁻¹) and E2A::HLF fusion (qualitative 1+) were noted on day +748. Dual CD19 (1×10⁸ cells)/CD22 (1.4×10⁸ cells) CAR-T cells were infused on day +804. Subsequent MRD and E2A::HLF results remained positive. The patient died after treatment discontinuation on day +902 (final MRD: 1.1×10⁻³; E2A::HLF qualitative 3+).

Pro-DLI (total dose: 5×10⁶/kg) was infused at post-HSCT day +220 in Patient 4. Sustained MRD (flow cytometry and NGS) and E2A::HLF fusion negativity were observed during follow-up.

Survival analysis

Among the four pediatric ALL patients with the E2A::HLF fusion gene who underwent allo-HSCT, two achieved long-term survival, while two succumbed to complications of HSCT (n=1) and primary disease relapse (n=1). With a median follow-up of 553.5 days (range, 104–902 days), the 1-year overall survival (OS) rate was 3/4.

Discussion

The E2A::HLF fusion gene gives rise to a distinct but unfortunately rare form of high-risk pro-B-ALL in adolescents. Previous studies have shown that this fusion gene encodes a chimeric protein in which the transactivation domain of E2A is linked to the basic leucine zipper dimerization and DNA-binding domain of HLF (10,11). E2A-HLF blocks apoptosis and p53 activation induced by the intrinsic mitochondrial pathway, which plays a central role in leukemogenesis and chemoresistance (5,12). This may be the key factor for the poor prognosis of ALL patients with the t(17;19) chromosomal translocation.

Hypercalcemia represents a clinically rare yet critical complication in pediatric leukemia patients. Consistent with prior reports associating hypercalcemia with acquired coagulopathy at disease onset (4), our study revealed concurrent hypercalcemia in 2/4 of patients and acquired coagulopathy (mainly manifested as hypofibrinogenemia) in 3/4 of patients during initial presentation. Compared with patients without E2A::HLF fusion, the clinical manifestations in this cohort lacked specificity, predominantly presenting as bone pain with variable white blood cell counts (elevated, reduced, or normal) at diagnosis.

Over the past 60 years, with the use of multiple drug combinations of intensive chemotherapy, risk stratification, and HSCT for pediatric ALL, survival rates have significantly improved and are currently approaching 90% for children with B-ALL (13). Patients with E2A::HLF fusion B-ALL consistently have dismal outcomes. Inukai et al. reported 14 pediatric E2A::HLF B-ALL patients, 13 of whom were treated with chemotherapy alone and experienced relapse or death within 2 years of diagnosis (4). Even with HSCT, the prognosis remains poor, with studies documenting immediate posttransplant relapses and 5-year mortality rates reaching 66–100% (14). Chen et al. also reported similar trends; among the 6 patients in their study, 5 achieved CR after induction chemotherapy (15). However, all patients relapsed, and even in 2 patients who received HSCT, the disease-free survival was only 8 months (15). Consistent with prior reports, 3/4 in our cohort achieved CR postchemotherapy. However, following HSCT, one patient experienced multiple relapses, and two patients died. The 1-year OS rate was 3/4. These observations reinforce the exceptionally poor prognosis of E2A::HLF-positive B-ALL, where HSCT fails to confer survival benefits.

Developing novel therapeutic strategies to improve outcomes for children with E2A::HLF-positive B-ALL represents a critical unmet need in current clinical practice. Owing to its potent graft-versus-leukemia (GVL) immune effect, DLI is widely used for post-HSCT treatment and prevention. It induces a polyclonal T-cell response that can target multiple antigens on malignant cells. Zhang et al. demonstrated that E2A::HLF B-ALL cells exhibit elevated DR4/DR5 expression and heightened sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity (5). These findings provide a theoretical rationale for DLI in the management of pediatric E2A::HLF-positive B-ALL. Hirai et al. reported the first case of E2A::HLF ALL in a 12-year-old male, who underwent HSCT while in NR status, followed by 16 pro-DLI cycles (starting on day +43) over 2 years, achieving durable molecular remission (16). In our cohort, three E2A::HLF B-ALL patients received pro-DLI after HSCT, with two maintaining continuous CR and one experiencing relapse (response rate 2/3). These findings may remind the potential efficacy of pro-DLI administration for relapse prevention in this high-risk subgroup.

CD19 CAR-T-cell therapy represents a promising treatment for relapsed/refractory (R/R) B-cell malignancies (17). However, limited data exist regarding its efficacy in E2A::HLF-positive B-ALL. Wu et al. reported that 4 E2A::HLF-positive B-ALL patients achieved CR after CAR-T-cell therapy, with 1 patient sustaining remission, 1 surviving after bridging HSCT, and 2 succumbing to disease progression (18). Chen et al. described 4 E2A::HLF-positive B-ALL patients: 2 received CD19 CAR-T cells, and 2 received dual CD19/CD22 CAR-T cells (15). After treatment, 3 patients achieved sustained CR, including 2 patients who subsequently underwent HSCT. In our study, 3 patients received CAR-T-cell therapy. Two patients underwent CD19 CAR-T-cell therapy before HSCT, and both of them maintained durable remission. One patient relapsed post-HSCT and exhibited a suboptimal response to CD19/CD22 CAR-T cells, ultimately leading to death due to progressive disease. These observations suggest that sequential chemotherapy, CAR-T-cell therapy, and HSCT bridging may optimize outcomes for TCF3::HLF-positive B-ALL patients, potentially by achieving deeper bone marrow remission before HSCT. Notably, bridging HSCT after blinatumomab has also shown survival benefits in small cohorts, although larger validation is warranted (19).

Conclusions

In conclusion, given the rarity and extremely poor prognosis of this disease, the combination of CAR-T-cell therapy before HSCT and pro-DLI treatment after HSCT may represent an effective strategy to improve outcomes in these pediatric patients. However, our findings were limited by the small cohort size and relatively short follow-up duration, necessitating further investigation to validate these observations.

Acknowledgments

We thank all of the patients and their families for their kind cooperation. We thank all of the members of clinical team who provided care for patients.

Footnote

Reporting Checklist: The authors have completed the AME Case Series reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-631/rc

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-631/prf

Funding: This work was supported by the Beijing Natural Science Foundation (No. 7254351) and National Science and Technology Key Projects (No. 2017ZX09304029001).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-631/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this case series were in accordance with the ethical standards of the Institutional Review Board (IRB) of Beijing Children’s Hospital, Capital Medical University ([2025]-E-155-R), and with the Helsinki Declaration and its subsequent amendments. Written informed consent was obtained from the patients’ parents or guardians for publication of this case series and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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