A multi-center clinical trial of allogeneic hematopoietic stem cell transplantation in transfusion-dependent thalassemia

Domain: chemistry
Published: 2026-02-23
Editorial outcome: minor_revision

Abstract
Allogeneic stem cell transplantation (allo-HSCT) has recently been approved as standard therapy for transfusion-dependent thalassemia (TDT) but remains limited to the use of HLA-matched sibling donors (MSDs), due to a lack of large-scale prospective studies evaluating the use of grafts from alternative donors. Here, we report the results of a non-randomised, interventional, phase 4 clinical trial evaluating allo-HSCT from alternative donors for the treatment of TDT. A total of 823 patients with TDT were transplanted with grafts from MSDs (n=331) or alternative donors, including matched unrelated donors (MUDs; n=352) and haploidentical related donors (Haplos; n=140). Conditioning was with busulfan, cyclophosphamide, fludarabine and anti-thymocyte globulin. Graft-versus-host disease (GvHD) prophylaxis was cyclosporine, methotrexate (MTX) and mycophenolate mofetil (MMF) for recipients of MSDs and tacrolimus, MTX, MMF for others. The primary endpoints were 2-year overall survival (OS) and event-free survival (EFS). Two-year OS for MSDs, MUDs, Haplos was 97.2% (95% CI, 95.4-99.0), 93.1% (90.5-95.9) and 95.4% (91.9-99.1); EFS was 97.2% (95.4-99.0), 92.9% (90.1-95.7) and 94.7% (90.9-98.6); GvHD-free, relapse-free survival (GRFS) was 91.4% (88.4-94.6), 77.0% (72.6-82.7) and 75.6% (68.5-83.4) respectively. Two-year OS and EFS for MUDs were lower than those for MSDs (both P<0.05) and were not significantly different with those for Haplos (both P>0.05). Transplant-related mortality was 4.4% (3.0-5.9) and graft failure rate was 0.5%. The incidence of grades 2-4 acute GvHD and moderate-severe chronic GvHD from alternative donors were higher than those from MSDs (28.9% [24.7-33.2] vs 7.5% [4.9-10.7], P<0.001; 12.3% [9.2-15.7] vs 5.0% [2.9-7.9], P<0.01). In summary, these findings may help expand the donor pool for patients with TDT lacking MSDs (ClinicalTrials.gov: NCT04009525).

Introduction
The hemoglobinopathies thalassemia and sickle cell disease (SCD) are common genetic diseases. Approximately 300,000 SCD infants and 40,000 β-thalassemia infants are born annually and these variants consume huge health-care resources, related to life-long iron chelation and blood transfusions. Over 80% of these patients live in low or middle-income countries, and less than 15% of patients with transfusion-dependent thalassemia (TDT) live beyond 35 years due to inadequate treatments. Thus, curative strategies, i.e., allogeneic hemopoietic stem cell transplantation (allo-HSCT) and/or gene therapy are needed.

Allo-HSCT with HLA-matched sibling donors (MSDs) has been standard therapy for TDT patients, event-free survival (EFS) reaching 80–90%. Unfortunately, MSDs could be found for only 25–30% of TDT patients. Alternative donors (matched unrelated donors [MUDs] and haploidentical related donors [Haplos]) have been explored, but allo-HSCT with alternative donors is characterized by high rates of graft failure (GF), and graft-versus-host disease (GvHD), yielding inferior overall survival (OS) and EFS compared with MSDs. Although alternative donor outcomes have been comparable with MSDs in hematological malignancies, their use for TDT is still investigational, and a lack of large-scale prospective studies prevents the application of alternative donor allo-HSCT for TDT.

We previously reported a 3-year EFS of 97% after allo-HSCT for 184 TDT patients with MSDs, using the protocol GX-07-TM. With support from the Chinese Red Cross and the Guangxi local government, we launched the project Severe Thalassemia Patients Treatment Plan in 2019, aiming to promote allo-HSCT for TDT and transplant patients within three years. We designed this prospective, multi-center trial to confirm our previous results and to assess outcomes using alternative donors.

Results
Patients
Between July 5, 2019, and July 31, 2023, 841 patients from 16 Chinese centers were screened. Eighteen patients met the exit criteria, and 823 patients were eligible for the final analysis, including 787 with β-TDT and 36 with α-TDT. Among them, 331 received transplants from MSDs and 492 from alternative donors, comprising 352 MUDs and 140 Haplos. Median follow-up after transplantation was 23 months (IQR 13–36, range 3–52). The last follow-up was October 31, 2023.

Engraftment and graft failure
The median time to neutrophil engraftment was 12 days (IQR 11–14), 12 days (IQR 11–13) and 12 days (IQR 11–14) in MSDs, MUDs and Haplos, respectively. The median time to platelet engraftment was 14 days (IQR 12–20), 12 days (IQR 11–15) and 14 days (IQR 12–17) in the three type donors. Neutrophil engraftment did not differ between MSDs, MUDs and Haplos. Compared with MSDs and Haplos, the median time to platelet engraftment was less in MUDs (p<0.01). Primary engraftment was achieved in 819 patients, 4 patients experienced GF (GF was 0.5%; 1 primary GF, 3 secondary GF, 2 died and 2 became transfusion dependent).

Primary endpoints
The primary endpoint was achieved. The observed 2-year OS was 95.2%, significantly exceeding the predefined reference value of 91.5% (P<0.001). Two-year OS and EFS were 95.2% (95% confidence interval (CI), 93.7–96.7), and 94.9% (93.4–96.5) for all patients, with both 97.2% (95.4–99.0) for MSDs, 93.1% (90.5–95.9) and 92.9% (90.1–95.7) for MUDs, 95.4% (91.9–99.1) and 94.7% (90.9–98.6) for Haplos. The 2-year GvHD-free, relapse-free survival (GRFS) was 82.7% (80.0–85.4) for all patients, 91.4% (88.4–94.6) for MSDs, and 77.0% (72.6–81.7) for MUDs and 75.6% (68.5–83.4) for Haplos. In a post hoc analysis, 800 patients with MUDs and Haplos had slightly lower OS and EFS, and higher transplant-related mortality (TRM) compared with MSDs recipients. There was no center-dependent effect observed for these results.

