| Part Ⅰ. Clinical study of interferon a-2b pre-emptive therapy in leukemia patients with relapsing tendencies after allogeneic hematopoietic stem cell transplantationObjective:To determine the efficacy and safety of IFN-a-2b pre-emptive therapy for leukemia patients with relapsing tendencies after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:Retrospectively analyzed 31 leukemia patients with relapsing tendencies after allogeneic hematopoietic stem cell transplantation (allo-HSCT) from Jan 1 st,2006 to Mar 31th,2014 in our hospital. After allo-HSCT, patients were periodically tested for leukemia-associated immunophenotype (LAIP), bone marrow, minimal residual disease, leukemia specific or related fusion genes, and donor cell chimerism through multi-parameter detection to evaluate disease remission. Of 98 patients who were evaluated and undergone therapy,31 patients received IFN-a-2b pre-emptive therapy once a relapse was detected,, and 67 patients received non-IFN-a-2b therapy.Results:For the 31 patients who received IFN-α-2b pre-emptive therapy, they received IFN-α therapy for a median time of 60 days (range:5-720) after transplantation and the median interferon treatment for all patients was 50 doses (5-600) (3 million U/dose). Twenty five patients had effective treatment and their disease reverted without hematological relapse (effective rate 80.6%). Two patients progressed to hematological relapse again after temporary response; three patients had no response and disease progressed to hematological relapse. For the 67 patients who received non-IFN-a-2b therapy,22 patients had a good response(effective rate 32.8%),45 patients had no response,among them 44 patients progressed to hematological relapse at a median time of 35 days (range:6-940), and one is living with continuous positive of TCR rearrangement. Efficient rates of two groups have a significant statistical significances(P=0.000). At the last follow up, all patients in IFN-a-2b group had a good toleration, and no patient terminated therapy due to side effects. In the middle of IFN treatment,18 patients(58.1%) developed GVHD, among them 6 patients(19.4%) developed aGVHD,4 patients(12.9%) having grade I-II; 2 patients (6.5%) had grade III-IV skin aGVHD,14 (45.2%) had limited cGVHD. The median follow-up time was 21 (4.5-78.5) months. In IFN-a-2b group,22 patients had disease-free survival, the 5-year overall survival rate (OS) was 47.0%±13.9%, and the leukemia-free survival rate (LFS) was 38.7%±13.1%; the 5-year OS of non-IFN-a-2b group patients was 14.5%±10.7%, and the LFS was 12.5%±9.4%, lower than IFN-a-2b group (P=0.000,P=0.002 respectively). GVHD patients had significantly greater treatment efficiency than patients without GVHD (88.9% vs 53.8%, P=0.043, P<0.05).Conclusion:IFN-a-2b pre-emptive therapy can revert disease progression for leukemia patients with relapsing tendencies after allo-HSCT, and can remove minimal residual disease, effectively prevent high-risk patients to the progress of hematology recurrence, and reduce the risk of recurrence.Part II. Clinical studies of the combination of IFN-a and donor lymphocyte infusion for the treatment of acute leukemia relapse post allo-HSCTObjective:To evaluate the efficacy and safety of the combination of interferon-a(IFN-a) and donor lymphocyte infusion (DLI) named as cytokine-activated donor lymphocyte infusion (aDLI) for the treatment of acute leukemia relapse post allo-HSCT.Methods:Retrospective study of 73 clinical cases of allo-HSCT postop patients with leukemia from Jan 1,2006 to Mar 31,2015 at our department. The study include regular examination of bone marrow after allo-HSCT, minimal residual disease, the specificity of the fusion gene, donor chimerism rate of cells, evaluation situation of the disease remission, once the disease recurrence, aDLI group treated with IFN-a followed by G-CSF-mobilized donor peripheral blood stem cell (G-PBSC) infused, tDLI only infused with G-PBSC. There are 53 cases for aDLI and 21 for tDLI treatments. Basic clinical features, curative effect in the treatment, survival condition and related adverse reactions were compared between the two groups.Results:1. There were no statistical differences in the clinical characteristics between two groups.2. The Overall complete remission (CR) rate in aDLI group was 76.9% and 23.8% in tDLI group (P=0.000). The CR rate of patients combined chemotherapy was 87.1% vs 25% (P=0.008) for aDLI group and tDLI group, respectively. In the group without chemotherapy, the CR rate of acute myeloid leukemia (AML), the CR rate in aDLI group reached 66.7% and 20% in tDLI group, respectively (P=0.128). According to the comparison of the diseases:The CR rate of acute myeloid leukemia(AML):aDLI group 80%, tDLI group 28.6%(P=0.005); The CR rate of acute lymphatic leukemia (ALL):aDLI group 77.8%, tDLI group 14.3% (P=0.004).3. The incidence of acute graft versus host disease (aGVHD) of aDLI group was higher than that of the tDLI group and there was a statistical significant difference between the two groups(51.9% vs 14.3%, P=0.003), while, neither the chronic graft versus host disease(cGVHD) nor