Role Of CCL2 And VEGF Secreted By Bone Marrow Stromal Cells On Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a malignancy of hematopoietic system,accounting for 60%-70% of adult acute leukemia.Although the therapeutic approaches has improved significantly with advances in medicine,the five-year survival rates is about 40% for the younger patients and even less than 5% for the older[1,2].Therefore,it is urgent to study the mechanism of development of acute myeloid leukemia for exploring new therapeutic targets,laying foundation for precision medicine.Bone marrow stromal cells(BMSCs)are the main constituent of bone marrow hematopoietic microenvironment.BMSCs play an important role in the support and regulation of all kinds of hematopoietic stem/progenitor cells.BMSCs involves in the development and progression of leukemia through the secretion of cytokines,growth factors,matrix proteins and so on [3].Some studies have shown that the expression levels of some molecules related to BMSCs in different stages of AML may be related to the occurrence,development,the response to treatment and prognosis of AML [4].Up to now,it is not clear yet that which kind of BMSCs-related molecules play a key role in regulating the biological effects of leukemia cells of AML.Therefore,studying thoroughly the expression changes of BMSCs-related molecules in the occurrence and development of AML and clarifying the mechanism are expected to provide new ways for the diagnosis and treatment of AML.Objective:To screen the expression level of BMSCs-associated molecules in different stages of AML and their clinical value.To study the effect of BMSCs-associated molecules on the leukemia cells at cellular level.Method:Part I: Through retrieving Pub Med,CNKI database,and so on,38 BMSCs-associated molecules related to AML(such as VCAM1,ICAM-1,CDH1,CD44,MMP-2,TMP3,DAPK1,Cx43,IL-6,IL-10,TNF-a,SDF-1)were detected.A total of 151 bone marrow samples were collected from 69 patients with AML at the time of initial diagnosis,complete remission(CR),relapse/ refractory disease,and 7 bone marrow samples from nonmalignant hematopathy patient(excluding aplastic anemia)were used as control;2-4ml bone marrow sample with EDTA anticoagulation from each sample was used to extract and culture BMSCs;RNA of BMSCs were extracted by Trizol.The m RNA expression levels of 38 molecules in all the samples were detected by PCR chip,and the BMSCs-related molecules that may be highly specific to the development of AML were screened out.We screened out statistically the chemokine 2(chemokine(C-C motif)ligand 2,CCL2)and vascular endothelial growth factor(VEGF)by their expression difference in different disease stages of AML.Part II: The relationship between CCL2,VEGF and AML was further validated at cell levels.BMSCs HS-5 cell was first transfected with null plasmid,or CCL2-or VEGF-overexpression plasmid.Then the co-culture system of BMSCs HS-5 and leukemic cells MOLM13 or HL60 were constructed to detect the influence of different expression levels of CCL2 and VEGF to their biological behavior.The viability and proliferation of leukemia cells was detected by CCK8 assay,invasion ability of MOLM13 and HL60 cells by Transwell assay.Result:1.Through clustering analysis of PCR chip results,it was found that in 38 BMSCsassociated molecules,the expression of CCL2 and VEGF in the bone marrow of AML during different disease periods showed significant difference and consistency in changes.The expression of CCL2 and VEGF was higher in the newly diagnosed patients than in the normal group(P<0.05).Its expression levels declined significantly when patient received CR.The difference was statistically significant when compared to results of newly diagnosed AML(P<0.05).But there was no significant difference when compared to control group(P = 1.000).when compared to the expression levels of CCL2 and VEGF for CR patients,their expression levels for recurrent disease were significantly increased(P<0.05)and their m RNA levels for refractory disease were also increased significantly(P<0.05).2.The overexpression plasmids of CCL2 or VEGF were verified by electrophoresis and Q-PCR.The co-culture system of HS-5 and MOLM13,HL60 were successfully constructed.When HS-5 overexpress the CLL2 or VEGF,the survival curve showed that the viability of MOLM13 or HL60 in co-culture system increased significantly(P<0.05),and Q-PCR results showed that the cell proliferation of MOLM13 and HL60 in co-culture system was 5.3 times and 5.8 times that of control group respectively at 72 hours.While HS-5 overexpressed CCL2 and VEGF at the same time,the cell viability of MOLM13 and HL60 was 7.3 times and 6.1 times that of control group(p <0.001,p = 0.008),with significant increase than expression