In univariate analysis, OS and EFS were significantly lower for MUDs compared to MSDs (hazard ratio (HR) 2.74, 95% CI 1.25–5.99; P=0.012 and HR 2.90, 95% CI 1.33–6.32; P=0.007, respectively), for splenectomy group compared to non-splenectomy (3.35, 1.66–6.76; P=0.001 and 3.12, 1.56–6.24; P=0.001), for patients older than 15 years (4.86, 1.91–12.36; P=0.001 and 4.48, 1.79–11.23; P=0.001), and for patients with grades 2–4 and 3–4 acute GvHD (aGvHD) compared to those without aGvHD (5.59, 2.91–10.72; P<0.001 and 5.08, 2.69–9.59; P<0.001 for 2–4 aGvHD, respectively; 10.39, 5.39–20.04; P<0.001 and 9.59, 5.00–118.16; P<0.001 for 3–4 aGvHD, respectively). OS and EFS were significantly lower in patients with infections (13.30, 1.73–101.93; P=0.013 and 7.68, 1.70–34.68; P=0.008, respectively), cytomegalovirus (CMV) reactivation (2.45, 1.27–4.74; P=0.008 and 2.54, 1.32–4.87; P=0.005, respectively) and hemorrhagic cystitis (HC) (2.46, 1.29–4.69; P=0.006 and 2.85, 1.52–5.35; P=0.001, respectively), compared to unaffected patients. Additionally, EFS was also lower in patients with veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) (3.62, 1.29–10.18; P=0.015) compared to those without VOD/SOS. The total number of CD34+ cells infused showed a protective effect; thus, OS were improved with higher CD34+ cell doses infused (0.89, 0.82–0.98; P=0.013). Factors that have a negative impact on OS and EFS are found in Supplementary Table 3. Post hoc multivariable analysis of risk factors for OS and EFS showed that grades 2–4 aGvHD (HR 5.24, 95% CI 2.70–10.20 and 4.56, 2.40–8.69), age >15 years (3.07, 1.06–8.88 and 2.88, 1.01–8.23), and previous splenectomy (3.36, 1.49–7.54 and 3.15, 1.41–7.02) were significant negative predictors for OS and EFS compared to patients without these risk factors.

Secondary endpoints
The cumulative incidence of grades 2–4, and 3–4 aGvHD was 7.5% (95% CI, 4.9–10.7) and 1.9% (0.8–3.9) in patients for MSDs, 27.6% (22.8–32.6) and 14.6% (10.8–18.9) for MUDs and 32.3% (24.2–40.6) and 14.7% (8.9–21.9) for Haplos. Two-year cumulative incidence of chronic GvHD (cGvHD) and moderate-severe cGvHD was 8.5% (5.7–12.0) and 5.0% (2.9–7.9) for MSDs, 19.1% (14.8–23.9) and 11.2% (7.9–15.2) for MUDs, and 22.6% (15.2–30.8) and 15.1% (8.9–22.9) for Haplos. Among the 668 patients with follow-up longer than one year, cGvHD occurred in 8.9% (5.9–12.5) of MSDs and 18.8% (14.3–23.8) for MUDs and 23.1% (15.5–31.7) for Haplos; moderate–severe cGvHD occurred in 5.4% (3.2–8.5) for MSDs, 10.7% (7.3–14.8) for MUDs and 14.7% (8.4–22.7) for Haplos, respectively. These analyses show a consistent pattern of higher cGvHD rates after alternative donors (MUDs and Haplos) than after MSDs. Of the 112 cases of cGvHD were recorded, 56% had prior aGvHD. The organ scores of cGvHD were shown in Supplementary Table 5.

In univariable analysis, MUDs (HR 4.09, 95% CI 2.62–6.37; HR 7.00, 95% CI 3.15–15.60) and Haplos (5.03, 3.06–8.27; 7.35, 3.05–17.70) increased risk for grades 2–4 and 3–4 aGvHD compared with MSDs. For moderate-severe cGvHD, the risks were significantly higher for patients with MUDs (2.21, 1.24–3.94) and Haplos (2.58, 1.30–5.10), compared to MSDs. History of aGvHD markedly influenced cGvHD risk (4.42, 2.74–7.14 for grades 2–4 aGvHD; 8.03, 4.74–13.60 for grades 3–4 aGvHD). The proportion of patients with moderate-severe cGvHD at 2 years is shown in Table 2.

Post hoc multivariable analysis revealed that the risk of grades 2–4 and 3–4 aGvHD was significantly higher in patients with MUDs (HR 3.38, 95% CI 1.74–6.58 for grades 2–4; 7.37, 2.12–25.56 for grades 3–4) and Haplos (4.89, 2.88–8.30 for grades 2–4; 7.59, 3.07–18.79 for grades 3–4), compared to MSDs. Additionally, grades 2–4 aGvHD were associated with an increased risk of developing moderate-severe cGvHD (3.80, 2.29–6.32).

Safety
Infectious complications exhibited notable variations among donor types. Haplo-patients had higher incidences of pneumonia (48.6%), CMV (52.1%) and Epstein-Barr virus (EBV) (32.9%) reactivation compared with those having MSDs and MUDs. Moreover, the rates of bacterial and fungal infections, septicemia, and posterior reversible encephalopathy syndrome (PRES) were similar between groups. VOD/SOS, diagnosed in 27 patients (3.3%), without difference between the subgroups. Hemorrhagic cystitis was observed in 190 (23.0%) patients, 48 (14.5%) were MSDs, 99 (28.1%) MUDs, and 43 (30.7%) Haplos, respectively, for all SAEs see Supplementary Table 8.

The overall 2-year TRM was 4.4% (95% CI, 3.0–5.9) for all patients, 2.5% (0.8–4.2) for MSDs, 6.3% (3.7–9.9) for MUDs, and 4.6% (0.9–8.1%) for Haplos. The main causes of death in 38 cases were infections (two patients died from the COVID-19 infection), GvHD and intracranial hemorrhage. Fifty-four (6.6%) patients developed mixed chimerism (MC) at a median of 133 (range 15–574) days. The 12.4% MC in patients with MSDs was higher than 3.1% in MUDs and 1.4% in Haplos. There was no significant difference in outcome for patients ages 2–7 and 7–15 years, but patients over 15 years exhibited lower two-year OS, EFS, and higher TRM than the younger cohort(s). After adjustment for age and hepatomegaly (>5cm), the 92 splenectomised patients still had inferior OS, EFS, and increased TRM, although they had faster neutrophil and platelet engraftment compared with non-splenectomised patients. The infection-related mortality in splenectomised group was higher than that in non-splenectomised patients (7.6% vs 2.5%, P=0.010).

