| Leukemia is a malignant disease of hematopoietic system with maturation disorder,excessive proliferation and blocked apoptosis in the process of hematopoietic stem cell differentiation.With the emergence of new clinical chemotherapeutic drugs and the adjustment of the dose of chemotherapeutic drugs,the complete remission（CR）rate of acute myeloid leukemia（AML）has been significantly improved,but there are still about 30%of patients who are difficult to reach CR,and 50%～80%of patients will relapse after CR eventually.Patients who are unable to obtain CR or relapse after CR are likely to develop refractory AML.Therefore,there is an urgent need to find new and more effective treatments.On the basis of traditional chemotherapy,the treatment of AML puts more and more emphasis on the molecular and immunological characteristics of the disease itself,as well as individual targeting and immunotherapy.Some refractory/relapsed AML may benefit from molecular targeted therapy,novel bispecific monoclonal antibodies（bispecific T-cell engagers,Bi TE）,immune checkpoint inhibitor,chimeric antigen receptor modified T cell（chimeric antigen receptor modified T cell,CAR-T）and hematopoietic stem cell transplantation,but their clinical application is limited because of the high cost of these treatments,long clinical trial cycle and many complications.There is no standard salvage treatment for elderly AML patients with poor physical fitness score,more complications and refractory/relapsed AML,so it is of great significance to explore a salvage treatment that is both efficient,economical and with few adverse reactions.The Chinese guidelines for refractory/relapsed AML（version 2017）and the 2020.v2 version of the National Comprehensive Cancer Network（NCCN）guidelines recommend clinical trials.The development of"new uses"with anti-leukemia effect from traditional drugs with low price and little adverse reactions has aroused widespread concern,in which the research on"new use of old drugs"is a starting point.AML recurrence is thought to originate from residual leukemic cells that have survived and have not been detected by routine morphological assessment.New or more sensitive methods can detect these cell populations,which are called measurable residual diseases after consolidation therapy.Persistent negative MRD is the best response category for patients with AML.Patients with persistent positive MRD should be treated with allo-Hematopoietic Stem Cell Transplantation,but it is difficult for some patients to perform allo-HSCT due to donor,economic and other reasons.For these patients,other treatments should be taken to reduce the level of MRD as much as possible to delay the recurrence of the disease.At present,recurrence is still the main obstacle for the long-term disease-free survival of AML patients.Considering the poor prognosis of recurrent AML patients,maintenance therapy for AML patients has been put on the agenda.The ideal maintenance therapy is a treatment that can be tolerated by patients,can bring long-term remission and effectively reduce the risk of recurrence.Interferon（IFN）has the role of immune regulation,and can activate a variety of immunoreactive cells such as NK,T cells,macrophages,and can regulate the production of antibodies,play a role in immune enhancement.In addition,it can also directly or indirectly inhibit the gene transcription of vascular endothelial growth factor（VEGF）and fibroblast growth factor（b-FGF）through influencing cell cycle arrest（including G0/G1 and S phase arrest）,thus inhibiting tumor angiogenesis and indirectly inhibiting tumor proliferation.Interferon can also up-regulate the expression of Fas L,thus induce apoptosis of tumor cells throμgh Fas/Fas L pathway,or induce apoptosis of tumor cells by increasing the activity of tumor necrosis factor（TNF）,activating caspase system,regulating the activity of interleukin and changing the expression of apoptosis-related genes.In the 1980s,some scholars used IFN to treat AML,IFN was used for induced remission of AML,maintenance therapy and salvage treatment of recurrence after allo-HSCT.Interleukin-2（IL-2）is an important immune regulatory factor in the body’s immune network,which plays an important role in the growth,proliferation and differentiation of many kinds of immune cells such as T cells and NK cells.IL-2alone can be used in the treatment of AML,but the effect of single drug treatment is poor.Thalidomide is an anti-angiogenic drug,which can not only reduce the production of VEGF and b-FGF,but also inhibit the expression of VEGF coreceptor Neuropilin-1;it can also change the tumor microenvironment and play a role in anti-tumor therapy.Thalidomide is also an immunomodulatory drug,which can enhance the activity of T,NK and dendritic cells,and regulate the secretion of cytokines such as TNF-α,IFN-γand IL-2.The results showed that the microvessel density in bone marrow of patients with acute leukemia was significantly increased,and high levels of VEGF and b-FGF could be detected in peripheral blood or bone marrow of patients with AML.In recent years,thalidomide has been reported to be effective in the treatment of refractory/relapsed AML.To sum up,interferon,IL-2 and thalidomide all have a certain curative effect on AML,but the effect of medication alone is limited.Then we assume that the combination of the three drugs can produce synergistic effect and enhance the therapeutic effect of anti-AML,which will be discussed in this study.The content of the study is:（1）the clinical efficacy of interferon,IL-2 and thalidomide in the treatment of patients with AML.