of CCL2 or VEGF alone(p< 0.05).When HS-5 overexpressed CCL2 or VEGF in Transwell co-culture system,the number and invasive ability of both MOLM13 and HL60 was improved;When HS-5 overexpressed both CCL2 and VEGF,the number of both cells is much bigger than that when overexpressing alone,and the invasive ability was enhanced more significantly.Conclusion:1.CCL2 and VEGF secreted by BMSCs are closely related to the development of AML disease.Their expression have regularity at different stages of AML,first diagnosis(increase),remission after treatment(reduce),relapse/refractoriness(increase again),which show significance to the evaluation of therapeutic effect and prognosis.2.Through in vitro experiments,we confirmed that CCL2 and VEGF secreted by bone marrow stromal cells can promote the growth of AML cells alone or synergistically.Hodgkin lymphoma(HL)is a malignancy of lymphoid system,which is more common in males.The main manifestation often presents as non-painful,gradually progressive enlarged lymph nodes in the neck.About 50% of patients are with mediastinal mass,25% with systemic symptoms.According to pathological characteristics,HL is divided into two types: classical Hodgkin lymphoma(CHL)and nodular lymphocyte predominant Hodgkin lymphoma(NLPHL)-the latter is rare.With the improvement of diagnostic techniques and treatments,CHL has become a disease with high rate of cure.In clinic,CHL has showed good responses to ABVD,Standford V,BEACOPP regimens et al.Most patients will gain a long-term remission,even cure,after first-line chemotherapy.The 5-year overall survival rate and progress-free survival rate can be as high as 90%.But there are still about 20% of patients with CHL failing to first-line therapy,or relapsing after remission.At present,the treatment of relapsed/refractory CHL(RR-HL)is still it is a challenge of clinical practice.How to improve the long-term survival of RR-CHL patients is an urgent problem.There are no standard protocols for the treatment of RR-CHL.Some clinical studies have shown that sequential autologous hematopoietic stem cell transplantation(ASCT)after high-dose chemotherapy has achieved good results in the treatment of patients with RR-CHL.Purpose: To observe the efficacy of ASCT in the treatment of RR-CHL.METHODS: We respectively reviewed 89 patients with RR-CHL in our hospital from January 2000 to December 2012,of whom 45 patients underwent ASCT,and 44 patients received chemotherapy or chemotherapy combined with involved-field radiotherapy(IFRT).According to receiving ASCT or not,patients were divided into ASCT group(n = 45)and non-ASCT group(n = 44).All patients in ASCT group received MOED+G-CSF to mobilize autologous peripheral blood stem cells,and CEAC as condition regimen.Finally,the results of two groups were analyzed.Results: All patients in ASCT group underwent hematopoietic reconstitution and no transplant-related deaths.Compared with non-ASCT group,the incidence of digestion symptoms,severe bone marrow suppression and febrile neutropenia of ASCT group was much higher(P <0.05).There was no significant difference in the incidence of other adverse events(P> 0.05).The median follow-up time was 54 months(12 to 156 months).In ASCT group,6 patients relapsed and 6 patients died with a mortality rate of 13.3%;the 3-year Progress Free Survival rate(PFS)was 80.0% ± 6.0%,and 3-year overall survival rate(OS)was 91.1% ± 4.2%.In non-ASCT group,8 patients relapsed and 21 patients died with a mortality rate of 47.7%;the median-survival time was 114.00(50.25 ~ 176.75)months,the median-relapse time was 36.00(12.179~59.821)months,and the 3-year PFS and OS were 59.1% ± 7.4% and 68.2% ± 7.0% respectively.The OS and PFS of ASCT group were significantly better than those of the non-ASCT group(P <0.01).The patients who received a complete remission(CR)evaluated by PET/CT before ASCT had a better OS and PFS than that of patients without CR(3-year OS: 100% versus 87.5%;3-year PFS: 100% versus 71.9%).Analysis of prognostic factors by Cox progressive regression showed that non-ASCT,high LDH value,extranodal infiltration,and bone marrow invasion were risk factors for PFS.Non-ASCT,advanced disease and bone marrow invasion were risk factors for OS.Conclusion: The patient with RR-CHL who received ASCT has much better 3-year OS and PFS than that of patients without transplantation,which demonstrates that ASCT shows a good efficacy in the treatment of RR-CHL.ASCT is relatively safe and adverse events did not increase significantly.The sates of disease evaluated by PET/CT before transplantation is closely related to long-term survival.ASCT has an exact efficacy for RR-CHL with good safety,and can be recommended to such patients,which deserves further clinical applications.