<scp>Flu/Cy</scp> Plus <scp>PTCy</scp> Conditioning Regimen in Haplo‐ <scp>HSCT</scp> of Severe Aplastic Anemia

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the primary curative option for severe aplastic anemia (SAA). Given the limited availability of fully matched donors, haploidentical transplantation (haplo-HSCT) has increasingly emerged as a first-line treatment strategy [1-3]. However, engraftment failure and graft-versus-host disease (GVHD) represent significant challenges in haplo-HSCT for SAA, both of which adversely affect patient outcomes [4]. Therefore, developing innovative pre-transplantation conditioning strategies to mitigate poor graft function (PGF) and GVHD is critical for improving the prognosis of SAA patients. Although the combination of anti-thymocyte globulin (ATG) and cyclophosphamide remains a preferred haplo-HSCT regimen for SAA in many centers, ATG-based approaches carry risks of severe allergic reactions, increased infections, and higher costs [5]. Fludarabine-based conditioning has recently been linked to a reduced acute graft-versus-host disease (aGVHD) incidence in haplo-HSCT [1]. Notably, successful fludarabine-based protocols for haploidentical SAA transplantation reported universally incorporate either total body irradiation (TBI) or busulfan (BU) within the conditioning regimen [1, 6]. Critically, both TBI and BU are associated with significant toxicities, including profound myelosuppression, elevated secondary malignancy risk, growth impairment (particularly in children), and potential long-term endocrine/organ damage. Our study presents a novel conditioning regimen for SAA patients undergoing haplo-HSCT, uniquely omitting both ATG and TBI/BU. This strategy aims to eliminate the immediate risks (e.g., allergy, infection) and long-term complications linked to ATG, while simultaneously avoiding the established long-term toxicities of TBI and BU, thereby prioritizing treatment safety without compromising efficacy. Eleven patients with SAA who underwent Flu/Cy plus post-transplantation cyclophosphamide (PTCy) haploidentical transplantation between January 2015 and April 2025 from the First Affiliated Hospital of Chongqing Medical University were enrolled. A suitable donor was defined as an available human leukocyte antigen (HLA) haploidentical relative of the patient, and the prioritization for selecting the donor-recipient relationship adheres to the order of children, siblings, father, mother, or collateral relatives [3, 7]. The presence of donor-specific antibodies (DSA) > 2000 via Luminex liquid chip technology was an exclusion criterion. This study has been approved by the Medical Ethics Committee of The First Affiliated Hospital of Chongqing Medical University. The last follow-up was conducted on April 2, 2025. All patients with SAA who underwent haplo-HSCT received a Flu/Cy base conditioning regimen, combined with a PTCy for GVHD prophylaxis. Fludarabine was administered intravenously at 30 mg/m2/day from day −6 to day −1, and cyclophosphamide was administered intravenously at 600 mg/m2/day over the same period. GVHD prophylaxis included PTCy (cyclophosphamide 50 mg/kg/day IV on day +3 and day +4), followed by cyclosporine A (CsA 3.0 mg/kg/day IV starting on day +5, with target trough levels between 150 and 250 ng/mL) or tacrolimus (Tac 0.05 mg/kg twice daily, adjusted to achieve trough level between 5 and 15 ng/mL), mycophenolate mofetil (MMF 0.5–1.0 g/day starting on day +5 until day +30) [8]. Since 2020, short-course methotrexate (MTX 15 mg/m2 IV on day +1 and 10 mg/m2 on days +3, +6, and +11) has been adopted. In the event of significant oral mucositis, the MTX dose scheduled for day +11 was omitted. In patients without evidence of GVHD, CsA or Tac was gradually tapered and discontinued after 1 year. Acyclovir, trimethoprim-sulfamethoxazole, and antifungal agents were administered for the prophylaxis of viral infections, Pneumocystis jirovecii pneumonia, and invasive fungal infections. The primary endpoint was overall survival (OS). The secondary endpoints included failure free survival (FFS), GVHD-free/relapse-free survival (GRFS), and engraftment. OS was defined as the time from the date of HSCT to death or the last follow-up. Death, graft failure, and relapse were regarded as treatment failure. FFS was