Mechanism Of Regimen-related Toxicity In Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation Role Of Cytokines/Chemokine And Th17 In Regimen-related Toxicity

Background and ObjectiveAllogeneic hematopoietic stem cell transplantation(allo-HSCT) can cure hematological malignancies and autoimmune diseases, espically for refractory leukemia with unattainable complete remission(CR) before transplantaion. However, conditioning regimen has become a critical element for the HSCT. The ideal conditioning regimen can effectively reduce the tumor burden, suppress the recipient's immune system and less toxicity.It has become a critical issue to solve the conditioning of composition and dose. However, so far, we have not find an ideal of conditioning regimen. Conditioning regimen usually include as follow:myeloablative conditioning regimen (the standard conditioning regimen):total body irradiation/ cyclophosphamide (TBI/CY) or busulfan/CY (Bu/CY)); Reduced intensity regimens (RIC); non-myeloablative conditioning regimen and the intensity regimen. In theory, the more intensity of conditioning can remove the more residual tumor cells and less relapse, but at the same time regimen-related toxicity (RRT) also is higher. Therefore, we need choose the appropriate conditioning regimen in clinical practice, according to type of disease, disease status, the age and complications.Researchers indicated:IL-6, IL-1βand TNF-a are the important inflammatory cytokines in the RRT. In recent years, many researches found that chemokines also play an important role in the RRT, the studies showed that:CXCL-10 is important in the RRT.Currently, researchers indicated that chemoradiotherapy damaged tissues to release of multiple cytokines, which can cause severe RRT. At the same time, the cytokines also can cause early aGVHD. So it is very important of confirming the mechanism of tissue damage by conditioning.Besides these inflammatory/chemokines, whether or not others involved the RRT? Th17 is a new CD4+ T cell subsets which found in recent years, following the Thl, Th2, Treg cells. Recent studies indicated that Th17 plays a very important role in inflammation, autoimmune diseases, cancer, transplantation rejection and graft versus host disease (GVHD). However, it has not been reported whether or not Th17 involves RRT. We have known that conditioning regimens can damage the tissues, cause inflammatory response, and the mechanism of RRT is an inflammatory response. So we speculated that Th17 and its main effector-IL-17A might be play a role in the RRT.Standard regimen, reduced intensity regimens and the intensity regimen are common in clinic. The aims of this study as follow:in order to better understanding of RRT and find out appropriate early diagnostic index to take preventive measures, to study whether or not the regimens (TBI/CY, BuCY, Bu/Flu, and intensity regimen) could cause cytokines to change, to study the relationship between Th17 and RRT and in order to detect early diagnostic indicators and reveal the mechanism of RRT. Method40 patients during 2009-2010 in Nanfang hospital were received the standard pretreatment (TBI/CY, BuCY), reduced toxicity regimens (Bu/Flu) and the intensity regimen (Flu/Ara-C+TBI/CY or TBI+CY+VP-16/VM-26),9,8,11,12 cases respectively. The blood sample were collected that 1 day before conditioning and 0 day before stem cells infusion in patients. The patients and control group were harvested morning peripheral venous blood of about 2ml, EDTA anticoagulation, peripheral blood mononuclear cells (PBMC) and plasma were harvested by standard Ficoll-Paque density centrifugation. Plasma kept at -20℃. Plasma concentrations of IL-1β, IL-6, TNF-α, CXCL-10 and IL-17A were measured by Enzyme-linked immunosorbent assay(ELISA). And For Th17 cell detection, PBMCs were stimulated firstly for phorbol myristate acetate(PMA), ionomycin (IM) and brefeldin A, upon harvest, cells were stained with antibodies, and measured by flow cytometry. We also investigate the incidence and severity of RRT and aGVHD, compare the relationship between the variance and the RRT or aGVHD.Statistical analysis was performed using SPSS version 13·0 for Windows software. Comparison between the two groups used two independent samples t test. Comparison of before and after conditioning used paired-samples t test. We used one-way ANOVA with multiple groups before conditioning; the comparison of after conditioning, if it is difference of multiple groups before conditioning, using ANCOVA; if not, then using one-way ANOVA. We compared with the incidence of RRT and aGVHD in different regimens by