Effects and long‐term follow‐up of using umbilical cord blood–derived mesenchymal stromal cells in pediatric patients with severe BK virus‐associated late‐onset hemorrhagic cystitis after unrelated cord blood transplantation

This is a retrospective study to evaluate the efficacy and safety of umbilical cord blood–derived mesenchymal stromal cells (MSCs) for the treatment of pediatric patients with severe BK virus–associated late‐onset hemorrhagic cystitis (BKV‐HC) after unrelated cord blood transplantation (UCBT). Thirteen pediatric patients with severe BKV‐HC from December 2013 to December 2015 were treated with MSCs. The number of MSCs transfused in each session was 1 × 106/kg once a week until the symptoms improved. The median follow‐up time was 1432 (89‐2080) days. The median frequency of MSC infusion was 2 (1‐3), with eight cured cases and five effective cases; the total efficacy rate was 100%. The copy number of urine BKV DNA was 4.43 (0.36‐56.9) ×108/mL before MSC infusion and 2.67 (0‐56.3) ×108/mL after MSC infusion; the difference was not significant (P = .219). There were no significant differences in the overall survival, disease‐free survival, and the incidence of relapse and acute and chronic graft‐versus‐host disease between the MSC infusion group and non‐MSC infusion group. There was also no significant difference in the cytomegalovirus, Epstein‐Barr virus (EBV), and fungal and bacterial infection rates between the two groups. Although umbilical cord blood–derived MSCs do not reduce the number of BKV DNA copies in the urine, the cells have a high efficacy rate and minimal side effects in treating severe BKV‐HC after UCBT among pediatric patients. MSCs do not affect the rates of relapse, long‐term infection, or survival of patients with leukemia.


| BACKG ROU N D
ing HCs both internationally and locally. 3,4 However, the average number of cases was relatively small, and the follow-up time was relatively short; further, there are no reports on MSCs from umbilical cord blood for the treatment of pediatric BK virus-associated HC. In this study, we investigated the treatment of severe pediatric BK virus-associated HC after HSCT with MSCs from umbilical cord blood in our transplantation center for the first time.

| Patients
From December 2013 to December 2015, 13

| Transplantation procedures
All patients were treated with a myeloablative conditioning regimen.

| Prevention and supportive treatment of infection
Infection prophylaxis consisted of the administration of oral fluconazole, cefprozil, and SMZco. Voriconazole or posaconazole was used to prevent fungal formation in patients with previous fungal infections. Intravenous acyclovir was added to prevent viral infections >1 day after transfusion of the cord blood. Ganciclovir or foscarnet was administered as the preemptive CMV therapy.
Heparin sodium and prostaglandin E1 were administered to prevent hepatic venous occlusion disease during the conditioning regimen. Heparin sodium was discontinued when the platelet count was below 20 × 10 9 /L. Granulocyte colony-stimulating factor was added at >6 days after transplantation until the leukocyte count reached >4×10 9 /L and became stable for 3 days. Red blood cells were transfused when the hemoglobin level was <60 g/L, and platelets were transfused when the platelet counts were <20×10 9 /L. Furthermore, mesna was administered at the same time as cyclophosphamide and after 3, 6, and 9 hours. The total dose of cyclophosphamide administered was 100%-120%.

| Diagnosis and grading of BKV-HC
BKV-HC was diagnosed mainly on the basis of the following two as- Late-onset HC is associated with the BKV when more than 10 6 copies of BKV DNA are found in the urine. 5

| Other definitions
Acute GVHD (aGVHD) was diagnosed and graded according to previously published criteria. 7 Chronic GVHD (cGVHD) was classified as mild, moderate, or severe according to the 2014 National Institutes of Health consensus criteria. 8 Relapse was defined by the morphological evidence of the disease in the peripheral blood, bone marrow, or extramedullary sites; the time to relapse was defined as the number of days from transplantation to the first diagnosis of relapse. 9 The overall survival (OS) was defined as the number of days from transplantation to death of any cause. Disease-free survival (DFS) was defined as the number of days from transplantation to the first diagnosis of relapse or death. 9 For infections occurring 100 days after transplantation, we

| Preparation of umbilical cord bloodderived MSCs
The umbilical cord blood-derived MSCs (UCB-derived MSCs) were purchased from Shandong Cord Blood Bank. The umbili- When the monolayer adherent cells were nearly 80% confluent at 10-14 days, they were digested with 0.25% trypsin and passaged at 1:2. Thereafter, the cells were continuously subcultured in vitro at 1:2 when they reached ~80% confluency with cell expansion. 2 When the cells were cultured to the third generation and were nearly 80% confluent, they were digested with 0.25% trypsin and collected. We use fresh MSCs to treat hemorrhagic

| Treatment of grade III to IV HC with umbilical cord blood-derived MSCs
Four patients had grade III to IV HC at onset. Nine patients had grade I to II HC at onset but advanced to grade III to IV HC after routine hydration, alkalization, antiviral therapy, and bladder irrigation. Once diagnosed with severe HC, the patients were infused with umbilical cord blood-derived MSCs once a week until their symptoms were relieved. The number of MSCs infused in each session was 1 × 10 6 /kg. Dexamethasone (2.5 mg) was administered to prevent anaphylaxis before infusion for approximately 30 minutes.

