| BackgroundAllogeneic hematopoietic stem cell transplantation(allo-HSCT)is a promising treatment for malignant and nonmalignant hematopathy,immune deficiency diseases,some genetic metabolic diseases and serious autoimmune diseases.Cord blood,since its unique advantages,such as rich source,convenient collection,long-term frozen storage,not easy to be infected by hepatitis virus,cytomegalovirus and EB virus,low HLA matching requirements,convenient to look up,and weak immunogenicity,low incidence of graft-versus-host disease(GVHD),especially chronic GVHD(cGVHD),and strong graft-versus-leukemia(GVL)effect,has become another important allo-HSCT source except bone marrow and peripheral blood.In the recent 20 years,our transplantation center has carried out unrelated umbilical cord blood transplantation(UCBT).In clinical practice,we found that patients after UCBT can have a series of clinical manifestations,such as non-infective fever,erythematous rash not caused by drugs,diarrhea and/or jaundice,and non cardiogenic pulmonary edema before neutrophil recovery,namely,pre-immune response(PIR),also known as pre-engraftment syndrome(PES).The reported incidence of PES ranges from 20%to 86.6%,and median onset time is 7(range,5-14)days.The main treatment for PES is methylprednisolone(MP).Our previous study found that mild PES can promote hematopoietic reconstitution and reduce primary graft failure after UCBT.However,the mortality of patients with severe PES is high,very easy to defer to severe acute GVHD(aGVHD),with the increasing of transplant-related mortality(TRM).Univariate and multivariable analysis showed that occurrence 5 to 6 days after UCBT and multiple clinical symptoms were two independent high risk factors affecting 180-day TRM in PES patients.The risk scoring system of PES after UCBT was established,one high risk factor was scored as 1.PES could be divided into mild-(PES-0),moderate-(PES-1),and severe-(PES-2)groups,and different doses of MP stratified intervention could significantly improve the prognosis of severe PES patients.ObjectiveThis study was divided into two parts.Part 1:A prospective,open,non randomized controlled clinical study was designed and carried out to further validate different doses of MP stratified treatment based on the risk score classification could control PES timely and effectively,and further improve the prognosis of severe PES patients without affecting cord blood engraftment.Part 2:In this study,based on the pre risk scoring system,Luminex protein liquid-phase chip detection technology and cytometric bead array(CBA)were used to screen and analyze biomarkers that may predict the occurrence and severity of PES.MethodsPart 1:MP stratified intervention based on risk classification in the treatment of PES after UCBT:a prospective,non-randomized,and open-label study(registration No.:ChiCTR-ONC-16009013).340 hematological malignancy patients who underwent UCBT from September 2016 to December 2018 were enrolled into this study.When patients were diagnosed PES,they were divided into mild,moderate,and severe according to two high risk factors,and treated with different doses of MP,0.5mg/kg/d for mild PES(PES-0),lmg/kg/d for moderate PES(PES-1)and 2mg/kg/d for severe PES(PES-2).Anti-CD25 monoclonal antibody(Basiliximab)would be used as second-line treatment if the symptoms progressed within 3 days,or the symptoms did not relieve within 5 days,or the symptoms only improved partially within 7 days(usage:12mg/m2,d1,d3,d7,d14;or dl,d4,d8,d15 for 4 times).Once symptoms were controlled,MP would be reduced by 10%per week gradually.The primary endpoint was 180-day TRM.The secondary endpoints were neutrophil engraftment rate,neutrophil engraftment time,platelet engraftment rate,platelet engraftment time,cumulative incidences of grade Ⅱ·Ⅲ and Ⅲ·Ⅳ aGVHD,cumulative incidence of 1-year cGVHD,cumulative incidence of 1-year relapse rate,1-year overall survival(OS),1-year disease free survival(DFS),1-year GVHD-free relapse-free survival(GRFS).Part 2:Screening and analysis of biomarkers for PES after UCBT.First,4 patients with hematologic malignancies who received UCBT were selected,including I without PES,1 