This is the first study on the use of PTIS in both related and unrelated donor transplantation for TDT. In this study, we introduced a novel program consisting of multidrug PTIS therapy and a modified myeloablative conditioning regimen. The safety and efficacy of this novel conditioning regimen in children with TDT was presented by the retrospective analysis. A notable finding of this study is that the novel conditioning regimen reduced the risk of fungal infection, mixed chimerism, and immune-related complications, while the outcome of unrelated donor transplantation is equivalent to matched sibling donor transplantation, despite the older age and higher risk of transplantation and higher probability of positive PRA titers.

Older transplant age and hepatomegaly have been shown to be associated with inferior outcomes. It has been suggested that patients older than 7 years and with hepatomegaly should be defined as a very high-risk subgroup [20,21,22]. A multicenter study [23] reported that, after adjusting for donor type and conditioning regimen, the 5-year OS were 84% for patients aged 7 to 15 years and 63% for those aged 16 to 25 years. In another Indian study [24], the estimated OS for recipients aged > 7 years was only 90%. It is recommended that HSCT should be performed before the age of 7 years or before the onset of organ damage caused by severe iron overload and other thalassemia-related complications [22, 25]. In this study, despite the older age of transplantation of median age of over 7 years in NCR group, the NCR has overcome the disadvantages of age and unrelated donor, achieving comparable prognosis as in MSD.

Furthermore, PRA is another risk factor affecting engraftment in patients with thalassemia, which has been associated with multiple transfusions before transplantation [26, 27]. Our previous study found that PRA had a negative effect on DNA synthesis and the colony formation of CD34+ cord blood cells in vitro [28]. Transplants in PRA-positive recipients had a higher transplant-related mortality associated with poor graft function and vascular complications [27, 29]. Taking up the challenge of the high-risk subgroup with older transplant age and strong positive PRA, our center is trying to optimize this preparative backbone by adding PTIS and reducing the intensity of the conditioning regimen. It was showed that exposure to fludarabine led to a sustained loss of STAT1, which was essential for cell-mediated immunity, contributing to the prolonged period of immunosuppression [30], while dexamethasone-mediated T cell suppression diminished immature T cells [31]. Rituximab or bortezomib were used to eliminate plasma cells and memory B cells responsible for the production of anti-HLA antibodies, while plasmapheresis contributed to the elimination of PRA. Prior HSCT, low-dose chemotherapy with fludarabine and dexamethasone, or plasmapheresis alone or in combination with rituximab, bortezomib, sirolimus, high-dose IVIG, splenectomy [32], or T-cell depletion [11, 12] can prevent AIHA and pure red cell aplasia in PRA-positive recipients. According to the results, PTIS suppressed the immunity and depleted the lymphocytes from the recipients, resulting in stable implantation, less AIHA, and less graft failure, while the immune reconstitution post-transplantation was not impaired.

TDT is characterized by hypercellular bone marrow, which may contribute to higher risk of graft failure [11, 12]. Therefore, the ideal conditioning regimen must enhance the suppression of marrow hyperplasia, as well as the heighten immune system, to achieve stable engraftment. Bu-Cy has been the cornerstone conditioning regimen in most studies, although it is associated with hepatic and vascular complications. Various centers have endeavored to optimize protocols to minimize organ damage and enhance graft success. In a recent international study, the TCI score was developed as a new tool to define and measure the intensity of the conditioning regimen [33]. The NF-08-TM protocol reduced the dose of both Cy and Bu, but added Thiotepa (TT), with a TCI score of 4.5. The reported OS and TFS for matched and mismatched unrelated donor HSCT were 93.6% vs. 84.6% (p = 0.058), 90.4% vs. 82.1% (p = 0.089) [34]. In a study conducted at the Guangzhou Women and Children’s Medical Centre, 257 patients underwent a modified NF-08-TM conditioning regimen for both fully-matched and mismatched donor grafts, with the high incidence of immune-related complications, including AIHA ranging from 19.17 to 25.37%, PRES from 6.74 to 11.94%, and PTLD from 0.52 to 1.49%. Notably, patients receiving grafts with two or more HLA-allele mismatches were more susceptible to develop AIHA (40.91% vs. 17.78%, p = 0.041) [7]. A study from India also reported a higher incidence of PRES and immune-mediated cytopenia in patients undergoing unrelated donor transplantation [24]. In this study, all transplants received a myeloablative conditioning regimen consisting of Bu, Cy, Flu, and ATG. Our previous study based on CCR had observed a high incidence of serious bacterial infections and fungal pneumonia, which could be related to the immunosuppression of high-dose ATG. In addition, Cy had shown dose-related cardiotoxicity [35]. Therefore, in the NCR group, the lower dose of Cy (120 mg/kg instead of 180–200 mg/kg) has the same TCI score as the NF-08-TM protocol. Considering that a lower dose of Cy could have a negative impact on the maintenance of donor engraftment [11, 12], PTIS plays the role of providing additional immunosuppression prior to conditioning regimen without compromising immune reconstitution. It’s worth noting that the incidence of immune-related complications decreased significantly. Interestingly, the proportion of mixed chimerism was significantly lower in the NCR group compared to the CCR group for MSD transplants, which might be attributed to the prolonged and intensive suppression of recipient immune cells before transplantation. Furthermore, URD transplantation was comparable to MSD transplantation for TDT under the novel conditioning regimen. In the setting of UD-HSCT, reducing the intensity of the conditioning regimen did not affect OS and TFS. The novel conditioning regimen is able to be applied cost-effectively and accessibly in developing countries.

In addition to addressing the early transplantation complications, we place significant emphasis on the long-term outcomes for pediatric transplant recipients, which includes monitoring and managing issues such as iron overload, endocrine function and growth development, as well as quality of life and psychological well-being post-transplantation. Each pediatric patients performed growth development and endocrinology assessments prior to transplantation, and it is recommended that they attend regular outpatient follow-ups to monitor their growth and development post-transplantation. Our group had revealed that thalassemia patients with iron overload were at an increased risk of developing abnormal glucose metabolism before transplantation [36]. Additionally, premature ovarian failure was observed in some patients. Numerous studies have documented a significant depletion of the primordial follicle pool following CTX treatment [37, 38], which partly underpins our decision to reduce the single dose of CTX in novel conditioning regimen. Therefore, we will continue to pay attention to the effect of preconditioning regimen on growth and development post transplantation in future follow-up.

In conclusion, URD transplantation in patients with TDT can achieve a comparable prognosis to MSD transplantation without increasing the treatment-related mortality or graft failure rate by PTIS and modified myeloablative conditioning regimen. Based on the favorable outcome in the high-risk subpopulation with mismatched unrelated donors in this study, mismatched unrelated donors PBSC transplantation may be an optimized alternative for patients with TDT in the absence of MSD or MUD.