| IntroductionCord blood transplantation (CBT) is an effective and potential curative treatment for pediatric patients with hematological malignancies who lack HLA-identical related or unrelated donors. Over the last25years, the field of umbilical cord blood (UCB) banking and transplantation has grown exponentially. Over600000UCB units have been stored for transplantation worldwide, and＞30000CBTs have been performed. UCB serves as an alternative stem cell source; only30%of patients who require an allograft will have a human leukocyte antigen (HLA)-matched sibling donor. Despite＞20million adult volunteer donors in the National Marrow Donor Program and affiliated registries, many patients, particularly patients of diverse racial/ethnic backgrounds, will not have a suitably matched, unrelated volunteer donor identified in the required time period. UCB has extended access to transplantation, especially to patients of racial and ethnic minorities and is rapidly available. Post-transplantation immune disorders, including pre-engraftment syndrome (PES), engraftment syndrome, and acute graft-versus-host disease (GVHD) are problematic in CBT. The complex and intricate pathophysiology of post-transplantation immune disorders is a consequence of interactions between the donor and host innate and adaptive immune responses. PES, a clinical entity of unknown pathogenesis, has been described in patients receiving CBT. Although a uniform definition is lacking, PES is commonly characterized by noninfectious fever and various other clinical findings before neutrophil engraftment, including skin rash, pulmonary infiltrates, and diarrhea, jaundice, or weight gain. To date, PES is an entirely clinical entity with no known pathognomonic histopathologic changes or biochemical markers. Kishi et al were the first to report a pre-engraftment immune reaction, which occurred in35of45adult recipients of reduced-intensity conditioning CBT. However, PES still remains poorly characterized and its clinical significance and the prognostic impact after CBT are unclear. The first part of the research is to address these issues, we retrospectively analyzed the incidence, risk factors, and clinical outcomes of PES in unrelated CBT recipients enrolled in a multicenter CBT trial. The second part of the research, we investigated that donor monocyte-macrophages were the key effector cells which expressed high level cytokines in PES patients. By target gene prediction+GO+pathway analysis, we suspected that the occurrence of PES were activated by monocytes and macrophages, and associated with three possible Toll, NOD, and RIG-I-like receptor signaling pathways.Methods and patientsBetween April,2000and December,2013,240consecutive patients with hematologic malignancies underwent unrelated CBT in Anhui Provincial Hospital. All of the240hematologic malignancies patients referred for CBT had one of the features associated with poor outcomes:ⅰ) Patients were in first complete remission (CR1) but have high risk factors at first diagnosis:ALL with adverse cytogenetics or molecular abnormalities [ph+chromosome (BCR-ABL positive), hypodiploidy,11q23abnormalities (MLL rearrangements)], or with high level of minimal residual disease (MRD)(1%or more after completion of6weeks of induction therapy); AML with adverse cytogenetics or molecular abnormalities [inv(3)(q21q26.2) or t(3;3)(q21;q26.2), t(6;9)(p23;q34),11q23abnormalities (MLL rearrangements),-5or del(5q),-7or del(7q), complex karyotype, normal cytogenetics with FLT3-ITD mutation], or with a prior history of myelodysplastic syndrome (MDS). ⅱ) CR2(but the length of CR1≤12months), CR3or more. ⅲ) Primary induction failure, or no remission after relapse with salvage chemotherapy.Cord blood units that were serologically matched for≥4of six HLA antigens and which contained at least3×107nucleated cells/kg and1.2×105CD34+/kg of recipient body weight before freezing were obtained from the cord blood bank at the China Cord Blood Bank Network. The units were not depleted of T lymphocytes. The choice to transplant one vs. two UCB units was based solely on cell dose criteria. If a UCB unit with the minimum cell dose was not available, the patient was transplanted with two partially HLA-matched UCB units, which needed to have a minimum cryopreserved dose of3.5×107nucleated cells/kg and2×105CD34+/kg, respectively. All patients received a myeloablative conditioning regimen of TBICY[total body irradiation (TBI, total12Gy,4fractions) and cyclophosphamide (CY,60mg/kg daily for2days)](age≥14years or primary induction failure or no remission after relapse) or BuCY2[busulfan (0.8mg/kg every6h for4days) and CY](age＜14years or prior radiotherapy which would presuppose a high risk of toxicity). Cytarabine (2.0g/m2every12h for2days) and/or carmustine (BCNU,250mg/m2) was added to the myeloablative conditioning regimen in order to penetrate the blood brain barrier to kill the CNS leukemia cells maximally (for some patients). Fludarabine (30mg/m2daily for4days) was combined with BuCY2to promote engraftment.For GVHD prophylaxis, all patients were given a combination of cyclosporine (Novartis, Stein, Switzerland) and