Methods
Studydesignandparticipants
Thiswasaprospective,multicenterclinicaltrialatsixteenhospitalsinChina(SupplementaryTable1).Inclusioncriteriawere:(1)diagnosisofTDTbyhemoglobinelectrophoresis,DNAanalysis,andbloodtransfusiondependence;(2)age2–20years;(3)availabilityofmatchedsibling-oralternativedonor;and(4)performancestatus(0–2,Karnofskyscore>70%,orLanskyscore≥60%forchildren).Exclusioncriteria:(1)cardialejectionfraction<50%,severecardiovascular/pulmonarydisorders(PaO2≤60mmHgatrest,roomair);(2)liverdysfunction(ALTorAST≥4timestheuppernormallimit[ULN]ofinstitutionalreference);(3)creatinine≥2timestheULNorcreatinineclearance<30mL/min;(4)uncontrolledactiveinfection;(5)CMVorEBV>400copies/mLinbloodbyquantitativePCR;(6)hepatitisCor-Bpositivity;(7)HIVpositivity;(8)previoustransplant;and(9)anyconditiondeemedbytheinvestigatortomakethepatientunsuitablefortrial.

Themedicalethicscommitteeateachhospitalapprovedthetrial,whichwasconductedaccordingtotheDeclarationofHelsinki.Allpatientsanddonorsprovidedwritteninformedconsent/assent.Consentwasobtainedfromtheparentsorlegalguardiansofallparticipatingchildrenpriortoenrollment.Foradolescentscapableofprovidingassent,age-appropriateinformationwasprovided,andtheirassentwasalsoobtainedwheneverapplicable.Theethicscommitteereferencenumberwas[MedicineEthics]2019No.(009).ThestudyprotocolandstatisticalanalysisplanareincludedinSupplementaryData1.

Procedures
PatientswereassignedtogroupswithMSDs(10/10or9/10atHLA-A,-B,-C,-DR,-DQ)andalternativedonors(MUDs[10/10or9/10matches]orHaplos)basedondonoravailability.MSDswerepreferred,followedbyMUDs.Intheabsenceofthese,Haploswereconsidered.

Allpatientsreceivedthesameconditioningregimen:IVbusulfan(BUSULFEX™,1mg/kgi.v.4×dailyondays−9to−6),cyclophosphamide(50mg/kgi.v.oncedaily,days−5to−2),fludarabine(50mg/m²i.v.oncedailyfromdays−12to−10),andanti-thymocyteglobulin(Thymoglobulin™,2.5mg/kgi.v.oncedaily,days−4to−1)(SupplementaryFig.4).Everyonereceived20–30mg/kghydroxyureaorallydailyfor2–3monthsbeforetransplantation.PatientswithMSDsandHaplosreceivedG-BM&PBSCs,orBMandcordblood(CB)grafts.PatientswithMUDsreceivedG-CSFmobilizedPBSCs.ForGvHDprophylaxis,patientswithMSDsreceivedCsA,MTX,andMMF.PatientswithalternativedonorsreceivedTac,0.03mg/kg/day),MTX,andMMF(250mg/dayfor90days).

Supportivecareroutinesareoutlinedinapublishedprotocol.Laboratorytests,andclinicalassessments,includingvitalsigns,wereperformedbeforetransplantation,every1–2dayswhilein-patient,afterdischargeweeklyforthefirst3monthspost-transplantation,biweeklyfromthefourthtosixthmonths,andthenevery4weeksuntilthestudywascompleted.

Outcomes
Primaryendpoints were 2-year OS and EFS, OS defined as time from transplant to death from any cause, or censored at time of last follow-up. EFS was defined as the time from transplantation to death from any cause, graft failure, or recurrence of transfusion dependence. The FAS included all patients who underwent transplantation and received at least one dose of the conditioning regimen, GF or recurrent TDT, similarly censored for patients alive. Secondary endpoints were aGvHD and cGvHD, neutrophil and platelet recovery, engraftment of donor cells, TRM, GF incidence, incidence of serious infections, reactivations of CMV, adenovirus, EBV, and MC. GRFS at 2 years was defined as free events including grades 3–4 aGvHD, cGvHD requiring systemic treatment, thalassemia relapse, or death from any cause. GvHD was classified by the Glucksberg and NIH classifications. Neutrophil and Platelet engraftment, TRM, GF, MC were defined as in prior studies. The diagnosis of VOD/SOS was according to McDonald. The number of adverse events and organism group (bacteria, virus, or fungus) were provided for infections.

Statisticalanalysis
Two-year OS after allo-HSCT was ~91.5% without distinguishing between donor types according to previous reports. Assuming a 2-year OS of at least 94% to be significantly different from the null targeted value of 91.5% for a two-sided α-level of 5%, power of 90% and a dropout rate of 15%, with recruitment time of 36 months and follow-up time of 12 months, ~800 patients were required.

