Triple stem cell infusion alleviated graft-versus-host disease and improves outcomes in unmanipulated haploidentical hematopoietic stem cell transplantation

Abstract

Haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) provides cure opportunity for patients requiring prompt allogeneic HSCT but failing to identify well-matched donor, but its outcomes are potentially impaired by increased transplant-related mortality (TRM). We performed haplo-HSCT using granulocyte colony-stimulating factor (G-CSF)-primed peripheral blood stem cells (PBSCs), umbilical cord mesenchymal stem cells (UC-MSCs) and third-party unrelated umbilical cord blood (UCB) stem cells. Modified “Beijing protocol” were performed in this study. All of the patients were transplanted by Busulfan or TBI-based regimen. Anti-thymocyte globulin were used to T-cell depletion in vivo. Cyclosporine, mycophenolate mofetil, and short course methotrexate were used to prevent graft-versus-host disease (GVHD). One hundred and sixty-five patients with hematological disorders undergoing haplo-HSCT from Jan 2021 to Nov 2023 were included in this study. The median time of neutrophil engraftment were 12 days (range: 9–25 days), and the median time of platelet engraftment were 13 days (range: 6–50 days). Full haploidentical donor chimerism were obtained within 30 days. No evidence of UCB chimerism was found. Twenty-five patients developed acute GVHD. The incidence of grade II-IV and grade III–IV acute GVHD was 12.73% and 6.67%, respectively. Twenty-eight patients developed chronic GVHD, 10 were limited (6.06%) and 18 were extensive (10.91%). The TRM is total of 26 deaths (15.8%) and the cumulative incidence of relapse (CIR) is total of 17 deaths (11.8%) occurred as of the statistical period. The 2 years overall survival (OS) rate is 72.96%. The median overall survival rate was not reached. Haplo-HSCT performed by PBSCs, UC-MSCs and UCB “triple-infusion” achieved excellent outcomes, and need to explored in a larger cohort.

Graphical abstract

Dear Editor

Haploidentical hematopoietic allogeneic stem cell transplantation (Haplo-HSCT) has emerged as a potentially curative modality for patients with an acute and compelling indication for allogeneic hematopoietic stem cell transplantation (allo-HSCT). These patients, regrettably, face the predicament of lacking a fully human leukocyte antigen (HLA)-matched donor, which is a crucial determinant for the success of traditional allo-HSCT. To surmount this critical challenge, various strategies are being explored, including T-cell depletion and the co-infusion of multiple sources of hematopoietic stem cell graft sources ¹ . Noteworthy among these is research into utilizing umbilical cord blood (UCB) stem cells or mesenchymal stem cells (MSCs) to enhance stem cell engraftment and graft-versus-host disease (GVHD) prevention^2,3. The functional limitations of peripheral blood stem cell (PBSC) can be addressed by UCB, prevent the occurrence of GVHD and promote the implantation of stem cells. Co-transplantation of unrelated UCB may further decrease risk of relapse due to its unique biologic properties ⁴ . Haplo-cord Hematopoietic Stem Cell Transplantation (HCT) acute myelocytic leukemia (AML) patients exhibited a faster cumulative incidence of neutrophil recovery and increased T-cell reconstitution in the early period posttransplantation ⁵ . The early repeated infusions of MSCs decreased the incidence and severity of chronic GVHD, and the incidence and severity of acute GVHD manifested as a better GVHD-free and relapse-free survival rate for patients after haplo-HSCT ⁶ . Given that several clinical trials have demonstrated the potential of MSCs ⁷ , MSCs may be a helpful tool for managing GVHD and maintaining the graft-versus-leukemia (GVL) effect, which is demonstrated by an increase in the rate of transplant recipients who have GVHD-free and relapse-free survival (GRFS)^6,8. In this study, we investigated the safety and effectiveness of PBSCs transplantation combined with third-party UCB and umbilical cord mesenchymal stem cell (UC-MSC) in terms of engraftment, GVHD prophylaxis and overall survival (Supplemental file).