defined as survival without treatment failure. GRFS was defined as survival without graft failure and grade III-IV aGVHD or extensive cGVHD. The clinical characteristics and treatment profiles of these 11 patients are summarized in Table 1. The median age of the patients was 28 years (range, 12–53 years), and that of the donors was 32 years (range, 21–60 years). Eight patients received a combination of bone marrow hematopoietic stem cells (BMSCs) and peripheral blood hematopoietic stem cells (PBSCs), whereas three received PBSCs alone. The infused CD34+ cell dose was (9.50 ± 4.40) × 106/kg, and the mononuclear cell (MNC) dose was (19.18 ± 6.18) × 108/kg. ABO match was observed in six cases, whereas mismatch occurred in five. All 11 patients achieved successful engraftment. The median time to neutrophil engraftment was 17 days (range: 15–21 days), whereas the median time to platelet engraftment was 14 days (range: 12–35 days). Notably, three patients experienced secondary PGF characterized by delayed platelet engraftment. Two developed bacterial bloodstream infections during transplantation, whereas one had cytomegalovirus (CMV) reactivation. All ultimately achieved successful platelet engraftment after anti-infective therapy. Three patients developed aGVHD, all limited to grade I skin involvement; no cases of grade III-IV aGVHD were observed. Chronic GVHD (cGVHD) developed in two patients—one with mild disease and the other with moderate lung involvement—both managed in the outpatient setting. CMV infection occurred in three patients, including one case of CMV disease. Epstein–Barr virus (EBV) infection was observed in two patients. All infections were effectively controlled. No post-transplant lymphoproliferative disorder (PTLD) or thrombotic microangiopathy (TMA) was reported. At the last follow-up (median, 4.3 years), 10 of 11 (90.9%) patients were alive. Both the one-year OS and one-year FFS were 90.9% (95% CI, 75.4%–100%) (Figure 1). Notably, the one-year GRFS was also 90.9% (95% CI, 75.4%–100%). One of the patients died following transplantation. This patient underwent a haploidentical transplantation from his 60-year-old father (7/12 HLA match) with BMSC and PBSC grafts. No cardiac, hepatic, or renal toxicity was observed during transplantation. Both neutrophil and platelet engrafted at +17 days. However, the patient subsequently developed severe pneumonia caused by bacterial and Aspergillus infections, along with CMV infection that progressed to CMV disease, and ultimately died. In recent years, various conditioning regimens have been optimized to enhance engraftment and improve OS as well as GRFS in patients with AA [9-11]. The combination of PTCy, calcineurin inhibitor (CNI), and MTX in the regimen was associated with high rates of neutrophil and platelet engraftment, low GVHD incidence, and favorable survival outcomes in patients with SAA and transfusion-dependent thalassemia [12, 13, 11]. However, these studies typically added busulfan (BU), with or without ATG, in contrast to the BU-, ATG-, and TBI-free conditioning regimen evaluated in our cohort. Our approach employs hyperfractionated cyclophosphamide to minimize non-hematological toxicity, whereas fludarabine provides the essential immunosuppressive effect required for sustained engraftment. PTCy effectively reduces the incidence of GVHD and associated mortality by eliminating antigen-exposed proliferating T cells and inducing immune tolerance [14, 15]. To strengthen immune tolerance without additional toxicity from ATG or TBI, we empirically introduced a short-course MTX [16] as an adjustment for GVHD prophylaxis in subsequent patients. This study investigated a haploidentical HSCT platform for SAA using Flu/Cy conditioning regimen, incorporating PTCy, MTX, CNI, and MMF as GVHD prophylaxis. The regimen demonstrated favorable engraftment, low GVHD incidence, and encouraging clinical outcomes, suggesting potential clinical utility, particularly in resource-limited settings. Given the limited sample size, prospective multicenter studies are warranted to further validate its feasibility and clinical significance. The authors have nothing to report. The authors have nothing to report. The data that support the findings of this study are available from the correspondingauthor upon reasonable request.