with moderate PES(PES-I)and 2 with severe PES(PES-2).The day of UCBT was Day 0.Serum samples were collected for 0d,+3d,+5d,+7d,+10d,+12d.The levels of interferon(IFN)-γ,interleukin(IL)-1β,IL-1Rα,IL-2,IL-4,IL-6,IL-8,IL-10,IL-17A,monocyte chemoattractant protein(MCP)-1,macrophage inflammatory protein(MIP)-1α,MIP-1β,tumor necrosis factor(TNF)-α were detected by milliplex multi-factor detection kit,to screen out the cytokines with great difference between patients with PES and those without PES.Secondly,1 5 cases were selected.There were no PES in 3 patients,PES in 12 patients,including PES-0 in 5 patients,PES-1 in 4 patients,and PES-2 in 3 patients.The levels of cytokines IFN-γ,IL-6,IL-8,IL-10,MCP-1 and MIP-1α were detected by milliplex multi factor assay kit at different time points.Finally,the levels of cytokines IFN-y,IL-2,IL-4,IL-6,IL-8,IL-10,IL-17A,MCP-1,MIP-laand TNF-α were detected in 65 patients with hematologica malignancies at different time points after UCBT.Results1.Part 11.1 Patients characteristics and risk classification of PES:340 hematological malignancy patients who underwent UCBT from September 2016 to December 2018 were brought into analysis,191 were males,149 were females.The median age was 20 years(range 0.75-70 years),and the median weight was 44 kg(range 8-95 kg).116(34.1%)patients had acute lymphocyte leukemia(ALL),154(45.3%)patients had acute myeloid leukemia(AML),37(10.9%)patients had myelodysplastic syndrome(MDS),17(5%)patients had chronic myeloid leukemia(CML),16(4.7%)patients had other hematological malignancies.All patients received a single unit UCBT,306(90%)patients received B u/Cy2 based myeloablative conditioning regimen,25(7.4%)patients received TBI based myeloablative conditioning regimen,and 9(2.6%)patients received reduced intensity conditioning regimen.1 00(29.4%)patients had no PES,the other 240(70.6%)occurred PES.In 240 PES patients,risk score was mild(PES-0)in 152 subjects(63.3%),moderate(PES-1)in 67(27.9%),and severe(PES-2)in 21(8.8%).All the patients were follow up until 10th July 2019,the median follow-up time was 534days(range 194-1 041 days).1.2 Comparative analysis of patients and transplantation characteristics without PES and with different risk of PES:The median age of patients in severe PES group was 14 years(range 1-50 years),and the median weight was 31 kg(range 8-82 kg),which were significantly lower than those in other groups.There were no significant differences in gender,disease type,disease status before transplantation,conditioning regimens,GVHD prevention,infused TNC,CD34+cells,CD3+cells,ABO compatibility and HLA antigen matching between the donors and recipients.1.3 The primary endpoint:There was no significant difference in 180d-TRM between patients without PES and those with PES(16.0%vs 18.8%,P=0.579).Subgroup analysis showed that 180d-TRM of severe PES(PES-2)patients was 33.3%,slightly higher than that of patients without PES(16%),mild PES(PES-0)(18.4%)and moderate PES(PES-1)(14.9%)(P=0.236).Compared with the data before MP stratification intervention(from April 2000 to February 2012 92 PES patients after UCBT in our center),for 180d-TRM,although there was no statistical difference between mild PES(PES-0)and moderate PES(PES-1)before and after MP stratification intervention(PES-0.17.1%vs 18.4%,P=0.84;PES-1,21.9%vs 14.9%,P=0.428),180d-TRM of severe PES(PES-2)patients was significantly lower after MP stratified intervention(33.3%vs 68.4%,P=0.03 1).1.4 Secondary endpoints1.4.1 Cumulative incidence of neutrophil engraftment rate and time:There was no significant difference in the cumulative incidence of neutrophil engraftment rate and time between patients without PES and patients with PES(neutrophil engraftment rate:89.0%vs 96.2%,P=0.161;median engraftment time:17 days vs 17 days,P=0.962),Subgroup analysis showed that the cumulative incidence of neutrophil engraftment rate was the lowest in non-PES group(89.0%),followed by mild PES(PES-0)group(94.7%).Moderate PES(PES-1)and severe PES(PES-2)groups were 100%and 95.2%,respectively,with statistical differences among two groups(P<0.001).The median engraftment time was 17 days in non-PES group,17 days in