Reduced-dose of Posttransplant Cyclophosphamide and Cotransplantation of Peripheral Blood Stem Cells and Human Umbilical Cord-derived Mesenchymal Stem Cells in Severe Aplastic Anemia

doi:10.21203/rs.3.rs-31898/v1

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Reduced-dose of Posttransplant Cyclophosphamide and Cotransplantation of Peripheral Blood Stem Cells and Human Umbilical Cord-derived Mesenchymal Stem Cells in Severe Aplastic Anemia

https://doi.org/10.21203/rs.3.rs-31898/v1

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posted 11 Jun, 2020

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Background Posttransplant cyclophosphamide (PTCY) as graft-versus-host disease (GVHD) prophylaxis is an effective strategie for patients receiving matched sibling donor hematopoietic stem cell transplantation (MSD-HSCT) and haploidentical HSCT (haplo-HSCT).

Methods We evaluated the effectiveness and safety of reduced-dose cyclophosphamide, 20 mg/kg for 13 patients in the MSD-HSCT group and 25 mg/kg for 22 patients in the haplo-HSCT group, on days +3 and +4 combined with cotransplantation of peripheral blood stem cells (PBSCs) and human umbilical cord-derived mesenchymal stem cells (UC-MSCs) for patients with severe aplastic anemia (SAA) retrospectively.

Results In the MSD-PTCY group, the times to neutrophil and platelet engraftment were significantly shorter than those in the MSD-control group (P<0.05), 11 (range 10 to 14) days and 12 (range 9 to 15) days. The cumulative incidence of acute GVHD (aGVHD) at day +100 (15.4%) was lower in the MSD-PTCY group than in the MSD-control group(P=0.050). No patient developed chronic GVHD (cGVHD). The 1-year overall survival (OS) and event-free survival (EFS) rates in the MSD-PTCY group were 100% and 92.3%. In the haplo-PTCY group, the times to neutrophil and platelet engraftment were significantly shorter than those in the haplo-control group (P<0.05), 12 (range 9 to 15) days and 11.5 (range 9 to 17) days. The cumulative incidences of aGVHD at day +100 and 1-year cGVHD in the haplo-PTCY group were 31.8% and 18.2%. The 1-year OS and EFS rates in the haplo-PTCY group were 81.8% and 66.9%.

Conclusions Reduced-dose PTCY and cotransplantation of PBSCs and UC-MSCs is feasible for patients with SAA, especially for overcoming the high incidences of aGVHD and cGVHD due to PBSCs.

Hematology

Reduced

PTCY

severe aplastic anemia

peripheral blood stem cells

umbilical cord-derived mesenchymal stem cells

Severe aplastic anemia (SAA), a life-threatening disease, can be cured by allogeneic hematopoietic stem cell transplantation (allo-HSCT) based on human leukocyte antigen (HLA)-appropriate donors. Nevertheless, patients undergoing HSCT always suffer from related complications, including graft-versus-host disease (GVHD) and infection, especially those lacking conventional HLA-identical donors. Thus, the use of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is limited by the incidences of graft failure and GVHD[1]. Graft sources are usually bone marrow (BM) and peripheral blood stem cells (PBSCs). PBSCs are increasingly used owing to the ease of obtaining cells and the high stem cell counts. However, the use of PBSCs is related to a higher incidence of acute GVHD (aGVHD) and chronic GVHD (cGVHD) than the use of BM[2], although the CIBMTR study demonstrated that there was no difference between BM and PBSCs in developing countries[3].

Mesenchymal stem cells (MSCs) possess the abilities of potential self-renewal, multilineage differentiation and regulation of immune function. Hence, MSCs reduce the risk of aGVHD and cGVHD[4]. It has been shown that MSCs can not only facilitate the engraftment of neutrophils and platelets but also promote donor chimerism[5]. In addition, human umbilical cord-derived MSCs (UC-MSCs) have a higher proliferation and differentiation ability than bone marrow MSCs[6]. Therefore, UC-MSCs play a considerable role in the clinical application of allo-HSCT for patients with SAA.