mycophenolate mofetil (Roche, Basel, Switzerland). Intravenous cyclosporine was started (2.5-3mg/kg/d) on day-1and continued until patients were able to take cyclosporine orally with target trough levels of200ng/mL at least1month. Mycophenolate mofetil (25mg/kg/d) was given on day+1. The rapidity of tapering was based on the presence or absence of GVHD, infectious disease, and relapse risks.PES was defined as unexplained fever＞38.3℃not associated with documented infection and/or an unexplained erythematous skin rash resembling that of acute GVHD, with either the fever or the rash occurring before or at neutrophil recovery. Specifically, fever attributed to PES was not associated with any clinical evidence of infection, with patients having both a negative infectious disease workup and a continued lack of response to broad-spectrum antimicrobial agents. Erythematous skin rash attributed to PES was not associated with any clinical suspicion of drug allergy.Weight gainwas defined as a3%increase in body weight between the day of CBT and the onset of PES. Noninfectious diarrhea was defined as passage of watery stools more than twice a day for at least3consecutive days with no evidence of infectious etiology.Variables related to the patient, the disease, and the transplantation procedures were compared with the use of the chi-squared test for categorical variables. The Cox regression model was used for multivariate analysis of clinical variables. The end-points were engraftment, graft failure, PES, acute and chronic GVHD, relapse, transplant-related mortality (TRM), malignancy-free survival (MFS), overall survival (OS). Time-to-event outcomes for neutrophil and platelet engraftment, PES, GVHD, TRM, and relapse were estimated using cumulative incidence curves. The difference in the cumulative incidence curves was based on Gray’s test. The Kaplan-Meier method was used to estimate the probabilities of the survival (OS and MFS) which were compared using the log rank test. p<0.05was considered statistically significant. All analyses were performed with SPSS (version17.0).The second part of the research, we investigated that donor monocyte-macrophages were the key effector cells which expressed high level cytokines in PES patients. Serum samples with known miRNA expression in patients with PES, using the difference in miRNA expression levels log2-ratio, Scatter plot diagram comparing the expressions. Difference between the two samples by miRNA expression was analyzed before and after PES. The detection of miRNA expression maybe found to play an important role in the pathogenesis of PES. This would also be helpful to find the molecular signaling pathways and the mechanism of PES.ResultsFrom2000to2012(the first period of the clinical research), a total of137patients received CBT, and73patients (53.3%) developed PES at a median of7days (range5-15days). The cumulative incidence of PES was53.3%(95%CI,45.9-65.4%). Comparing the patient demographics and graft characteristics of the PES patients and the non-PES patients, we found that myeloblative conditioning and double CBT were the high risks for the development of PES. But there were no significant differences between the2groups in terms of age, sex, weight, underlying malignancy, BM status at CBT, HLA match, ABO compatibility or conditioning approach. There also were no between-group differences in total infused TNC dose, total CD34+cell dose, total CD3cell dose, and total CD56cell dose. Total of14patients developed primary engraftment failure,2patients recovered with autologous hematopoisis, and the other12patients salvaged with haploid-transplantation. We found that patients with no PES was the high risk for primary engraftment failure (p＜0.001).64patients had non-infectious fever (87.6%) within14days,59patients with red rash (80.8%),16patients with non-infectious diarrhea (21.9%),11patients with weight gain (≥3%)(15.1%),9patients with elevated liver transaminases (12.3%),5cases with elevated bilirubin (6.8%),4cases of renal damage (5.5%), and5patients with hypoxemia or pulmonary edema (6.8%). Rash and non-infectious fever are the most specific diagnostic symptoms, but weight gain, non-infectious diarrhea, liver and kidney dysfunction and other syndromes were not the specific characters of PES.The incidence of acute GVHD in the PES group was higher than the non-PES group patients (p=0.019):Grade II-IV acute GVHD developed in25(34.2%) patients of the73PES group patients, and the100-day cumulative incidence of grade II-IV acute GVHD was35.6%(95%CI,29.3-43.8%); Only9patients developed II-IV acute GVHD in the64non-PES group patients, and100-day cumulative incidence of grade II-IV acute GVHD was15.2%(95%CI,8.5-22.6%).The cumulative transplant-related mortality was30.6%(95%CI:20.3%-40.9%) in PES patients, and23patients (27%) died due to the transplant-related mortality (TRM). TRM was developed in28patients (43.8%) in the64non-PES group patients, and the cumulative transplant-related mortality was43.5%(95%CI:34.5%-50.2%) Although the transplant-related mortality was higher in the non-PES group than the PES group patients, but