From January 2021 to November 2023, a total of 165 patients with hematological malignancies undergoing Haplo-HSCT were included in this study. Triple stem cells were utilized for allo-HSCT, including PBSCs from granulocyte colony-stimulating factor (G-CSF) mobilized donor, MSCs derived from UCB and third-party UCBs. The protocol for donor selection was based on the expert consensus of the European Society for Blood and Marrow Transplantation (EBMT) in 2020 version, as well as the guidelines from the Chinese Medical Association in the 2021 version^9,10. The characteristics of the included patients are presented in Table 1. A modified “Beijing protocol” was implemented in this study. All patients received transplantation via a busulfan or TBI (total body irradiation)-based regimen. Anti-thymocyte globulin (ATG) or anti-T lymphocyte globulin (ATLG) was administered for in vivo T-cell depletion. Cyclosporine, mycophenolate mofetil, and a short course methotrexate were used to prevent GVHD. All donors were mobilized by G-CSF (Filgrastim, 5–7.5 μg/kg/day) for 4–6 days, the PBSCs were collected by apheresis using Fresenius Kabi on day 5–7 based on the concentration of CD34 cells in peripheral blood. The PBSCs were immediately transfused to patients without ex vivo T-cell depletion. The human UCB was uniformly prepared from Sichuan cord blood bank, all the UCBs was collected from the healthy and full-term cesarean deliveries in our hospital and processed within 24 hours after receiving signed informed consent by the third-party birth parent in hospitals in Sichuan province. After obtaining signed informed consent from the third-party birth parent, UC-MSCs were uniformly prepared from the General Hospital of Western Theater Command. The MSCs were derived from the UCB, which was from healthy and full-term cesarean deliveries at our hospital. Within 24 hours, the process was completed, and 5–6 generations of cell culture were carried out (1 × 10⁵/kg). UC-MSCs were subsequently infused, and 1 week later, another dose was administered (provided by Cell Laboratory, Department of Hematology, the General Hospital of Western Theater Command). Third-party UCB (provided by Sichuan cord blood bank) was infused on the following day.

Table 1.

The characteristics of included patients.

Characteristics	N = 165
Median age (range)	37 (8–64)
<18 years	16 (9.7%)
≥18 years, <50 years	112 (67.9%)
≥50 years, <60 years	37 (22.4%)
Gender
Male	89 (53.9%)
Female	76 (46.1%)
Underlying disease
AML	73 (44.2%)
ALL	49 (29.7%)
MDS	16 (9.7%)
SAA	20 (12.1%)
MAL	2 (1.2%)
CML	3 (1.8%)
CMML	1 (0.6%)
Duration from diagnosis to HSCT (months; median and range)	6 (0.34–240)
Conditioning regimen
Bu-based	(82.4%)
TBI/TMIL-based	(13.9%)
Other	(3.7%)
MNC (108/kg) (median, range)	(8, 3.33–16)
CD34+ cells (106/kg) (median, range)	(5.49, 1.68–13.57)
Haplo-donor HLA match score
5/10	(7.3%)
6/10	(4.2%)
7/10	(1.2%)
8/10	(0.6%)
6/12	(37.6%)
7/12	(28.5%)
8/12	(14.5%)
9/12	(1.2%)
10/12	(3.6%)
11/12	(0.6%)
12/12	(0.6%)
Haplo-donor/recipient blood type
Matched	(61.2%)
Mismatched	(38.8%)
UCB donor HLA match score
3/6	(6.7%)
4/6	(68.5%)
5/6	(24.8%)
Organs involved in aGVHD	N (%)
Gastrointestinal tract	16 (64%)
Gastrointestinal tract and skin	2 (8%)
Liver	1 (4%)
Skin	6 (24%)
Organs involved in cGVHD	N (%)
Oral mucosa	5 (25%)
Skin	6 (24%)
Lung	1 (5%)
Kidney	1 (5%)
Joint	2 (10%)
Skin and eye	1 (5%)
Oral mucosa and eye	1 (5%)
Lung and skin	1 (5%)
Lung and oral mucosa	1 (5%)
Oral mucosa, skin, liver, and lung	1 (5%)
Transplant-related mortality rate	N (%)
TMA	1 (3.8%)
Infection	13 (50%)
Secondary poor implantation	1 (3.8%)
Hematencephalon	1 (3.8%)
COVID-19 infection	6 (23.1%)
Primary graft failure	1 (3.8%)
Disease status of patients before HSCT	N (%)
PR/NR	30 (20.8%)
CR1	89 (61.8%)
CR2	22 (15.3%)
CR3	2 (1.4%)
CR4	1 (0.7%)

ALL, acute lymphoblastic leukemia; AML, acute myelocytic leukemia; ATG, antithymocyte globulin; Bu, busulfan; Cy, cyclophosphamide; HLA, human leukocyte antigen; MDS, myelodysplastic syndrome; SAA, severe aplastic anemia; TBI, total body irradiation; TMIL, total marrow lymphoid irradiation; UCB, umbilical cord blood.