Currently, strategies for reducing GVHD involve the use of antithymocyte globulin (ATG) and posttransplant cyclophosphamide (PTCY) in haplo-HSCT. However, the high cost of ATG is a major obstacle to routine application. In recent years, PTCY as a haploidentical T cell-depleting protocol has become an effective alternative strategy for patients with nonmalignant hematological disorders in the haplo-HSCT setting[7-8]. PTCY has displayed a better outcome as GVHD prophylaxis than ATG in mismatched unrelated donor (MMUD) HSCT[9]. Several studies have obtained encouraging results after evaluating the efficacy of PTCY at 50 mg/kg in the haplo-HSCT setting[10-12]. Biju G et al considered that PTCY at 50 mg/kg as the sole GVHD prophylaxis was practical in patients with SAA undergoing matched sibling donor HSCT (MSD-HSCT)[13]. Then, a study revealed that a lower dose of 40 mg/kg was available and safe as aGVHD prophylaxis in MMUD-HSCT as an alternative to ATG[14]. Nevertheless, patients with SAA in the allo-HSCT setting usually are at greater risk of cardiotoxicity due to duplicate adverse effect of cyclophosphamide (CY) in the conditioning regimen and PTCY except for anemia, and assessments of lower PTCY doses are lacking up to now. Herein, we evaluated the effectiveness and safety of a reduced-dose PTCY protocol combined with cotransplantation of PBSCs and UC-MSCs for patients with SAA.

Patients

The study aim was to analyze patients with SAA who underwent MSD-HSCT and haplo-HSCT with cotransplantation of PBSCs and UC-MSCs at the Department of Hematology, Affiliated Cancer Hospital of Zhengzhou University from March 2017 to June 2019. We compared the engraftment time, cumulative incidences of aGVHD and cGVHD, incidence of infection and outcome (1-year event-free survival (EFS) and 1-year overall survival (OS)) in our MSD-PTCY group and haplo-PTCY group with those contemporary in a cohort of 20 patients and in a cohort of 15 patients receiving CSA and short-course methotrexate (MTX) as GVHD prophylaxis, respectively. The study was approved by the Institutional Review Board (IRB) of the institution and registered in the Chinese Clinical Trial Registry (ChiCTR1900026462). All patients and/or guardians signed informed consent forms approved by the review board of the institution. In this study, 35 patients with SAA were enrolled, including 13 patients who underwent MSD-HSCT and 22 patients who underwent haplo-HSCT.

Transplantation procedure

Thirteen patients who underwent MSD-HSCT received a conditioning regimen consisting of fludarabine (FLU) 30 mg/m²/day for 5 days given on days -6 to -2, ATG 2 mg/kg/day for 4 days given on days -4 to -1, and CY 24 mg/kg/day for 5 days given on days -6 to -2. Twenty-two patients who underwent haplo-HSCT received a conditioning regimen consisting of FLU 30 mg/m²/day for 5 days given on days -6 to -2, ATG 2.5 mg/kg/day for 4 days given on days -4 to -1, CY 24 mg/kg/day for 5 days given on days -6 to -2, and a single 2.5~3 Gy of total body irradiation (TBI) on day -1. Two patients who underwent haplo-HSCT without TBI suffered graft failure and underwent secondary transplantation. The second conditioning regimen included busulfan 0.8 mg/kg, q6h on days -3 to -2, and 3 Gy TBI on day -1. CSA was administered with from day +5 and PTCY 20 mg/kg was administered on days +3 and +4 for patients who underwent MSD-HSCT. CSA combined with mycophenolate (MMF) was administered from day +5 and PTCY 25 mg/kg was administered on days +3 and +4 for patients who underwent haplo-HSCT. The consecutive patients in the control groups received short-course MTX at a dose of 10 mg/m² on day +1 followed by a dose of 7 mg/m² on days +3, +6 and +11, with CSA and/or MMF used as GVHD prophylaxis. The graft sources for all patients were PBSCs and MSCs in the PTCY groups. Intravenous infusions of UC-MSCs were given to all patients with PTCY on day 0 at a dose of 1×10⁷/kg.