We summarized data using medians and interquartile ranges, or proportions, and made comparisons with Kruskal–Wallis test, Wilcoxon rank-sum test, Chi-square test or Fisher Exact test, as appropriate. For the OS, EFS, GRFS, and TRM, time-to-event rates were estimated using the Kaplan-Meier method, and hazard ratios were estimated using the Cox proportional hazards model with centers as a random-effect. For complications such as MC, VOD/SOS, HC, PRES and infections, the odds ratio was estimated using the Logistic model. In the presence of competing risks, the Fine and Gray model was used for GvHD. The risk factors included in the multivariate analysis were predetermined according to their importance and presumed relevance to outcome. SPSS v23.0 and R v4.2.1 were used for analysis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I commend the authors for a large prospective transplant trial in TDT. There are big numbers and there is no centre effect in\noutcomes and these are important. Transplanting patients at a young age is also important\nThere are many patients, including those receiving MSD, MUD and haplo-identical donors\nThe TRM is low\nIt is a well written manuscript. The separation of methods (including references) from the rest of the manuscript was\nconfusing to me\nMajor comments\n1) The conditioning regimen is Flu Cy ATG and Busufan. This must appear in the abstract and not simply reference the trials\nacronym.\na) this is descrobed as intensive conditioning- the authors should clarify that this is intensively immunosuppressive but not\nmyelosuppresive\nb) the authors should clarify if busulfan levels were used and what the target pK was if so\nc) the authors should make clear that in the absence of pK the low incidence of VOD etc might simply reflect low exposure to\nbusulfan\n2) There is low graft failure. The authors comment that graft failure is due to the immune suppression and that that intensive\nT-cell immune suppression of the recipient facilitates engraftment. Achieving engraftment in transplant is facilitated by\nintensive myeloblation, and by the immune action of donor cells. Part of the engraftment is therefore facilitated by aGVHD\n(27 and 32% in the MUD and haplo) and chronic GVHD (19 and 22% respectively). Of course patients with GVHD will have\ndonor cell engraftment.\nLate \"graft failure\" in TDT is usually autologous reconstitution and reflects inadequate myelosuppression rather than\nrejection as such, and retransplant can be performed with the same donor, and with success. This further argues that this is\nnot a T-cell phenomenon, or not simply.\n3) The acute and the chronic GVHD rates are high and this must be an important message of the article and must appear in\nthe abstract. Gvhd free, DFS is the most important endpoint, and in Non malignant diseases like TDT then this must be the\ngoal of transplant\na) The GVHD prophylaxis for MSD in TDT including T-cell depleting serotherapy is standard and should be stated as such\nb) GVHD will be risked by usng PBSC etc in the MUD\nc) The GVHD prophylaxis in the haplosetting is far from standard and most institutions would use PTCy or αβ-T cell\ndepletion to achieve engraftment without GVHD, The authors should address why they used a similar GVHD prophylaxis in\nthe mismatched setting, and what would be their recommendations going forward\nd) what is the quality of life in those with cGVHD. In TDT there is medical alternative therapy and perhaps this might be\npreferred and have a higher quality of life than those with cGVHD", "reviewer_2": "I read with interest this paper reporting results from a multicentre study. It is a well written exhaustive report. Obviously the\nfact that it is an uncontrolled trial marks the limits and this should be acknowledged as a limitation in the Discussion\nparagraph.\nI have no major issues but there are some inconsistencies between the protocol and the paper. For example events for EFS\nare defined in a different way and the FAS is defined twice in the protocol with different definitions. Authors should solve\nthese inconsistencies.\nMoreover sample size was determined to assess a difference between 2-years overall survival rate and the fixed value of\n91.5% but nothing is reported about this statistical testing that will be quite significant, I guess. Please start the Result\nparagraph reporting if the primary endpoint was either met or not.\nIn figure 1 the total number of alternative donor is 492.\nDiana Giannarelli", "reviewer_3": "Liu et al provide the results of a prospective multi-center trial of allogeneic stem cell transplantation for thalassemia from\nChina. The study is distinguished by its large sample size, uniform treatment protocol, and comparison of multiple donor\nsources for stem cells. There has been a persistent effort to expand the availability of alternative-donor SCT to patients\nlacking matched-sibling donors in countries with high prevalence of thalassemia major, particularly Thailand, India, and\nChina.\nThe barriers to the adoption of alternative donors for thalassemia are transplant-related mortality, graft failure, and cGVHD.\nWith an observed EFS 93-97%, the study team has easily overcome the first two challenges. Among recipients of alternative\ndonors, cGVHD was observed in approximately 20%, with moderate to severe cGVHD in 11-15% of recipients.\nComments:\n1. The difference in EFS between the MSD and MUD/HID groups is small, but there is a notable gap in GVHD and relapse-\nfree survival (GRFS, 91% vs 77%). To appreciate the significance of this observation, please describe the health status of\npatients with cGVHD at the last follow-up.\na. Under ‘Secondary Endpoints’ on cGVHD, please provide information on functional status and immunosuppression.\nb. The observed incidence of cGVHD may underestimate the true incidence due to the short post-transplant period for some\npatients. Can the authors provide the incidence of cGVHD in patients with follow-up greater than 1 year?\n2. The use of post-transplant iron chelation in patients following engraftment is uncommon, as phlebotomy is generally\npreferred to treat iron overload. Please add the rationale for recommending chelation therapy.\n3. The study protocol uses the same intensive conditioning for MSD and alternative donors. Could some patients in the MSD\ngroup be treated with less immunosuppressive regimens?\n4. The impact of splenectomy on post-transplant outcomes is a significant observation. Please discuss whether infection-\nassociated mortality differed between splenectomized and non-splenectomized groups.\n5. Please provide genotypes of patients with alpha thalassemia in Table 1.\na. The anemia in many forms of hemoglobin H disease responds to splenectomy. Was this considered as an option before\ntransplant, where applicable?