mild PES(PES-0)group,15 days in moderate PES(PES-1)group,and 20 days in severe PES(PES-2)group,with statistical differences among three groups(P=0.015).1.4.2 Cumulative incidence of platelet engraftment rate and time:There was no significant difference in the cumulative incidence of platelet engraftment rate between patients without PES and patients with PES(84.2%vs 84.0%,P=0.239),but the median platelet time of patients without PES was faster than that of patients with PES(32 days vs 37.5 days,P=0.024).The results of subgroup analysis showed that there was no significant difference in platelet engraftment rate and time(P=0.313,P=0.078,respectively).1.4.3 Cumulative incidence of aGVHD:The cumulative incidence of grade Ⅱ to Ⅳand Ⅲ to Ⅳ aGVHD was significantly higher in PES group than that in non PES group(32.5%vs 18.0%,P=0.009;26.7%vs 13.0%,P=0.007).Subgroup analysis showed that the cumulative incidence of grade Ⅱ to Ⅳ and Ⅲ to Ⅳ aGVHD was the lowest(1 8%and 13%,respectively)in patients without PES.With the increase of PES severity,the cumulative incidence of aGVHD increased.The highest cumulative incidence of grade Ⅱ to Ⅳ and Ⅲ to Ⅳ was found in severe PES(PES-0 26.3%,21.1%;PES-138.8%,32.8%;PES-2 57.1%,47.6%;P<0.001)1.4.4 Cumulative incidence of 1-year cGVHD:There was no significant difference in the cumulative incidence of 1-year cGVHD and extensive cGVHD between patients without PES and patients with PES(12.0%vs 11.7%and 10.0%vs 8.3%,P=0.946 and P=0.658).Subgroup analysis showed that there was no significant difference in the cumulative incidence of 1-year cGVHD and extensive cGVHDin patients with PES at different risks(PES-0 9.9%和 6.6%;PES-1 13.4%和 9.0%;PES-2 19.0%和 19.0%;P=0.602和P=0.282)1.4.5 Cumulative incidence of 1-year relapse rate:There was no significant difference in 1-year relapse rate between patients without PES and those with PES(1 3.0%vs 8.3%,P=0.239).Subgroup analysis showed that there was no significant difference in l-year relapse rate of PES patients with different risks(PES-0 10.5%:PES-I 4.5%;PES-2 4.8%;P=0.245).Compared with the data before MP stratification intervention(from April 2000 to February 2012,92 PES patients after UCBT in our center),there was no statistical difference in the cumulative incidence of 1-year relapse rate of mild PES(PES-0)and severe PES(PES-2)before and after MP stratification intervention(PES-0,4.9%vs 11.1%,P=0.68;PES-2,5.3%vs 11.1%,P=0.68;PES-2,5.3%vs 4.8%,P=0.93),the cumulative incidence of 1-year relapse rate of patients with moderate PES(PES-1)after MP stratified intervention was significantly lower than that before MP stratified intervention(5.6%vs 18.8%,P=0.01 5)1.4.6 Probabilities of 1-year OS,DFS and GRFS:Probabilities of 1-year OS,DFS and GRFS were 75%,69%and 55%in non-PES group,72.9%,67.9%and 51.2%in PES group,no significant difference(P>0.05).Subgroup analysis showed that there was no significant difference in OS and DFS in PES patients with different risks(OS:PES-0 73.0%,PES-177.6%,PES-2 57.1%,P=0.225;DFS:PES-0 66.4%,PES-174.6%,PES-2 57.1%,P=0.374).However,1-year GRFS in the patients with severe PES(PES-2)were significantly lower than those with moderate and mild PES(28.6%vs 50.7%vs 54.6%,P=0.015).Compared with the data before MP stratification intervention,MP stratified intervention significantly improved the l-year OS,DFS and GRFS of severe PES(PES-2)patients(OS:56.7%vs 21.1%,P=0.021;DFS:56.70%vs 21.1%,P=0.017;GRFS:27.8%vs 5.3%,P=0.035),and significantly increased the 1-year DFS(72.7%vs 56.2%,P=0.028)of moderate PES(PES-1)patients;there was no significant difference in OS,DFS and GRFS for mild PES(PES-0)patients before and after intervention(OS:70.7%vs 71.9%,P=0.795;DFS:70.7%vs 65.1%,P=0.864;GRFS:48.8%vs 53.4%,P=0.84).2.Part 22.1 Screening of cytokines in patients with and without PES:Four patients were diagnosed as hematologic malignancies,including 1 case of CML,1 case of AML,I case of ALL,I case of MDS.I male,3 females,with a median age of 16 years(range,9-50 years).All patients received single-unit UCBT,with the conditioning regimen of FLU/BU/CY,and CSA combined with MMF as GVHD prophylaxis.According to the clinical diagnostic criteria and risk