Supportive Care

Patients were hospitalized in a laminar flow ward with positive pressure and high-efficiency particulate air filtration until neutrophil recovery. Standard infection prophylaxis with a quinolone or berberine, trimethoprim-sulfamethoxazole and nystatin was begun on day -7. Standard Pneumocystis jiroveci prophylaxis was given to patients after engraftment for 1 year. Patients received ganciclovir at 5 mg/kg in the conditioning period. Broad-spectrum antibiotics, antifungals were used for agranulocytosis or fevers. Granulocyte colony-stimulating factor (G-CSF) was given intravenously starting on day +5 at 5 µg/kg/day until the peripheral blood absolute neutrophil count (ANC) was ≥0.5×10⁹/L for 3 days. Rituximab was given as prophylaxis for Epstein-Barr virus(EBV) on day +5 in the haplo-PTCY group. Blood products were irradiated with 25 Gy. Cytomegalovirus(CMV) reactivation was monitored by quantitative PCR twice a week until at least day +100. Preemptive therapy with ganciclovir or foscarnet was applied. EBV was also monitored weekly by quantitative PCR.

Posttransplantation complications

Post-HSCT complications mainly involve aGVHD, cGVHD, infections and other toxicities. GVHD was graded in accordance with the consensus criteria for aGVHD and cGVHD [15-16]. The diagnosis of GVHD depended on clinical symptoms and/or skin, oral mucosa, liver and gut biopsies. The grade of nonhematologic and noninfectious toxicity was diagnosed according to the Common Terminology Criteria for Adverse Events (CTCAE, v4.0) of the US National Cancer Institute and National Institutes of Health from day 0 to day +360.

Engraftment and chimerism

Neutrophil engraftment was defined as the first of three consecutive days with an absolute ANC ≥0.5×10⁹/L. Platelet engraftment was defined as the first day of seven consecutive days with a platelet count≥20×10⁹/L. Primary graft failure was defined as failure to achieve an ANC of 0.5 ×10⁹/L by day +28. Secondary graft failure was defined as an ANC<0.5 x10⁹/L after initial engraftment that was independent of infection, medications and insensitive to G-CSF. Full donor chimerism was defined as ≥95% leukocytes of donor origin in peripheral blood and/or bone marrow samples [17].

Statistical analysis

Median values and ranges were expressed for continuous variables and percentages for categorical variables. Continuous variables were analyzed by an independent samples T test. Differences in categorical variables were compared by Fisher’s exact test. Cumulative incidences of aGVHD and cGVHD were estimated using competing risk model with early death being a competing event. OS and EFS were performed with the Kaplan-Meier method and log-rank tests, while median survival was estimated with a 95% confidence interval. A two-sided P-values less than 0.05 were considered statistically significant. Analyses were performed using GraphPad Prism for Windows, version 8.4.1 and SAS version 9.4 (SAS Institute, Cary, NC).

Patients

The clinical characteristics of patients in the MSD-HSCT and haplo-HSCT groups are summarized in table 1 and table 2. No significant difference was observed between the PTCY groups and the control groups in terms of baseline demographics except for stem cell source. Eight patients (40%) of MSD-control group and all patients of haplo-control group received PBSCs and BM, while all patients of PTCY groups received PBSCs and UC-MSCs, except for two patients who suffered from primary graft failure received extra BM from a secondary donor in the haplo-PTCY group. There were 6 males (46.2%) and 13 males (59.1%) in the MSD-PTCY and haplo-PTCY groups and the median ages were 20 (range 9 to 35) and 14 (range 3 to 31) years respectively. One patient of each PTCY group was treated with anti-human lymphocyte immunoglobulin pretransplantation. The remaining patients had previously received treatment with CSA and/or steroids. In addition, two patients (9.1%) had hospital admissions for pulmonary infection requiring treatments prior to admission in the haplo-PTCY group, and one of the two patients had additional fungal sinus infections. Fortunately, the infections of the two patients were controlled after engraftment.