This study comprised 165 patients with hematologic disorders undergoing haplo-HSCT, with an average age of 37 years. Statistical analyses were performed using SPSS 25, The cumulative incidence of grade I to IV aGVHD, meanwhile, the cGVHD contained limited and extensive cases. The OS rate was estimated using Kaplan–Meier analysis and were expressed as percentages with 95% confidence intervals (CI). Neutrophil engraftment was successfully achieved in 162 out of 165 patients, occurring at a median 12 days (range: 9–25 days). Platelet engraftment was successfully achieved in 155 patients, with a median of 13 days (range: 6–50 days). Full haploidentical donor chimerism was attained within 30 days, and no evidence of UCB chimerism was detected. Among the study cohort, 25 patients experienced the onset of acute GVHD (aGVHD), with the incidence of grade II–IV and grade III–IV aGVHD at 12.73% and 6.67%, respectively. Chronic GVHD (cGVHD) was present in 28 patients, including 10 limited cases (6.06%) and 18 extensive cases (10.91%) (Figure 1). Patients experiencing aGVHD received intravenous methylprednisolone (1–2 mg/kg/day) and tapering off as manifestation under control. For steroid insensitivity or steroid-dependent aGVHD, second-line treatments such as basiliximab or ruxolitinib were prescribed. Since UCB contains naïve T lymphocytes that might contribute to the generation of antigen-specific T-lymphocyte immunity in the early posttransplantation period, and considering the prophylaxis with letermovir, our study demonstrated a lower level of cytomegalovirus (CMV) and Epstein–Barr virus (EBV) viremia, which was in line with similar studies^11,12. The data not shown. The 2-year OS rate for all patients was 73.0% (95% CI, 65.2%–81.6%), For leukemia patients specifically, the 2-year OS rate was 73.1% (95% CI, 64.1%–83.3%), the median OS rates for both groups have not yet been reached.

Figure 1.

(a) Time to neutrophil engraftment (days). (b) Probability of overall survival (%). (c) Incidence of aGVHD. (d) Time to platelet engraftment (days). (e) Probability of overall survival of leukemia. (f) Incidence of cGVHD.

Haplo-HSCT has emerged as a viable alternative for patients lacking full matched donor. It offers a stronger GVL effect and a lower risk of relapse compared to matched sibling donors ¹³ . Despite advancements in haplo-HSCT, transplant-related complications such as GVHD continue to impact overall survival (OS). Ex vivo T-cell depletion has shown efficacy in GVHD prevention ¹⁴ . Recent studies suggest that haplo-HSCT with unrelated UCB transplantation may decrease GVHD incidence, enhance 5-year OS and 3-year progression-free survival (PFS), and reduce cumulative incidence of recurrence (CIR) in patients undergoing haplo-HSCT. In addition, this strategy may boost posttransplant T-cell recovery and improve AML patient survival without raising the risk of adverse events^3,15. Moreover, recent evidence indicates that haploid PBSCs with UCB transplantation results in faster platelet engraftment, lower relapse rate, and better disease-free survival and OS compared to haploid PBSCs combined with bone marrow transplantation ⁵ . This underscores the favorable impact of UCB in combined transplantation.

Nowadays, MSCs are wildly used in various clinical contexts, with their immune-regulatory effects widely acknowledged. Mesenchymal stem cells infusion has demonstrated success in both preventing and treating GVHD^6,10. In a recent study, 20 haplo-HSCT recipients received infusions of haploidentical peripheral blood cells, bone marrow cells, MSCs, and UCB stem cells, resulting in high rates of donor engraftment, low rates of GVHD, and high OS rates ⁴ . In our study, comparable results were observed, with “triple infusion” transplantation leading to alleviation of both acute and chronic GVHD. Furthermore, this marrow free strategy simplifies the transplantation process by eliminating the need for bone marrow aspiration. Our study demonstrates that haplo-HSCT utilizing PBSCs, MSCs, and UCB “Triple-infusion” approach yields favorable outcomes, with reduced complications and transplant-related mortality (TRM) attributed to the rapid engraftment of stem cells and a low incidence of acute and chronic GVHD. Prospective trials with larger sample sizes will be necessary to confirm the superior efficacy of the triple-infusion approach.

Supplemental Material

sj-tiff-1-cll-10.1177_09636897251359786 – Supplemental material for Triple stem cell infusion alleviated graft-versus-host disease and improves outcomes in unmanipulated haploidentical hematopoietic stem cell transplantation

Supplemental material, sj-tiff-1-cll-10.1177_09636897251359786 for Triple stem cell infusion alleviated graft-versus-host disease and improves outcomes in unmanipulated haploidentical hematopoietic stem cell transplantation by Fang Hua, Shan Zhang, Xiaomei Zhang, Yan Deng, Ying Han, Sihan Lai, Ying He, Lei Ma, Xupai Zhang, Dan Chen, Yi Su, Jian Xiao, Ling Zhang, Hui Yang, Rong Huang, Haiyan Hu, Mingli Chen, Guangcui He, Hao Yao and Hai Yi in Cell Transplantation