（2）the mechanism research of interferon,IL-2 and thalidomide on AML.The purpose of this study is to clarify the efficacy of the combination of three drugs in patients with AML and clarify the mechanism of action,and provide a safer,more effective and non-chemotherapy treatment for elderly patients,poor physical fitness score,complications and refractory/relapsed AML,persistent positive MRD after consolidation therapy and maintenance therapy for AML.PartⅠ:The clinical efficacy of interferon,interleukin-2 and thalidomide in the treatment of patients with AMLObjectivesTo observe the efficacy and adverse reactions of interferonα-1b,interleukin-2combined with thalidomide（"ITI"regimen）in the treatment of patients with AML who are refractory/relapsed or in poor physical condition or MRD（+）after consolidation treatment or in maintenance therapy.Methods1.From January 2014 to June 2017,68 AML patients with refractory/relapsed and poor physical condition were treated with"ITI"regimen from 11 hospitals,including the affiliated Cancer Hospital of Zhengzhou University,and its effective rate（including complete remission rate（CR）+complete remission with hematological incomplete recovery（CRi）+partial remission（PR））and survival condition were observed.2.From January 2014 to June 2017,18 AML patients（17 from the Affiliated Cancer Hospital of Zhengzhou University and 1 from the First People’s Hospital of Pingdingshan City）who were in hematological remission but were MRD positive and could not receive allogeneic hematopoietic stem cell transplantation were treated with different doses of"ITI"regimen to monitor their MRD levels.3.From January 2014 to June 2017,88 MRD-negative AML patients from 11 hospitals including the Affiliated Cancer Hospital of Zhengzhou University were treated with maintenance therapy,81 MRD-negative AML patients were not treated with maintenance therapy at the end of consolidation therapy in the same period served as the control group,and the recurrence rate and recurrence time were observed.4.The routine therapeutic dose and application method of"ITI"regimen:the initial dose of thalidomide is 100mg,per day before going to bed,and it can be gradually increased to 200mg,per day according to the patient’s tolerance,and compound salvia miltiorrhiza tablets or aspirin tablets are given to prevent deep vein thrombosis.rh IFNα-1b injection 60μg,subcutaneously once every other day,30 min before each application,ibuprofen granule was given orally to prevent drug-induced fever,and if it was tolerable in the later stage,antipyretic drugs could be discontinued.IL-2 injection 1 million IU,subcutaneous injection,once every other day.There is one treatment cycle every 4 weeks.Patients with acute myeloid leukemia with positive FLT3-ITD mutation were given sorafenib 400mg twice a day.5.The"ITI"regimen was used to increase the therapeutic dose:thalidomide 200mg/day,rh IFNα-1b 60μg,subcutaneously once a day,and IL-2 1 million IU,subcutaneously once a day,the rest as above.6.Statistical analysis:Using SPSS 21.0 statistical software,the measurement data satisfy the normal score.Cloth is expressed by mean±standard deviation and skewness distribution by median±quartile spacing.Kaplan-Meier method was used to analyze survival time,and Log-rank was used to test the difference of survival curve（P﹤0.05）.Results1.The CR+CRi of 48 AML patients with refractory/relapsed was 14.58%,the PR was 8.33%,and the total effective rate was 22.91%,the median OS of 48 patients was 6.5（from 1.0 to 60.0）months.2.The CRi of 12 AML patients with poor physical condition and intolerant to chemotherapy was 25%,the PR was 25%,andthe total effective rate was 50%,the median OS of 12 patients was 8.0（from 1.0 to 14.0）months.3.The total effective rate of the 18 AML patients with MRD-positive was 72.2%.The response rate was 57.1%and 81.8%in those who presented the MRD≥1.0%and<1.0%,respectively.4.The recurrence rate of 88 MRD-negative AML patients with maintenance therapy was 12.5%（11/88）,and the median recurrence time of 11 patients was 20 months.In the control group,the recurrence rate of 81 MRD-negative AML patients without maintenance therapy was 54.3%（44/81）,and the median recurrence time of 44 patients was 6 months.PartⅡ:The mechanism research of interferon,interleukin-2 and thalidomide on AMLObjectives1.To observe the effects of interferon and thalidomide on apoptosis and proliferation of AML cells and explore the mechanism.2.To observe the effects of interferon,interleukin-2 and thalidomide on immune function in patients with AML.3.To establish a mouse model of AML xenotransplantation,to observe the effects of interferon,interleukin-2 and thalidomide on AML in vivo,and to explore the mechanism.Methods1.CCK-8 assay was used to detect the proliferation inhibition of AML cell lines U937,THP-1 and OCI-AML3 before and after treatment with interferon and thalidomide.2.The apoptosis,cycle and mitochondrial membrane potential of AML cell lines U937,THP-1 and OCI-AML3 treated with interferon and thalidomide were detected by flow cytometry.3.Western blot was used to detect the changes of apoptosis and cycle signal pathway proteins in AML cell lines U937,THP-1 and OCI-AML3 cells treated with interferon and thalidomide.4.From March 2016 to May 2019,68 