Engraftment

In the MSD-PTCY group, all patients successfully engrafted. The median numbers of infused MNCs and CD34+ cells between the MSD-PTCY and control groups were similar (MNCs: 16.36 (range 12.57 to 23.91) x10⁸/kg vs 14.04 (range 6.33 to 24.95) x10⁸/kg; CD34+ cells: 7.39 (range 4.35 to 18.4) x10⁶/kg vs 5.39 (range 1.02 to 13.37) x10⁶/kg). However, We observed that the times to neutrophil and platelet engraftment were shorter in the MSD-PTCY group than in the control group (11 days (range 10 to 14) vs 13 days (range 10 to 23) and 12 days (range 9 to 15) vs 14 days (range 10 to 45)). The difference in graft rejection between the MSD-PTCY and control groups was not significant(7.7% vs 15.0%). The rates of chimerism at day +30 in the MSD-PTCY and control groups were 100% and 90% respectively, and there was no significant difference.

In the haplo-HSCT groups, the median numbers of infused MNCs and CD34+ cells between the haplo-PTCY and control groups were similar (MNCs: 18.73 (8.41-32.35) x10⁸/kg vs 23.43 (2.02-31.72) x10⁸/kg; CD34+ cells: 7.60 (4.21-19.45) x10⁶/kg vs 8.95 (1.76-22.53) x10⁶/kg). Two patients who suffered from primary graft failure experienced successful engraftment after receiving transplants from haploidentical donors in the haplo-PTCY group. In the haplo-control group, secondary graft failure developed in one patient at 1.5 months after transplantation, and this patient sustained engraftment after undergoing MMUD-HSCT. The times to neutrophil and platelet engraftment were shorter in the haplo-PTCY group than in the control group (12 days (range 9 to 15) vs 13 days (range 11 to 19) and 11.5 days (range 9 to 17) vs 14 days (range 11 to 62)). One patient from each group suffered graft rejection.

Acute and chronic GVHD

The cumulative incidence of aGVHD at day +100 in the MSD-PTCY group was 15.4% (95% CI: 2.2-39.9) compared to 50.0% (95% CI: 26.3-69.8) in the control group (P=0.050) (Fig. 1a). The cumulative incidence of grade II-IV aGVHD in the MSD-PTCY group was lower than that in the control group, 7.7% (95% CI: 0.4-30.3) vs 30.0% (95% CI: 11.8-50.7), but the difference did not reach statistical significance (P=0.122) (Table 3). No patient developed cGVHD in the MSD-PTCY group. In contrast, the 1-year cumulative incidence of cGVHD in the control group was 15.0% (95% CI: 3.5-34.1)(Fig. 1b).

The cumulative incidence of aGVHD at day +100 in the haplo-PTCY group was lower than that in the control group(Table 4), 31.8% (95% CI: 13.8-51.6) vs 60.0% (95% CI: 29.9-80.6), but we observed no significant difference (P=0.165) (Fig. 2a). The cumulative incidences of grade II-IV aGVHD in the haplo-PTCY group was 27.3% (95% CI: 10.8-46.9) compared to 53.3% (95% CI: 24.8-75.3) in the control group(P=0.170). Notably, one patient with grade IV liver aGVHD had intracranial aGVHD, and one patient with aGVHD developed skin cGVHD in the haplo-PTCY group. The 1-year cumulative incidence of cGVHD in the haplo-PTCY and the haplo-control groups was no difference(P=0.600), 18.2% (95% CI: 5.5-36.8) vs 26.7% (95% CI: 7.7-50.7) (Fig. 2b). There was no extensive cGVHD in the haplo-PTCY group, while one patient developed extensive cGVHD in the control group.

Regimen-Related Toxicities

The incidence of pulmonary infection in the MSD-PTCY group was obviously lower than that in the control group (P=0.022), 7.7% vs 50.0%. Similarly, the incidences of CMV and EBV reactivation in the MSD-PTCY group were significantly lower than those in the control group (CMV: 23.1% vs 65.0%; EBV: 0 vs 60.0%), and there were significant differences between the two groups (P: 0.032 and 0.001, respectively). Although the incidence of hemorrhagic cystitis was lower in the MSD-PTCY group than in the control group (7.7% vs 35.0%), the difference was not significant (P=0.108). One patient suffered from pure red cell aplasia (PRCA) after ABO-compatible HSCT, and hematopoietic recovery occurred eighteen months after transplantation. In the control group, four of twelve patients with EBV reactivation developed posttransplantation lymphoproliferative disorders (PTLD) and three patients were cured through combination therapy with immunosuppressor withdrawal, rituximab and donor lymphocyte infusion (DLI). Another patient experienced an epileptic seizure because of intracranial EBV infection. Furthermore, two patients developed unexplained seizures that were relieved after expectant treatment. Cardiotoxicity and thrombotic microangiopathy (TMA) each developed in one patient. No patient had veno-occlusive disease (VOD).