Kruskal-Wallis H test. And comparison the correlations of variance and RRT or aGVHD used Spearman correlation analysis. The significance level was set at P< 0.05.Result1. The basic level of Cytokines:Plasma concentrations of IL-6, IL-1β, TNF-a, CXCL-10 had no significant difference between control and patients before conditioning (P>0.05)2. comparison of cytokines range regimens before conditioning:IL-1β, IL-6, TNF-a were no significant differences (P> 0.05); CXCL-10 had significant difference among the groups(P= 0.006).3. Conditioning influence on cytokines:3.1 Comparison of the levels of cytokines in all patients between pre-and post-conditioning, the results suggested:The level of TNF-a was significantly higher in the post-conditioning (P<0.001);The mean of IL-6 pre-and post-conditioning were (2.19+1.24)pg/ml vs (6.05+12.15)pg/ml(P= 0.054), the level of IL-6 was also higher post-conditioning; Otherrs were no significant difference pre-and post-conditioning.3.2 The levels of cytokines post-conditioning: (Ⅰ)The levels of IL-1β, IL-6 and TNF-αwere no significant difference among the groups after conditioning; (Ⅱ)The levels of CXCL-10 was ignificant difference among the groups after conditioning, combined with the adjusted mean, BuCY group was higher than the other groups.3.3 The levels of cytokines between pre and post-conditioning:(1) The level of IL-1βwas no significant difference between pre and post-conditioning of regimens respectively (P> 0.05);(2) The level of IL-6 was no significant difference between pre and post-conditioning of regimens respectively (P>0.05), but the mean of IL-6 pre-and post-conditioning is as follow:BuCY (2.63±1.78) pg/ml vs (11.32±15.70) pg/ml, intensity regimens (2.41±1.09) pg/ml vs (8.32±17.55) pg/ml, TBI/CY (2.30±1.32) pg/ml vs (3.80±3.53) pg/ml, respectively.Bu/Flu was no significant differences. (3) The level of TNF-αbetween pre-and post-conditioning:the means of pre-and post-conditioning is as follow:Bu/Flu (3.91±2.54) pg/ml vs (6.88±3.38) pg /ml(P=0.035), intensity regimens (5.45±3.85) pg/ml vs (8.80±3.27) pg/ml (P= 0.004), BuCY (8.67±6.34) pg/ml vs (13.60±7.77) pg/ml(P=0.066), TBI/CY (5.12±3.85) pg/ml vs (8.17±6.14) pg/ml(P=0.237), respectively.(4) The levels of CXCL-10 between pre-and post-conditioning:the mean pre-and post-BuCY were (321±139)pg/ml vs (2488±381)pg/ml respectively; And intensity regimens had no significant variation.4.RRT4.1 RRTs were graded according to Bearman's criteria. The study cohort comprised 40 patients. The incidence of RRT was 82.5%. TBI/CY 77.8%, BuCY 75%, Bu/ Flu 81.8%, intensity regimens 91.7%, respectively. There were not significant difference among the groups (P=0.831). RRT was more common in the gastrointestinal tract and oral stamatitis,35%,57.5%, respectively. Oral and gastrointestinal tract RRT were nor significant difference among the four groups. Organ toxicity is summarized as follow:bladder 10%, heart 7.5%, liver 15%,5% of the lung, kidney 2.5%. Significant central nervous system (CNS) toxicity was not observed. Besides one patients developed grade III toxicity, others were gradeⅠ～Ⅱtoxicity. No patients died of an RRT.4.2 The correlation of RRT and cytokines:The levels of IL-1βdid not variance between pre-and post-conditioning, this does not consider it. The correlation of IL-6, TNF-a and CXCL-10 with overall RRT incidence or difference regimens suggested that except the ratios of TNF-a(post-/pre-conditioning) had moderate positive correlation with the RRT incidence in TBI/CY (r=0.756, P=0.030), others had no a significant correlation(all P>0.05). 5. The incidence of aGVHD+30 days after allo-HSCT5.1 The incidence of aGVHD was (18/40,47.5%). TBI/CY 5/9(55.6%), BuCY2/8 (37.5%), Bu/Flu 4/11 (36.4%), intensity regimens 7/12(58.3%), respectively. There were not significant difference among the groups (P=0.654). Besides one patients developed gradeⅢ°aGVHD, others were degreeⅠ-Ⅱ°No patients died of an aGVHD+30 days after allo-HSCT.5.2 The correlation of aGVHD and cytokines:It had no a significant correlation between the correlation of IL-6, TNF-a and CXCL-10 and aGVHD (all P>0.05).6. The relationship of Th17 with RRT6.1 The percentage of Th17 and the levels of IL-17 A had no significant difference between control and patients before conditioning.6.2 The percentage of Thl7 was no significant differences pre-or post-conditioning, and so do IL-17 A (both P>0.05)).6.3 Comparison of Thl7 and IL-17A between pre-and post-conditioning:①The percentage of Thl7was significantly high post-conditiong(P<0.001), but the absolute number of Th17 in peripheral blood decreased significantly (P<0.001), and IL-17A was no significant difference (P=0.503);②The mean of Thl7 pre-and post-conditioning is as follow:BuCY (0.75±0.40)% vs (4.11±3.92)%(P=0.035), intensity regimens (1.40±1.06)% vs (3.42±2.76)%(P= 0.048); TBI (1.15+0.89)% vs (3.60+3.65)%(P=0.068); Bu/Flu (1.39±1.16)% vs (2.52±2.11)%(P=0.178); the number of Th17 all of four groups significantly decreased (P<0.05). The level of IL-17A was significant difference in intensity group, pre-and post-conditioning were (3.15±2.17) pg/ml vs (4.08±2.81) pg/ml (P=0.021), other groups were no significant difference.6.4 It had no a significant correlation between the correlation of Th17, IL-17A and and RRT, aGVHD (both P> 0.05)).7. The incidence of RRT and aGVHD had no a significant correlation(r=-0.235, P=0.145).Discussion and Conclusion1. The researches indicated that the levels of IL-1β, IL-6 and TNF-a were the important inflammatory cytokines in the RRT. These significantly increased post-conditioning. In our study, the levels of IL-1βwas no significant changes between pre-and post-conditioning.The result did not consist with the major former study,, but there are also study consistented with ours, but there are also have some research results that consisted with ours. Combination of means and statistics, the levels of IL-6, TNF-αwere increased, which indicated that they might be the early indicators of RRT.2. Except Bu/Flu group, the levels of IL-6 were increased in post-conditioning.. The results showed Bu/Flu conditioning might be have smaller influence on IL-6, and intensity group had no significant difference with conventional conditioning. This is consistent with other reports.the In our study, The levels of TNF-αincreased post-conditioning among the groups. TNF-αmay be a common factor in the RRT, which was consistent with other reports.The levels of CXCL-10 was higher in the BuCY group than others. The former researches indicated the levels of CXCL-10 increased in the post-conditioning included TBI, our study suggested CXCL-10 was related to RRT, but the role of CXCL-10 in different groups must be further clarified.3. In this study, the overall incidence and severity of RRT were lower than the literatures. The incidence of early aGVHD was consisted with others. And the incidence of RRT and aGVHD had no significant difference in four kinds of conditioning. But incidence of RRT was higher in the intensity group than others. The results might be obtain disparity by enlarging the cases. In our study, the results showed that it had no correlation between cytokines and RRT or aGVHD. The better preventive measures could interrupt the natural course might be the reason, but we did not find the supported reports, that need to be further confirmed.4. The relationship between conditioning and Th17:Several animal studies indicated that Thl7 was closely related to inflammation and GVHD. Because the conditioning can provoke inflammation, and RRT is related to aGVHD, we hypothesized that RRT might be associated with Th17. In this study, the relative percentage of th17 was significantly increased post-conditioning, but the number of Th17 in peripheral blood was extremely decreased. The levels of IL-17A increased after intensity group, and the number of Th17 fewer than others, which indicated that Th17 might be activated and involved the RRT. This is consisted with our hypothesis. The levels of IL-17A had no significant difference between pre-and post-other groups, the reason might be the balance of the conditioning destroyed Th17 or activitied them. So far, we have not find the supported reports, so further research is needed.In this study, Based on seneral variables in the four conditioning (TBI/CY, BuCY, Bu/Flu, and intensity) changes pre-and post-conditioning, we obtain the results as follow:(1) The plasm levels of IL-6, TNF-a were increased, which supported that they may be an early indicators of RRT; (2) Bu/Flu group had smaller influence on IL-6. And TNF-αmight be a common factor in the RRT; CXCL-10 might be associated with the RRT; (3) The incidence of RRT and early aGVHD was no significant difference among four regimens.There had no close correlation between the cytokines and RRT or aGVHD; (4) The number of Th17 was decreased, but the relative percentage of Th17 was increased through conditioning.Summary:The aim of this study:Role of cytokines and Th17 in regimen-related. The study indicated that cytokines played important role in the RRT; Reduced toxicity regimens might be less influence on cytokine, and the toxicity of intensity conditioning was fairly conventional ones; RRT may be related to Th17.