| Statistical analysis
The two independent samples t test and Fisher's exact test were used to compare the continuous variables between the two groups.
The paired samples t test was used to compare the continuous variables before and after treatment. The chi-square test was used to compare the data on the classifications. The Kaplan-Meier method was used to compare the survival curves and the log-rank test to compare the survival rates between them. SPSS 22.0 was used for the statistical analysis. The rates of relapse and aGVHD were calculated using R software (version 2.11.1), with death as a competitive risk factor.

| Effect of MSCs
The median follow-up time in the 13 pediatric patients was 1432  (Table 2).

| Rapid side effects
No fever, rash, or other allergies occurred in all patients during MSC infusion; there were also no cardiovascular or cerebrovascular events.

| Recent and long-term infections
The recent infection rate after MSC infusion was compared with that before MSC infusion and within 3 months after MSC infu- Because many late infections were defined as those occurring

days after transplantation and MSC infusion was conducted
within 100 days, the long-term viral, bacterial, and fungal infection rates will be compared between the MSC infusion group and the non-MSC infusion group after 100 days (Table 3). Fungal infections included suspected, clinically diagnosed, and confirmed infections. The diagnostic criteria used were in accordance with the literature. 11-13

| aGVHD and cGVHD
Two patients had grade III to IV aGVHD after transplantation in the MSC group; the incidence was 15.4%. Both patients had aGVHD before MSC infusion. No new aGVHD developed after MSC infusion, and the original aGVHD cases were not aggravated. The incidence of aGVHD in the non-MSC infusion group was 10.8%. There was no significant difference between the two groups (P = .624).
cGVHD occurred in two patients in the MSC group after transplantation, with an incidence of 19.2%. The incidence of cGVHD in the non-MSC infusion group was 21.0%. There was no significant difference between the two groups (P = .978) (Figures 1 and 2).

| Relapse
In the MSC infusion group, one patient relapsed; thus, the 3-year relapse rate was 10%. In the non-MSC infusion group, the 3-year relapse rate was 26.0%. There was no significant difference between them (P = .240; Figure 3).

| Survival
In the MSC infusion group, two patients died of grade III to IV aGVHD, one patient died of relapse and another patient died of infection. All other patients survived disease-free. The OS was 69.2%.
The 3-year OS in the non-MSC infusion group was 59.0%. There was no significant difference between the two groups (P = .517).
The 3-year DFS was 68.1% in the MSC infusion group and 58.8% in the non-MSC infusion group. There was no significant difference between the groups (P = .681) (Figures 4 and 5). can be detected simultaneously in patients with HC. The sensitivity of the blood BKV DNA is poor and was not detected in many patients with HC. Most patients with HC had negative findings;

| D ISCUSS I ON
however, once BKV DNA is detected, it may be associated with a poor prognosis of the patients. 1 The sensitivity of urine BKV DNA is good; it is often used for the early diagnosis of BKV-related HC.
However, the BKV can be found in some patients after transplantation or even in normal human urine; the copy number is low under normal conditions. The BKV copy number is usually more than 10 6 in the urine, which is considered to be associated with HC. 5 Due to the immune regulation function of MSCs, they can be used in the treatment of aGVHD, cGVHD, engraftment failure or delay, and bronchiolitis obliterans, and many products are used in other diseases besides hematological diseases. 24  fusion. Therefore, we did not find any aggravation of infection in our study in the short term.
Mesenchymal stromal cells have a rapid effect in the treatment of BKV-related HC, greatly reducing the pain of patients. We found that these cells do not have an anti-BKV effect, as there was no significant difference in the urine BKV DNA before and after MSC treatment. To assess whether there is an increase in the incidence of long-term infections after MSC infusion, 13 patients who received MSC infusion were classified into the MSC infusion group and those who received UCBT at the same time into the non-MSC infusion (control) group. The median follow-up time of the patients was >3 years. The infection rates of bacteria, fungi, and viruses were compared between the two groups, and no significant difference was found. These results indicate that MSCs have the function of tissue repair and immune regulation and have no obvious immunosuppressive effect. 28 Thus, we also did not find any aggravation of infection in our study in the long term.
The increased proportion of patients with HC complicated with aGVHD is attributed to the increased viral activation owing to enhanced immunosuppressive therapy in the patients with aGVHD.
However, MSCs also have a good therapeutic effect on GVHD.
MSC infusion can enhance the repair of pathological tissues and organs in patients with GVHD and can also prevent the occurrence of GVHD in patients who underwent HSCT. At present, MSC infusion is conducted in transplantation centers before HSCT as a preventive strategy for GVHD. Thus, there was no difference in the incidence of aGVHD and cGVHD between the MSC group and control group after neutralization of the two factors. There was also no significant difference in the OS and DFS in the survival analysis between the two groups, indicating that MSC infusion had no significant effect on survival.

| CON CLUS IONS
In conclusion, although umbilical cord blood-derived MSCs cannot reduce BKV replication, these cells are effective in treating BKV-related HC because of their good tissue repair and immune regulation functions. The treatment efficiency is high, and the symptoms of HC can be rapidly improved. MSCs did not increase the incidence of infection, recurrence rates of aGVHD and leukemia, or rate of survival.