\nb. The discussion (Line 294, page 11) on splenectomy prior to transplant should distinguish between genotypes. I agree with\navoiding splenectomy in beta thalassemia syndromes. However, splenectomy is an important option for patients with alpha-\nthalassemia who require regular transfusions."}, "author_response": "Dear Reviewers,\nWe are grateful for the opportunity to submit the revised version of our manuscript, entitled “A\nMulti-center Clinical Trial of Allogeneic Hematopoietic Stem Cell Transplantation in Transfusion\nDependent Thalassemia”, along with a detailed point-by-point response to the reviewers’ comments.\nFollowing the reviewers’ constructive and insightful feedback, we have undertaken substantial\nrevisions to strengthen the manuscript. Key improvements include:\n1. Clarification and expansion of the conditioning regimen, including explicit description in the\nAbstract and a more detailed explanation of the immunosuppressive versus myeloablative\ncomponents.\n2. Addition of busulfan pharmacokinetic context, incorporating evidence from Chinese HSCT\ncohorts and acknowledging the lack of routine therapeutic drug monitoring in most centers.\n3. A comprehensive revision of GvHD-related sections, including incidence, prophylaxis\nrationale, comparison across donor types, and long-term health status of patients with chronic\nGvHD.\n4. Enhanced analyses addressing reviewer concerns, such as follow-up duration, chronic GvHD\nincidence after one year, rationale for post-transplant iron chelation, impact of splenectomy on\ninfection-related mortality, and α-thalassemia specific considerations.\n5. Clarification of methodology and endpoints, including alignment of definitions for EFS/FAS\nand explicit confirmation that the primary endpoint was met.\n6. Strengthened discussion of limitations, including non-randomized design, short follow-up for\nlate effects, and generalizability across ethnic groups.\n7. Supplemented the required materials, including reporting summary and source data.\nWe believe these revisions substantially improve the scientific rigor, clarity, and translational\nrelevance of the manuscript. Importantly, our prospective multicenter dataset-the largest to date in\ntransfusion-dependent thalassemia-provides high-quality real-world evidence supporting safe and\neffective allogeneic transplantation across donor types, particularly in resource-limited regions.\nWe thank the reviewers for their careful evaluation and insightful recommendations, which have\nsignificantly improved the quality of our work. We hope that the revised manuscript now meets the\nhigh standards of Nature Communications.\nWe remain at your disposal for any further information.\nSincerely,\nYongrong Lai, MD, PhD\nDepartment of Hematology,\nthe First Affiliated Hospital of Guangxi Medical University, Guangxi, China\nPoint-by-Point Response to the Reviewers’ Comments\nReviewer #1 (Remarks to the Author):\nComment 1\nThe conditioning regimen is Flu Cy ATG and Busufan. This must appear in the abstract and not\nsimply reference the trials acronym.\nResponse:\nWe appreciate the reviewer’s suggestion. We have now revised the Abstract to explicitly specify the\nconditioning regimen rather than referring only to the trial acronym. The regimen consisted of\nbusulfan (Bu), cyclophosphamide (Cy), fludarabine (Flu) and anti-thymocyte globulin (ATG).\nRevised text (Abstract):\nConditioning was with busulfan, cyclophosphamide, fludarabine and anti-thymocyte globulin.\nGraft-versus-host disease (GvHD) prophylaxis was cyclosporine, methotrexate (MTX) and\nmycophenolate mofetil (MMF) for recipients of MSDs and tacrolimus, MTX, MMF for others.\na) this is described as intensive conditioning- the authors should clarify that this is intensively\nimmunosuppressive but not myelosuppressive.\nResponse:\nWe are grateful for the thoughtful comments provided by the reviewers. According to the Transplant\nConditioning Intensity (TCI) scale, our regimen scored 4.5, indicating that it is a highly\nmyeloablative regimen. This regimen also exhibits a strong immunosuppressive effect,\nincorporating fludarabine, cyclophosphamide, and antithymocyte globulin (ATG, 10 mg/kg).\n(Bacigalupo et al., Bone Marrow Transplant, 2009; Giralt S, et al., Biol Blood Marrow Transplant,\n2009；Gyurkocza B, et al. Blood. 2014; Mulas O, et al., J Clin Med. 2022.). We have clarified this\npoint in both the Methods and Discussion sections.\nReference:\n1. Bacigalupo A, et al.Defining the intensity of conditioning regimens: working definitions. Biol\nBlood Marrow Transplant. 2009 Dec;15(12):1628-33.\n2. Giralt S, et al. Reduced-intensity conditioning regimen workshop: defining the dose\nspectrum.Report of a workshop convened by the center for international blood and marrow\ntransplant research. Biol Blood Marrow Transplant. 2009 Mar;15(3):367-9.\n3. Gyurkocza B, et al. Conditioning regimens for hematopoietic cell transplantation: one size does\nnot fit all. Blood. 2014 Jul 17;124(3):344-53.\n4. Mulas O, et al. Conditioning Regimens in Patients with β-Thalassemia Who Underwent\nHematopoietic Stem Cell Transplantation: A Scoping Review. J Clin Med. 2022 Feb\n9;11(4):907.\nRevised text ( Discussion)\nThe GX-07-TM protocol is an intensive myeloablative/immunosuppressive regimen, administering\nbusulfan (Bu), cyclophosphamide (Cy), fludarabine (Flu) and anti-thymocyte globulin (ATG).\nb) the authors should clarify if busulfan levels were used and what the target pK was if so\nResponse:\nWe appreciate the reviewer’s insightful comment. Busulfan PK was not routinely monitored due to\nmulticenter constraints; however, dosing followed body surface area-based guidelines, consistent\nwith established pediatric protocols. In some thalassemia patients transplants conducted at our\ncenter, Bu was administered intravenously at 1.0 mg/kg per dose, four times daily. PK assessment\nconsistently showed busulfan plasma concentrations within the range required for effective\nmyeloablation (target AUC ≈ 900–1,500 µM·min). Second, Bu PK have been evaluated in several\nstudies involving Chinese patients. A pharmacokinetic analysis conducted at the Blood and Marrow\nTransplantation Center of Ruijin Hospital enrolled 45 patients with hematologic malignancies\nundergoing allogeneic HSCT. In this study, patients received intravenous Bu at a dose of 1.6\nmg/kg. The findings showed that intravenous Bu exposure in Chinese patients was generally lower\nthan that reported in Caucasian populations, contributing to the Bu plasma concentrations in our\ncohort.\nTable. Bu PK from some thalassemia patients transplants was conducted at our center.