The incidence of pulmonary infection in the haplo-PTCY group was lower than that in the control group (40.1% vs 60.0%), while the incidence of CMV reactivation in the haplo-HSCT group was higher than that in the control group (81.8% vs 53.3%), but the differences were not statistically significant (P: 0.325, 0.080). The median times to CMV infection were +37 days (range 29 to 46) and +35 days (range 25 to 53) in the haplo-HSCT and the control groups respectively. Two of eighteen patients with CMV infection in the haplo-HSCT group and one of eight patients with CMV infection in the control group developed cytomegalovirus retinitis. The incidence of EBV reactivation in the haplo-HSCT group (22.7%) was obviously lower than that in the control group (80.0%), and the difference was significant (P=0.001). There were no PTLD in the haplo-PTCY group, but three patients developed PTLD based on their seropositive EBV status in the control group. The incidences of hemorrhagic cystitis with BKV seropositivity were similar between the haplo-HSCT group and the control group (31.8% and 26.7%). In the haplo-PTCY group, one of seven patients who were seropositive for BKV progressed to BKV encephalitis and suffered from intracranial fungal infection. In addition, one patient had TMA at day +50. Moreover, posterior reversible encephalopathy syndrome (PRES) developed in four patients in the haplo-PTCY group and in two patients in the control group. None of all patients developed VOD in the two groups.

Survival

The median follow-up time of the MSD-PTCY group was similar to that in the control group, 22 months (range 10 to 36) vs 21.5 months (range 2 to 36). The median follow-up of the haplo-HSCT and the control groups were 11.5 months (range 3 to 27) and 12 months (range 1.5 to 36), respevtively.

All thirteen (100%) patients in the MSD-PTCY group are now alive with normal hematopoiesis. Interestingly, one female is now three months pregnant. The 1-year OS of the MSD-PTCY group was 100% compared to 75.0% (95% CI: 50.0-88.7)% in the MSD-control group (P=0.057) (Fig. 3a). The 1-year EFS of the MSD-PTCY group was 92.3% (95% CI: 56.6-98.9)% compared to 65.0% (95% CI: 40.3-81.5)% in the MSD-control group (P=0.071) (Fig. 3b). In the control group, three patients died from serious pulmonary infection at 2, 3 and 5 months, one patient died of intracranial EBV infection at day +70, and one patient died of TMA at 4 months. In the MSD-PTCY group, twelve of thirteen patients had a follow-up period of more than 12 months at the time of writing. All the patients began to reduce immunosuppressor treatment at 6 months and these agents were withdrawn before 9 months except for the patient with PRCA.

In the haplo-PTCY group, two patients died from intractable pulmonary infection at 3 and 9 months, one patient died of cerebral hemorrhage at 3 months, and one patient died of intestinal GVHD at 7 months. In the control group, two patients died from pulmonary infection at 4 and 6 months, and one patient died of severe intestinal GVHD at 12 months. The cumulative incidence of 1-year OS was 81.8% (95% CI: 58.5-92.8) in the haplo-PTCY group compared to 77.1% (95% CI: 44.2-92.1) in the haplo-control group (Fig. 4a). The 1-year EFS in the haplo-PTCY group vs the control group was 66.9% (95% CI: 39.1-84.2) vs 40.8% (95% CI: 20.5-71.2) (Fig. 4b). However, there was no significant difference in 1-year OS or EFS between the groups. In the haplo-PTCY group, eleven of eighteen living patients had a follow-up period of more than 12 months at the time of writing, those patients began to reduce immunosuppressor treatment at 9 months after HSCT.