there was no significant difference (p=0.10). Total of20patients developed disease recurrence (14.5%). The cumulative recurrence rates were13.5%(95%CI:6.6%-19.2%) and15.8%(95%CI:9.5%-22.5%) in PES and non-PES group patients, and there was also no significant difference (p=0.74).5-year OS and5-year MFS did not differ depending on the presence or absence of PES after CBT [63.9%(95%CI,58.6%-72.2%) vs59.2%(95%CI,52.1%-66.2%),59.2%(95%CI,50.8.5%-64.3%) vs56.8%(95%CI,48.6%-62.5%), respectively; p=0.65,0.82]. By the time of the occurrence of PES, the maximum temperature, clinical symptoms, cyclosporine concentrations, methylprednisolone (MP) initial dose, the days from the effectiveness of MP, the days of maintains of MP, ineffectiveness of MP, we found that PES occurred in7days (＜7days), over three or more clinical symptoms, poor effectiveness of MP (ineffectiveness within in one week) were the poor prognostic risk factors of PES.We made a PES scoring system according to3high risk factors which had been described, and investigated the impact of high risk factors to the PES patients prognosis. The existence of one risk factor was defined as1point, and no risk factors with0score. Our study showed that the cumulative incidence of180-days transplant-related mortality (TRM) was higher in patients with at least one risk factors (43.6%vs14.8%, p=0.021); and180-days transplant-related mortality (TRM) was also higher in patients with more than one risk factors (2+3) than patients with one risk factor (60.8%vs19.4%, p=0.018);180-days TRM was also higher in patients with three risk factors (3scores) than patients with two risk factors (82.6%vs33.9%, p=0.015). For1-year OS, there were significant differences during the4groups patients (0point:82.8%;1point:79.2%;2points:48.7%;3points:0%)(p＜0.001).From March2012to December2013((the second period of the clinical research)),103patients with hematologic malignancies underwent UBCT, and79patients developed PES. We used methylprednisolone (MP) intervention according to the different PES score system. Patients with1high risk factor were treated with MP1mg/kg/d, and Patients with2or more high risk factors were treated with MP2mg/kg/d. After the intervention of MP, there were no significant statistical differences between patients with0score (n=26patients) and with at least1score (1+2+3points)(n=53patients), and there were also no significant statistical differences between patients with1score (n=27patients) and with at least2scores (2+3points)(n=26patients)(p=0.16; p=0.39). After the treatment of MP according to the PES score system, the prognosis of severe PES was significant improved by1-year OS between the two different periods (April2010to February2012and March2012to December2013)(25.5%vs72.3%)(p=0.019).According to our center experimental results and the clinical manifestations of PES, we found that the major effector cells of PES are donor monocyte-macrophages. We also found that donor monocyte-macrophages are the key effector cells which expressed high level cytokines in PES patients. Using HiSeq high-throughput sequencing technique of microRNA, we detected the serum miRNAs in3PES patients with periods of pre-PES, at the time of PES, and the remission of PES. About39obviously expressed miRNAs, such as hsa-let-7i-5p、hsa-mir-98-5p、 hsa-mir-152-3p、hsa-mir-223-3p, et al, were detected. By target gene prediction+GO+pathway analysis, we suspected that the occurrence of PES were activated by monocytes and macrophages, and associated with three possible signaling pathways: Toll, NOD, RIG-I-like receptor signaling pathways.ConclusionsIn conclusion, PES seems to be common after CBT and may be associated with enhanced engraftment, and patient with no PES was the high risk for primary engraftment failure. Rash and non-infectious fever are the most specific diagnostic symptoms. Although PES is closely associated with acute GVHD, PES after CBT is not associated with significant morbidity and is easily manageable with intravenous steroids. PES occurred within7days (<7days), over three or more clinical symptoms, poor effectiveness of MP (ineffectiveness within in one week) were the poor prognostic risk factors of PES. We made a PES scoring system according to these3high risk factors, and after the treatment of MP according to the PES score system, the prognosis of severe PES was significantly improved. We found that the major effector cells of PES are donor monocyte-macrophages. We also found that donor monocyte-macrophages are the key effector cells which expressed high level cytokines in PES patients. By target gene prediction+GO+pathway analysis, we suspected that the occurrence of PES were activated by monocytes and macrophages, and associated with three possible Toll, NOD, and RIG-I-like receptor signaling pathways. Because failure to recognize PES in CBT recipient risks unnecessary complications of this syndrome and unnecessarily long, empirical treatment, physicians should be aware of the possible occurrence of PES after CBT, especially in patients who have relevant risk factors for developing PES. |
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