\nRevised text (Discussion)\nThe other limitation is that we did not monitor the intravenous Bu levels for most patients because it\nwas unavailable at most Chinese HSCT centers, although the Bu pharmacokinetic data in small\nnumber TDT patients showed plasma concentrations within the effective myeloablative range.\nPrevious studies indicate that intravenous Bu exposure is lower in Chinese than in Caucasian\npatients33, suggesting ethnic metabolic differences. Thus, the GX-07-TM protocol may require dose\nadjustment for different ethnic populations to ensure optimal exposure and safety. Despite these\nlimitations, the prospective multi-center design and standardized treatment approach provide\nvaluable real-world evidence on allo-HSCT outcomes in TDT across multiple donor types.\nc) the authors should make clear that in the absence of pK the low incidence of VOD etc might\nsimply reflect low exposure to busulfan.\nResponse\nWe thank the reviewer for this valuable comment. The incidence of VOD/SOS in this study (3.3%)\nwas substantially lower than in our previous report (10.4%), which may be attributed to the use of\nintravenous BU and extensive clinical experience from over 1,300 thalassemia transplants at our\ncenter. Early recognition of VOD, optimized calcineurin inhibitor management, and effective\nhepatic protection strategies also contributed to this improvement. These above Bu PK assessments\nconfirmed exposure within the myeloablative range, while the low VOD incidence indicates that\nexcessive BU exposure was effectively avoided under the GX-07-TM protocol.\nRevised text (Discussion)\nThe incidence of VOD/SOS in this study (3.3%) was substantially lower than in our previous report\n(10.4%), which may be attributed to the use of intravenous Bu31 and our rich experience of\nidentifing early symoptoms of VOD/SOS, management of adverse drug reaction such as calcineurin\ninhibitors , azoles and so on.\nComment 2\nThere is low graft failure. The authors comment that graft failure is due to the immune suppression\nand that that intensive T-cell immune suppression of the recipient facilitates engraftment. Achieving\nengraftment in transplant is facilitated by intensive myeloblation, and by the immune action of\ndonor cells. Part of the engraftment is therefore facilitated by aGVHD (27 and 32% in the MUD and\nhaplo) and chronic GVHD (19 and 22% respectively). Of course patients with GVHD will have\ndonor cell engraftment.\nLate \"graft failure\" in TDT is usually autologous reconstitution and reflects inadequate\nmyelosuppression rather than rejection as such, and retransplant can be performed with the same\ndonor, and with success. This further argues that this is not a T-cell phenomenon, or not simply.\nResponse\nWe appreciate this insightful comment. We agree that engraftment reflects the combined effects of\nconditioning intensity, donor immune activity, and host suppression. While our regimen emphasized\nimmune suppression, it also provided sufficient myeloablation to prevent autologous reconstitution.\nThe low rate of late graft failure (<1%) supports the adequacy of marrow ablation. We have\nclarified that our regimen balances both immunologic and myeloablative mechanisms rather than\nrelying solely on T-cell suppression.\nRevised text (Discussion):\nIn addition, late graft failure in TDT often reflects autologous reconstitution from insufficient\nmyeloablation rather than immune rejection. Our regimen’s strong myeloablative component,\ntogether with high CD34+ doses and early chimerism-guided interventions, likely minimized this\nrisk and contributed to the exceptionally low GF rate.\nComment 3\nThe acute and the chronic GVHD rates are high and this must be an important message of the article\nand must appear in the abstract. Gvhd free, DFS is the most important endpoint, and in Non malignant diseases like TDT then this must be the\ngoal of transplant.\nResponse\nWe thank the reviewer for highlighting this important issue. We have revised the abstract to include\nthe incidence of acute and chronic GVHD and the 2-year GRFS.\nRevised text (Abstract)\nTwo-year OS, EFS and GvHD-free, relapse-free survival (GRFS) were 97.2%, 97.2% and 91.4%\nfor MSDs, 93.1%, 92.9% and 77.0% for MUDs, 95.4%, 94.7% and 75.6% for Haplos. Two-year OS\nand EFS for MUDs were lower than those for MSDs and were not significantly different with those\nfor Haplos. The transplant-related mortality was 4.4% and graft failure rate was 0.5%. The\nincidence of grades 2-4 acute GvHD and moderate-severe chronic GvHD from alternative donors\nwere higher than those from MSDs（28.9% vs 7.5%，P<0.001; 12.3% vs 5.0%, P<0.01).\na)The GVHD prophylaxis for MSD in TDT including T-cell depleting serotherapy is standard and\nshould be stated as such.\nResponse:\nWe thank the reviewer for this helpful suggestion. We agree that the GvHD prophylaxis used for\nMSD transplants in TDT—comprising a calcineurin inhibitor, methotrexate, and mycophenolate\nmofetil combined with ATG or other T-cell depletion could replace a more traditional GvHD\nprophylaxis regimen for patients with non-malignant disorders from MSDs.\nRevised text (Discussion):\nIn our opinion, the GvHD prophylaxis regimen of CsA, MTX, MMF and ATG or other T-cell\ndepleting serotherapy could replace a more traditional GvHD prophylaxis regimen for patients with\nnon-malignant disorders from MSDs.\nb) GVHD will be risked by usng PBSC etc in the MUD.\nResponse:\nWe appreciate this important comment. We agree that the use of granulocyte colony-stimulating\nfactor–mobilized PBSCs in MUD transplants is associated with an increased risk of both acute and\ncGvHD compared with bone marrow grafts. In China, however, the national donor registry provides\nonly PBSC products for unrelated donor transplants; therefore, all MUD recipients in this study\nreceived PBSC grafts in accordance with the national policy.\nReferences:\n1. Anasetti C, et al. Peripheral-blood stem cells versus bone marrow from unrelated donors. N\nEngl J Med. 2012 Oct 18;367(16):1487-96.\n2. Eapen M, et al. Peripheral blood grafts from unrelated donors are associated with increased\nacute and chronic graft-versus-host disease without improved survival. Biol Blood Marrow\nTransplant. 2007 Dec;13(12):1461-8.\nc) The GVHD prophylaxis in the haplosetting is far from standard and most institutions would use\nPTCy or αβ-T cell depletion to achieve engraftment without GVHD, The authors should address\nwhy they used a similar GVHD prophylaxis in the mismatched setting, and what would be their\nrecommendations going forward.\nResponse and Revised text (Discussion)\nWe thank the reviewer for this important comment. Optimizing GvHD prophylaxis regimens for\npatients receiving transplants from alternative donors remains essential to further improve outcomes.\nRecent studies suggest that PTCy, αβ–T cell depletion, or the addition of CD25 antibody to our\nprophylaxis protocols can promote durable engraftment while reducing the risk of GvHD.