Effective therapies for SAA patients usually involve immunosuppressive therapy (IST), allo-HSCT and eltrombopag. The application of IST is limited by late sequelae and the high cost of ATG[18]. Although the effective rate of eltrombopag is 20% to 40%, the main problems are relapse and secondary clonal disease[19]. The key challenge of allo-HSCT lies in minimizing complications and maximizing survival. Recently, PTCY has gradually come to be the predominate treatment worldwide due to its favorable prognosis in the allo-HSCT setting. However, a high dose of PTCY is accompanied by cardiotoxicity. Therefore, some studies have reported the outcomes of reduced doses of PTCY in malignant and nonmalignant hematological disorders[14,20]. Furthermore, UC-MSCs are popular in allo-HSCT for SAA because of several beneficial effects, including facilitating engraftment and decreasing GVHD. At present, there are few evaluations of reduced-dose PTCY for SAA patients receiving cotransplantation of PBSCs and UC-MSCs. Therefore, the aim of this prospective study was to demonstrate the influence of reduced-dose PTCY on early outcomes after cotransplantation of UC-MSCs and PBSCs for patients with SAA receiving transplants from either matched or haploidentical donors.

GVHD is a main barrier in patients with SAA undergoing allo-HSCT. The occurrence of GVHD is not needed for SAA. PTCY is an attractive option because it eliminates early proliferating alloreactive T cells to reduce impaired immune reconstitution in the haplo-HSCT setting[21,22]. The doses of PTCY were reduced to 20 mg/kg and 25 mg/kg from the conventional 50 mg/kg dose for MSD-HSCT and haplo-HSCT, respectively. The results showed that the cumulative incidence of aGVHD at day +100 in the MSD-PTCY group was 15.4% (95% CI: 2.2-39.9) compared to 50.0% (95% CI: 26.3-69.8) in the control group, and the P value was 0.050. In addition, the cumulative incidence of grade II-IV aGVHD was lower in the MSD-PTCY group than in the control group, 7.7% (95% CI: 0.4-30.3) vs 30.0% (95% CI: 11.8-50.7), but we did not observe a significant difference. No patient in the MSD-PTCY group developed cGVHD. George B et al reported that 30 patients with SAA received high-dose PTCY as the sole GVHD prophylaxis in PBSC MSD-HSCT. The incidences of grade II-IV aGVHD and cGVHD were 22.0% and 22.7%, respectively[13]. In MSD-HSCT for malignant hematologic disorders, high-dose PTCY has been associated with the occurrence of grade II-IV aGVHD (17% to 45%) and cGVHD (7% to 14%)[23-25]. A multicenter study showed that the cumulative incidences of grade II–IV aGVHD and cGVHD at 1 year were 33.7% and 22.4%, respectively, for SAA patients receiving BM and PBSCs in the haplo-HSCT setting[26]. In this study, the cumulative incidence of grade II-IV aGVHD at day +100 and 1-year cumulative incidence of cGVHD in the haplo-PTCY group were 27.3% (95% CI: 10.8-46.9) and 18.2% (95% CI: 5.5-36.8), respectively. Although there were no significant differences in either the cumulative incidence of aGVHD and grade II–IV aGVHD or the 1-year cumulative incidence of cGVHD, reduced-dose PTCY and cotransplantation of PBSCs and UC-MSCs probably overcame the high incidences of aGVHD and cGVHD due to PBSCs. Encouragingly, the 1-year OS in the MSD-PTCY group was 100%. We observed no difference in the 1-year OS and EFS values between the haplo-PTCY and haplo-control groups, but we believe that the use of PBSCs and UC-MSCs leads to decreased donor pain and improves donation rates compared to the use of routine PBSCs and BM.

The times to neutrophil and platelet engraftment in the PTCY groups were shorter than those in the control groups (P<0.05). The time to neutrophil and platelet engraftment in the haplo-PTCY group were 12 days (9-15) and 11.5 days (9-17), respectively. Moreover, the times to engraftment in the haplo-HSCT group were heterogeneous, with the time to neutrophil engraftment being 12 to 19 days, and the time to platelet engraftment being 13 to 21 days[26-29]. The application of UC-MSCs was also special in this study. Various abilities of MSCs in the HSCT setting involve facilitating engraftment, remedying graft dysfunction and providing GVHD prophylaxis[30]. A retrospective study demonstrated that UC-MSCs were likely to decrease the occurrence of severe aGVHD, which influenced life quality and prognosis for patients undergoing haplo-HSCT[31]. Another study reached the same conclusion and verified that cotransplantation of UC-MSCs with haplo-HSCT was feasible and safe for patients with SAA[33]. There is controversy regarding the engraftment function of MSCs, but most studies have shown that the role of MSCs in allo-HSCT is worthy of consideration[31-33].