treatment related mortality (TRM) reached the statistical significant difference, but aDLI group have a obvious high incidence rate versus the limit III-IV GVHD in tDLI group, which show 34.6% vs 9.5%(P=0.030), and 3 of sufferers died for critical GVHD finally under the circumstance of hematologic remission although.4. Treatment related adverse reaction:The incidence of fever for aDLI group is apparently higher than tDLI group,57.7% vs 23.8%(P=0.009), there is no more marked difference for the related toxicity of influence, hematologic toxicities, gastrointestinal reaction.5. As to the follow-up time, the 5-year OS of aDLI group and tDLI group were 30% vs 8.9%(P=0.001), the 5-year LFS of the two groups were 14.8% vs 7.1%(P=0.001). No matter OS or LFS, both of them show significant differences.Conclusion:1. This Single-center study showed that aDLI could increase remission rate of relapsed acute leukemia patients post HSCT compared with the traditional tDLI,. No matter combination with chemotherapy or DLI alone, AML or ALL which almost had no response to tDLI, aDLI significantly prolonged the 5-year OS and LFS for relapsed patients.2. Comparing with treatment-related toxicities between aDLI and tDLI, the incidence of fever of aDLI group is apparently higher than group tDLI, while there is no more marked differences for the related toxicities of infection, hematologic toxicities, gastrointestinal reaction etc. There wasn’t a statistical significance in TRM.Part Ⅲ. Preliminary Mechanistic Study of the combination of IFN-a and donor lymphocyte infusion for the treatment of acute leukemia relapse post allo-HSCTObjective:To define the relationship between dendritic cells (DC)、IFN-DC and specific CTL cells in aDLI group and enhancement of anti-leukemia effects.Methods:From January 2011 to march 2015,36 patients in our hospital with acute leukemia relapse post allo-HSCT receiving the treatment of aDLI and 6 patients who received the treatment of tDLI were enrolled in the study by collecting the peripheral blood EDTA anticoagulant at pretreatment,+l week,+2 weeks,+3 weeks,+4 weeks.1. The percentages of DC, IFN-DC and WT1+CTL, the immunophenotypes of DC and lymphocytes,and the secretion level of perform and granzyme B were analyzed by flow cytometry (FCM); 2.The mRNA expression of perform and granzyme B were detected by Real Time PCR; 3.The level of IL-12, IL-4 and IFN-gamma in serum were determined by enzyme-linked immunosorbent assay (ELISA); 4.Comparing the differences between the aDLI and tDLI group, response and non response group in aDLI group with the above-mentioned indicators.5.the leukemia-associated immunophenotype (LAIP) of the leukemia cells in bone marrow was detected by FCM before and after the treatment of aDLI..Result:1. Dynamic detection of the number of IFN-DC1.1 Comparison between aDLI and tDLI groups at the same timeThere were significant statistical differences (p<0.05) at +2w-+4w, but no significant statistical differences before treatment and+1w.1.2 Comparison at different time point of the aDLI groupThere were statistical significances in the number of IFN-DC between Pre-DLI and +1w-+4w (P< 0.05), and reached maximal levels 3 weeks post aDLI.1.3 Comparison between response and Non response groupsThere were significant statistical differences (p<0.05) at+lw-+4w, but no significant statistical difference Pre-aDLI.2. Dynamic detection of the number and phenotypes of activated DC2.1 Comparison at different time points of the aDLI groupFor the patients of aDLI group, the number of DC increased significantly at+1w-+4w post aDLI (P<0.05).The expression of CD83, CD86 and HLA-DR had statistical significances at+2w-+4w post DLI.2.2 Comparison between aDLI and tDLI groups at the same timeThere were no statistical significances in the number of DC at at+lw post DLI and pre-DLI, statistical significances were found between+2w-+4w and pre-DLI+1w(P< 0.05). The expression of CD83,CD86 and HLA-DR had statistical significances at+2w-+4w post DLI.2.3 Comparison between response and Non response groupsWe compared 25 effective patients with 11 ineffective patients of aDLI group, the result showed that:the number of DC had significant differences at+1-+4w post aDLI(P <0.05); there were statistical differences between two groups in CD83、CD86、HLA-DR at +2w+4w post aDLI(P< 0.05).3. Dynamic detection of the number of WT1-specific T cells for HLA-A*0201-positive patientsA total of 4 patients in aDLI group were enrolled, all these patients had good responses to the treatment, and a significant reduction in WT1 expression after aDLI. The number of WT1-specific T cells of three patients reached maximal levels at+4 w,+3 w, +3 w post aDLI respectively.Only one patient nearly had no response.4. Dynamic detection of the lymphocyte subsets and their activations4.1 Comparison between aDLI and tDLI groups at the same timeThe number of CD3+T effector cells in aDLI groups were higher at +3w-+4w than tDLI groups, CD3+CD8+T effector cells had statistical significances at +1-+3w (P<0.05), NK cells had statistical significances at +2-+4w (P<0.05). CD3+TCRγδ+ cells only had statistical significance at +2w. CD3-HLADR+ (B lymphocyte cell) had statistical significances at+1-+4w (P<0.05).Though CD3+ CD4+ and CD4+ CD25+(Treg cell) cells had some changes, no statistical significances appeared between two groups. Phenotypes of activated lymphocytes:the level of activations of CD3+CD25+and CD3+HLADR+ showed statistical significancesat at+l-+4w (P<0.05); CD3+CD69+had statistical significances at +2-+4w post aDLI.4.2 Comparison at different time point of the aDLI group(a)CD3+、CD3+CD8+T effector cells、CD3-HLADR+、NK cells all increased post aDLI, and had statistical significances at +2-+4w post DLI. CD3+TCRγδ+ cells had statistical significance at +2w-+3w. Although CD3+CD4+ T effector cells cells also increased to certain extent, but did not reach statistical significance(P>0.05). (b) CD4+ CD25 +cells appeared a reduce trend after the treatment without statistical significance; (c)Phenotypes of activated lymphocytes:the level of activations of CD3+CD25+, CD3+CD69+ and CD3+HLADR+ increased post aDLI, showing statistical significances(P<0.05).4.3 Comparison between response and Non response groupsThe number of CD3+ T effector cells in both effective patients and ineffective patients increased post aDLI, and had statistical significances at +3-+4w post aDLI; Meanwhile the number of CD3+CD8+ had statistical significances at +1-+4w (P<0.05). CD3+TCRγδ+ cells had statistical significances at +2w-+3w. CD3+CD25+and CD3+HLADR+ showed statistical significances at +1-+4w(P<0.05); Meanwhile the number of NK, CD3+CD69+ and CD3-HLADR+ had statistical significances at +2-+4w (P<0.05). Though CD3+ CD4+ and CD4+ CD25+(Treg cell) cells had some changes, no statistical significance appeared.5. Dynamic detection of the mRNA expression of perform and granzyme B5.1 Comparison between aDLI and tDLI groups at the same timeDetection by flow cytometry technique(FCM):CD3-G and CD3-P had statistical significances at+1w-+4w(P<0.05); meanwhile CD8-G,CD56-G, CD8-P and CD56-P showed statistical significances at+2w-+4w(P<0.05). The mRNA expression of perform and granzyme B detected by Real time quantitative PCR was basically in accord with detection by FCM.5.2 Comparison at different time point of the aDLI groupCD3-G, CD56-G and CD3-P increased post aDLI to certain extent,and the differences were statistical significant (P< 0.05); CD8-G, CD8-P and CD56-P increased obviously at +2w-+4w(P<0.05). The mRNA expression of perforin and granzyme B detected by Real time quantitative PCR was basically in accord with detection by FCM.5.3 Comparison between Response and Non response groupsCD3-G, CD56-G and CD3-P had statistical significances at +1w-+4w(P<0.05); CD8-G, and CD56-P had statistical significances at +2w-+4w(P<0.05); meanwhile, CD8-P reached statistical significances at +3-+4w(P<0.05). The mRNA expression of perforin and granzyme B detected by Real time quantitative PCR was basically in accord with detection by FCM.6. Dynamic detection of the secretions of IL-12,IL-4 and IFN-γ levels in serum6.1 Comparison between aDLI and tDLI groups at the same timeThe level of IL-12、IFN-γ increased significantly post DLI, aDLI group were more apparent, and had statistical significances at +2w-+4w post DLI, reaching maximal levels 2 weeks post aDLI.; while the level of IL-4 decreased after the treatment, at+4w, the level of IL-4 of aDLI group was lower significantly than tDLI group(P<0.05).6.2 Comparison at different time point of the aDLI groupThe level of IL-12、IFN-γ increased significantly at +2w-+4w post aDLI, while the level of IL-4 decreased at +4w(P<0.05).6.3 Comparison between Response and Non response groupsCompared 25 effective patients with 11 ineffective patients of aDLI group,the level of IL-12 had statistical significances at +2-+4w (P<0.05); the level of IL-12、IFN-γ had statistical significances at +2w,+4w post aDLI; while the level of IL-4 had statistical significances at +3w-+4w post aDLI.7. the immunogenicity of leukemia cellsA total of five patients detected by minimal residual disease(MRD), we found that the fluorescence intensity of MHC Ⅰ/Ⅱ (HLA-ABC, HLA-DR), costimulatory molecules (CD40L, CD28) and adhesion protein (ICAM) expressed on the leukemia cells increased after compared to baseline in three of five patients, and these markers increased at different levels post aDLI,while other two patients didn’t appear significant changes.Conclusions:1. IFN-a induced the generation of IFN-DC and promoted the maturation andactivation of DC, which then lead to the proliferation and activation of CD8+CTL directly or indirectly. The cytokines, perform and granzyme B may play an important role in mediating the GVL effects.2. aDLI promoted the proliferation and differentiation of immune effector cells such as CTL, NK cells and γδT cells, and increased the secretion levels of perform and granzyme, then enhanced the effect of cell killing activity.3. IFN-a could make leukemia-associated antigens easier identified and presentedthrough upregulating the immunogenicity of leukemia cells and overcome the behavior of tumor escape in order to enhance graft versus leukemia effect. |
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