\nd) what is the quality of life in those with cGVHD. In TDT there is medical alternative therapy and\nperhaps this might be preferred and have a higher quality of life than those with cGVHD.\nResponse:\nWe thank the reviewer for this important point. Chronic GvHD is a major determinant of\nlong-term quality of life (QoL) after transplantation, particularly in nonmalignant disorders such as\nTDT. In our cohort, most cGvHD cases were mild to moderate. We have added a description of\nQoL outcomes in the Discussion section.\nRevised text (Discussion):\nWe previously reported the quality of life for TDT patients after allo-HSCT using the GX-07-TM\nprotocol. The results shown chronic GvHD was independently associated with lower health-related\nquality-of-life scores and patients more than 4 years after transplantation achieved an health-related\nquality of life equal to that of their healthy peers32.\nReviewer #2 (Remarks to the Author):\nComment 1\nI read with interest this paper reporting results from a multicentre study. It is a well written\nexhaustive report. Obviously the fact that it is an uncontrolled trial marks the limits and this should\nbe acknowledged as a limitation in the Discussion paragraph.\nResponse:\nWe thank the reviewer for this constructive comment. We fully agree that the absence of a\nrandomized control arm is an important limitation of this multicenter study. Our trial was designed\nas a pragmatic, prospective cohort study to evaluate the real-world applicability of the GX-07-TM\nregimen across diverse donor types in TDT. While the large sample size and standardized treatment\nprotocol reduce selection bias, the lack of randomization may limit direct comparisons with other\nconditioning regimens or control populations. We have now explicitly acknowledged this limitation\nin the Discussion section.\nRevised text (Discussion):\nLimitations include the non-randomized design, relatively short follow-up, which precludes full\nassessment of long-term engraftment, late complications, or secondary malignancies, though\nongoing surveillance continues.\nComment 2\nI have no major issues but there are some inconsistencies between the protocol and the paper.\nFor example events for EFS are defined in a different way and the FAS is defined twice in the\nprotocol with different definitions. Authors should solve these inconsistencies.\nResponse:\nWe thank the reviewer for pointing this out. We have carefully reviewed the protocol and\nmanuscript to ensure complete consistency in the definitions of both the EFS endpoint and the FAS.\nIn the revised version, EFS is consistently defined as the time from transplantation to the first event,\nincluding death from any cause, graft failure, or recurrence of transfusion dependence. The FAS is\nnow clearly defined as all enrolled patients who underwent transplantation and received at least one\ndose of conditioning therapy. These corrections have been harmonized across the Methods and\nResultssections to match the protocol.\nRevised text (Methods, Outcomes):\nEFS was defined as the time from transplantation to death from any cause, graft failure, or\nrecurrence of transfusion dependence. The FAS included all patients who underwent transplantation\nand received at least one dose of the conditioning regimen.\nComment 3\nMoreover sample size was determined to assess a difference between 2-years overall survival rate\nand the fixed value of 91.5% but nothing is reported about this statistical testing that will be quite\nsignificant, I guess. Please start the Result paragraph reporting if the primary endpoint was either\nmet or not.\nResponse:\nWe thank the reviewer for this important observation. The sample size of our study was calculated\nbased on detecting a 2-year OS rate significantly higher than the reference value of 91.5%, with 80%\npower and a one-sided α of 0.025. In the final analysis, the observed 2-year OS was 95.2% (95% CI,\n94.0–97.0; P<0.001 vs. 91.5%), confirming that the primary endpoint was met with statistical\nsignificance. We have now included this explicit statement at the beginning of the Results section.\nRevised text (Results, Primary endpoints):\nThe primary endpoint was achieved. The observed 2-year OS was 95.2%, significantly exceeding\nthe predefined reference value of 91.5% (P<0.001).\nComment 4\nIn figure 1 the total number of alternative donor is 492.\nResponse:\nWe thank the reviewer for noting this point. The number of alternative donor transplants was\ncorrectly 492, comprising 352 MUDs and 140 Haplos transplants. We have clarified this explicitly\nin both the Results section and the Figure 1 legend to ensure consistency across the manuscript.\nRevised text (Results, Patients):\nEighteen patients met the exit criteria, and 823 patients were eligible for the final analysis, including\n787 with β-TDT and 36 with α-TDT. Among them, 331 received transplants from MSDs and 492\nfrom alternative donors, comprising 352 MUDs and 140 Haplos.\nRevised figure legend (Figure 1):\nPatient distribution by donor type: MSDs (n=331), MUDs (n=352), and Haplos (n=140).\nReviewer #3 (Remarks to the Author):\nComment 1\nThe difference in EFS between the MSD and MUD/HID groups is small, but there is a notable gap\nin GVHD and relapse-free survival (GRFS, 91% vs 77%). To appreciate the significance of this\nobservation, please describe the health status of patients with cGVHD at the last follow-up.\nResponse:\nWe thank the reviewer for this valuable comment. In our study, the GRFS endpoint included relapse,\ndeath, and clinically significant GvHD. Although the incidence of GvHD remained higher in the\nalternative donor group, leading to a lower GRFS compared with MSDs, GvHD-related\ncomplications were effectively managed. Consequently, relapse and mortality rates were low,\nresulting in favorable EFS outcomes. Among patients who experienced cGvHD, no relapse was\nobserved by the last follow-up; two deaths occurred—one due to bronchiolitis obliterans with\npulmonary infection and one due to an accident.\na. Under ‘Secondary Endpoints’ on cGVHD, please provide information on functional status and\nimmunosuppression.\nResponse:\nWe thank the reviewer for this suggestion. We have now added information on the functional\nrecovery and immunosuppressive status of patients with cGvHD in Supplementary Table 4. The\norgan scores of chronic GvHD showed the information on functional organs status in from 112\npatients with cGvHD.\nRevised text (Results):\nThe organ scores of cGvHD were shown in Supplementary Table 4.\nb. The observed incidence of cGVHD may underestimate the true incidence due to the short\npost-transplant period for some patients. Can the authors provide the incidence of cGVHD in\npatients with follow-up greater than 1 year?