AML patients who were initially intolerant to chemotherapy or relapsed/refractory or received maintenance therapy and voluntarily received interferonα-1b,interleukin-2 combined with thalidomide regimen from 11 hospitals,including the Affiliated Cancer Hospital of Zhengzhou University,were enrolled in the treatment group.Peripheral blood samples were collected at least one day before treatment and 3 months after standardized treatment.The lymphocyte subsets in peripheral blood and the expression levels of Granzyme-B and Perforin from NK cells were detected by flow cytometry.The expression levels of VEGF,INF-γ,TNF-αand IL-6 in the plasma of the patients were detected by microsample multiple quantitative technique（cytometric bead array,CBA）.The control group consisted of 35 healthy subjects from the physical examination center of the Affiliated Cancer Hospital of Zhengzhou University.5.Leukemia cell line MOLM-13 was used to establish AML disease model in severe immunodeficiency mice（NCG）.The changes of leukemia load in mice and the survival time of mice were observed before and after treatment.The changes of T lymphocyte subsets,the expression of granzyme B and perforin in T lymphocytes and the changes of DC in peripheral blood of mice treated with drugs were observed.6.Using SPSS 21.0 statistical software,the measurement data satisfy the normal distribution with the table of mean±standard deviation.The results showed that the skewness distribution was expressed by median±quartile interval,paired t-test was used to compare the experimental data before and after drμg treatment,analysis of variance was used to compare multiple means,Kaplan-Meier method was used to analyze survival time,and Log-rank was used to test the difference of survival curve（P﹤0.05）.Results1.Interferon and thalidomide could inhibit the proliferation of U937,THP-1 and OCI-AML3 cells,and the inhibitory effect was enhanced by the combination of interferon and thalidomide.2.Interferon and thalidomide alone could promote the apoptosis of U937,THP-1and OCI-AML3 cells in a concentration-dependent manner,and the apoptosis-promoting effect was further enhanced by the combination of the two drugs.3.The AML cell cycle was arrested in G1 phase after the combination of interferon and thalidomide.4.Interferon and thalidomide could decrease the mitochondrial membrane potential of U937,THP-1 and OCI-AML3 cells,and the effect was further enhanced by the combination of interferon and thalidomide.5.After the combination of interferon and thalidomide,the expression of anti-apoptotic proteins Bcl-2 and Mcl-1 decreased,the expressions of pro-apoptotic proteins Bak,cf-PARP and cytochrome C increased,the expression of cyclin P21increased and the expression of cyclin c-Myc decreased in U937,THP-1 and OCI-AML3 cells.6.Compared with the control group,the proportion of CD4+/CD8+and NK cells and the expression levels of Perforin and Granzyme-B of NK cells in the patients before treatment were lower than those in the control group,while the levels of VEGF,IL-6 and TNF-αcytokines in the peripheral blood plasma were higher than those in the control group,and the difference was statistically significant.The level of INF-γcytokines was lower than that in the control group,but the difference was not statistically significant.7.After 3 months of treatment,the proportion of CD4+/CD8+,the proportion of NK cells,the expression levels of Perforin and Granzyme-B of NK cells in peripheral blood,the level of INF-γcytokines in peripheral blood plasma increased and the level of VEGF cytokines decreased,and the difference was statistically significant.The lymphocyte ratio of CD3+CD4+and CD3+CD8+in peripheral blood and the level of TNF-αcytokines in plasma were higher than those before,while the level of IL-6 was lower,the difference was not statistically significant.8.MOLM-13 cells were injected into NCG mice throμgh tail vein,and the transplantation model of AML mice was established successfully.Interferon,interleukin-2 and thalidomide had anti-leukemia effect and prolonged the survival time of mice,respectively.After combined action,the anti-leukemia effect was further enhanced.9.After treated with interferon,interleukin-2 and thalidomide alone,the number of CD3+CD4+,CD3+CD8+T lymphocytes and DC in peripheral blood of mice was higher than that of control group,and the expression levels of Perforin and Granzyme-B in peripheral blood T lymphocytes of mice were higher than those of PBMC group,and the effect of"ITI"combined group was more significant,which was statistically different from that of single drug group and control group.Conclusion1.The"ITI"regimen is effective in reversing AML with positive minimal residual Disease,and has a certain curative effect on some refractory/relapsed and newly treated patients with AML,and can make some patinets get remission,prolong the life of the patients.2.The"ITI"regimen can be used for maintenance treatment of AML patients and prolong their survival.3.The"ITI"regimen enhances the anti-leukemia ability of patients with AMLby increasing cellular immunity and reducing the secretion of VEGF cytokines.4.Interferon and thalidomide can synergistically inhibit the proliferation and induce apoptosis of AML cells,and can arrest the cell cycle in G1 phase.5.The combined effect of"ITI"can significantly reduce the tumor load and prolong the survival time of AML xenograft mice. |
PDF Full Text Request