scoring system of PES,one patient did not occur PES,and three patients had PES,including one patient with PES-1 and two with PES-2.The results showed that the levels of IFN-γ,IL-6,IL-8,IL-10,MCP-1 and MIP-1αwere significantly different between patients with PES and those without PES.2.2 The selected cytokines were further verified by Luminex protein liquid chip platform:From September 2016 to February 2017,15 patients(9 males and 6 females)with hematological malignancies undergoing single-unit UCBT were enrolled in this study,with a median age of 10 years(range,9 months to 35 years).There were 8 cases of ALL,6 cases of AML and 1 case of juvenile myelomonocytic leukemia(JMML).According to the clinical diagnostic criteria and risk scoring system of PES,3 patients did not occur PES,12 patients had PES,of which 5 were PES-0,4 were PES-1,and 3 were PES-2.The levels of IFN-γ,IL-6,IL-8,IL-10,MCP-1 and MIP-1α at different time points were detected by milliplex multifactor detection kit.The results showed that the level of IL-8 on Day 5 in patients with PES was significantly higher than that in patients without PES(32.50 ± 13.81 vs 10.76 ± 5.44,P=0.021).Subgroup analysis showed that the level of IL-8 on Day 5 in PES-0 group was significantly higher than that in patients without PES(39.63±10.91 vs 10.76±5.44,P=0.048).IL-10 levels on Day 10 and Day 12 in PES-2 group were significantly higher(311.44±60.57 and 370.23±136.61,P=0.05 and P=0.011,respectively).2.3 The selected cytokines were further verified by CBA method:From July 2017 to January 2020,63 patients(32 males and 31 females)with malignant hematologic diseases who received single-unit UCBT in our transplantation center were selected.The median age was 16 years(range,1-55 years),and the median weight was 44kg(range,8-88kg).There were 16 cases(25.4%)of ALL,35 cases(55.5%)of AML,4 cases(6.3%)of myelodysplastic syndrome(MDS),2 cases(3.2%)of chronic myeloid leukemia(CML)and 6 cases(9.6%)of other hematological malignancies.22 cases(34.9%)did not occur PES,41 cases(65.1%)had PES.According to the PES risk scoring system for the 41 PES patients,22 patients(53.6%)with mild PES(PES-0),13(31.7%)with moderate PES(PES-1),and 6 patients(14.7%)with severe PES(PES-2).The levels of cytokines were detected by CBA at different time points.Results showed that,on Day 5 after UCBT,the levels of IL-6 and IL-10 in patients with PES were significantly higher than those in patients without PES(IL-6,42.98 ± 52.63 vs 15.14 ±17.03,P = 0.010;IL-10,21.82±40.95 vs 3.91 ± 4.01,P=0.015),and the highest level was found in PES-2 patients;on Day 7 after UCBT,the levels of IL-2,IL-6,IL-8 and IL-10 in patients with PES were significantly higher than those in patients without PES(IL-2,3.27 ± 2.82 vs 1.75 ± 1.51,P=0.002;IL-6,36.78 ± 44.36 vs 15.27 ± 10.19,P=0.017;IL-8,72.12±74.43 vs 15.53±5.55,P=0.025;IL-10,37.8±39.53 vs 6.25±5.92,P=0.000),and they were also the highest in PES-2 patients.However,the levels of MIP-1 a in patients with PES on Day 0 and Day 7 after UCBT were significantly lower than those without PES(6.94 ± 5.09 vs 29.56±38.43.P=0.000;+7d,8.63±8.11 vs 20.36 ± 38.20,P=0.001)Conclusions1.PES is a common immune response after UCBT.In our transplantation center,we performed myeloablative conditioning regimen without ATG,the incidence of PES was as high as 70%.According to the two high risk factors(early onset time,more clinical symptoms)that affect 180-day TRM of PES patients,the risk scoring system of PES after UCBT was established.The level of cytokines showed that the increase of IL-6,IL-8,and IL-10 in early stage of transplantation might predict the occurrence and severity of PES.However,more samples need to be further defined2.This prospective clinical study further confirmed that PES was benefit for cord blood engraftment.Different doses of MP stratified intervention could significantly reduce 180-day TRM and improve 1-year OS,DFS and GRFS in severe PES patients.3.However,the prognosis of severe PES patients is still the worst.It is necessary to find new interventions to further improve the survival and prognosis of severe PES patients. |
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