\nResponse:\nCox regression analysis based on a competing risk model was performed, taking time variables into\naccount. Since most patients in this study had a follow-up period exceeding one year, the analysis of\ncGvHD incidence and intergroup comparisons among patients with follow-up longer than one year\nshowed no obvious difference from the overall results. The evaluation of cGvHD occurrence in the\n668 patients with follow-up longer than one year using the competing risk model is summarized in\nthe Supplementary Table 3.\nMatched Sibling Alternative Donora\nVariable Total Donor\nTotal Alternative Matched Unrelated Haploidentical\nN=668 N = 285\nDonor Donor Related Donor\ncGvHDc, n 102 27 75 51 24\nCumulative 15.2% (12.6-18.1) 8.9% (5.9-12.5) 20.0% (16.1-24.3) *** 18.8% (14.3-23.8) ** 23.1% (15.5-31.7) ***\nincidence(95%CI)\nHR (95% CI) -- Ref 2.19 (1.41-3.41) 2.06 (1.29-3.29) 2.54 (1.47-4.39)\nModerate-severe 60 17 43 29 14\ncGvHDc,n\nCumulative 9.0% (6.9-11.4) 5.4% (3.2-8.5) 11.8% (8.7-15.4) * 10.7% (7.3-14.8) * 14.7% (8.4-22.7)*\nincidence(95%CI)\nHR (95% CI) -- Ref 2.00 (1.14-3.51) 1.87 (1.03-3.40) 2.37 (1.17-4.77)\nNote: * denotes compare with Matched Sibling Donor: *means p＜0.05,**means p<0.01,***means p<0.001.\nAbbreviations: cGvHD, chronic graft versus host disease; HR, hazard ratio.\nRevised text（Results, Secondary endpoints)：\nAmong the 668 patients with follow-up longer than one year, cGvHD occurred in 8.9% of MSDs\nand 18.8% for MUDs and 23.1% for Haplos; moderate–severe cGvHD occurred in 5.4% for MSDs,\n10.7% for MUDs and 14.7% for Haplos, respectively. These analyses show a consistent pattern of\nhigher cGvHD rates after alternative donors (MUDs and Haplos) than after MSDs.\nComment 2\nThe use of post-transplant iron chelation in patients following engraftment is uncommon, as\nphlebotomy is generally preferred to treat iron overload. Please add the rationale for recommending\nchelation therapy.\nResponse:\nWe thank the reviewer for this important comment. Although phlebotomy is commonly used for\npost-transplant iron reduction, we did not recommend it in our cohort because most donors were\nthalassemia carriers and many recipients had early post-transplant hemoglobin levels around 100\ng/L, making phlebotomy poorly tolerated and potentially unsafe. Instead, iron chelation was\nselected as a safer early strategy. In addition, our team has registered two clinical studies evaluating\ndeferoxamine and deferasirox after HSCT, which demonstrated that post-transplant chelation is\nfeasible and well tolerated (data not yet published, ChiCTR2000031229, ChiCTR2000031225).\nComment 3\nThe study protocol uses the same intensive conditioning for MSD and alternative donors. Could\nsome patients in the MSD group be treated with less immunosuppressive regimens?\nResponse:\nWe agree that immunosuppression can be tailored by donor type. Our conditioning regimen ensured\nstable engraftment with a very low incidence of graft failure. Previous studies have shown that graft\nfailure rates are generally lower in transplants from matched sibling donors compared with\nalternative donors. Whether less immunosuppressive regimens could be safely applied to MSDs\nrecipients remains an important question for future investigation.\nComment 4\nThe impact of splenectomy on post-transplant outcomes is a significant observation. Please discuss\nwhether infection-associated mortality differed between splenectomized and non-splenectomized\ngroups.\nResponse:\nWe appreciate this important point. In our cohort, splenectomized patients were generally older and\npresented with greater hepatomegaly and higher iron overload before transplantation, which\npartially explains their inferior overall and event-free survival. The infection-related mortality in\nsplenectomized group was higher than that in non-splenectomized patients (7.6% vs 2.5%, P=0.010)\n(Supplementary Table 9).\nRevised text (Results,Discussion):\nThe infection-related mortality in splenectomized group was higher than that in non-splenectomized\npatients (7.6% vs 2.5%, P=0.010). (Supplementary Table 9).(Results)\nOur findings indicate that splenectomy was associated with inferior overall outcomes, although\nengraftment of neutrophils and platelets occurred more rapidly in splenectomized patients.\nInfection-related mortality was significantly higher in the splenectomized group compared with\nnon-splenectomized patients. (Discussion)\nComment 5\nPlease provide genotypes of patients with alpha thalassemia in Table 1.\nResponse:\nThere were 36 cases of α-thalassemia in the dataset, which have been categorized according to\ndifferent genotypes and incorporated into the description of Table 1.\na. The anemia in many forms of hemoglobin H disease responds to splenectomy. Was this\nconsidered as an option before transplant, where applicable?\nResponse:\nWe sincerely thank the reviewer for raising this important point. We fully agree that splenectomy\ncan ameliorate anemia in many forms of HbH disease, particularly in older patients or in those with\npronounced splenomegaly or hypersplenism. This distinction between α- and β-TDT is clinically\nmeaningful. In our cohort, however, nearly all α-TDT cases were young children with severe\ntransfusion-dependent phenotypes, for whom splenectomy is generally not recommended. Only two\nα-TDT patients had undergone splenectomy before transplantation, and the small number precludes\nany definitive conclusions regarding its impact on transplant outcomes. We agree that the role of\nsplenectomy in α-TDT warrants further study.\nb. The discussion (Line 294, page 11) on splenectomy prior to transplant should distinguish between\ngenotypes. I agree with avoiding splenectomy in beta thalassemia syndromes. However,\nsplenectomy is an important option for patients with alpha-thalassemia who require regular\ntransfusions.\nResponse:\nWe appreciate the reviewer’s valuable suggestion and concur with the comment. For patients\npresenting with marked splenomegaly before transplantation, splenectomy is recommended prior to\nHSCT. If transfusion independence is achieved after splenectomy, HSCT is generally not advised.\nRESPONSE TO REVIEWERS’ COMMENTS\nWe thank the reviewers for their careful evaluation and insightful recommendations.\nWe hope that the revised manuscript now meets the standards for publication in Nature\nCommunications.\nSincerely,\nYongrong Lai, MD, PhD\nDepartment of Hematology,\nthe First Affiliated Hospital of Guangxi Medical University, Guangxi, China"}, {"round": 2, "reviewer_comments": {"reviewer_2": "I am happy with the Authors' answers. No more issues to raise.", "reviewer_3": "I thank the authors for addressing the comments and revising the manuscript."}, "author_response": ""}]

Decision letter