The incidence of viral infections is another problem for allo-HSCT protocols using PTCY. In this study, the viral infection rates were lower in the MSD-PTCY group than in the control group, both for CMV and EBV reactivation (P<0.05). It is worth noting that no patient underwent EBV reactivation in the MSD-PTCY group. Patients with CMV reactivation especially have a risk for developing CMV-related diseases, including CMV pneumonia, retinitis and enteritis. Two patients and one patient with CMV seropositivity in the haplo-PTCY and haplo-control groups developed CMV retinitis, respectively. As a result, they developed varying degrees of visual impairment. However, the incidence of CMV reactivation in the haplo-HSCT group was higher than that in the haplo-control group, although there was no significant difference, which may be due to the additive immunosuppressive effects of ATG and PTCY. How to reduce the incidence of CMV reactivation is an outstanding problem. In the haplo-HSCT group, the incidence of EBV reactivation was significantly lower than that in the haplo-control group (P<0.05), which was attributed to the use of rituximab at day +5. Bacigalupo, A et al showed that patients received rituximab at day +5 after alternative donor HSCT had significantly lower rates of EBV-DNA and lower maximum median EBV copies than the control group (P<0.05). Furthemore, rituximab at day +5 had no influence on infections[34]. Pulmonary infection is one of the key life-threatening complications. The incidence of pulmonary infection in the MSD-PTCY group was obviously lower than that in the control group (P<0.05). It is worth mentioning that the total dose of ATG was reduced from 10 mg/kg to 8 mg/kg in the MSD-PTCY group. Though no difference, the incidence of pulmonary infection in the haplo-PTCY group was lower than that in the haplo-control group, 40.1% vs 60.0%.

There are some limitations in the study. First, the small sample size and short follow-up period were weaknesses. Thus, a prospective study with a large sample size is necessary. In addition, further studies are required to evaluate the clinical utility of reduced-dose PTCY and UC-MSCs in preventing and alleviating GVHD, promoting engraftment and improving outcome. Despite these limitations, the protocol not only overcomes the high incidence of aGVHD and cGVHD due to PBSC but also decreases difficulties for donors. In conclusion, It is feasible for patients with SAA to receive reduced-dose PTCY and cotransplantation of PBSCs and UC-MSCs.

ATG, anti-thymocyte globulin; p-ALG, porcine anti-human lymphocyte globulin; PBSC, peripheral blood stem cells; BM, bone marrow; UC-MSCs, umbilical cord-derived mesenchymal stem cells; MNC, mononuclear cell; ANC, absolute neutrophil count; aGVHD, acute graft-versus-host disease; cGVHD, chronic graft-versus-host disease; EBV, Epstein-Barr virus; CMV, cytomegalovirus; OS, overall survival; EFS, event-free survival

Acknowledgments

We extended our gratitude to all who contributed to this paper, especially for Lanwei Guo who gave a hand regarding statistical methods.

Authorship Contributions

Jian Zhou and Yongping Song designed the research; Yingling Zu analysed the data and wrote the manuscript; Chengjuan Zhang did statistical analysis; Other authors provided patients; All authors gaved final approval for the manuscript.

Funding

This work was partly supported by the Henan Medical Science and Technique Foundation (Grant No. SBGJ2018085).

Availability of data and materials

All data in this study are included in this publication and related data files.

Ethics approval and consent to participate

This study was reviewed and approved by the Medical Ethics Committee of Affiliated tumor hospital of Zhengzhou university, Zhengzhou, China.

Consent for publication

All authors have agreed to publish this manuscript

Competing interests

The authors declare that they have no conflicts of interest.

Authors details

¹ Department of Hematology, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou 450000, China

²Center of Bio-Repository, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou 450000, China

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Reduced-dose of Posttransplant Cyclophosphamide and Cotransplantation of Peripheral Blood Stem Cells and Human Umbilical Cord-derived Mesenchymal Stem Cells in Severe Aplastic Anemia

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