Study Of Cytopenia Status After Hematopoietic Stem Cell Transplantation For Thalassemia Major

BackgroudP-thalassemia major (TM), an inherited anemia that is associated with reduced or absent β-globin synthesis, results in an imbalanced accumulation of a-globin chains and ineffective erythropoiesis with hemolysis. The optimization of both erythrocyte transfusion and iron chelating therapy has resulted in a remarkable improvement in the life expectancy of patients with TM. However, complications related to iron overload cannot be completely managed through chelating therapy, and compliance with a chronic transfusion regimen is difficult to maintain throughout a lifetime. The only curative therapy remains the replacement of the defective erythropoiesis by allogeneic hematopoietic stem cell transplantation (HSCT) now. We used a novel NF-08-TM transplant protocol included prophylaxis regimen for acute graft versus (vs.) host disease (aGVHD), which improve outcomes of unrelated donor (UD) peripheral blood stem cell (PBSC) transplant (PBSCT) and HLA well-matched sibling transplant (MST) through to overcome high rejection in TM-HSCT. However, we also observed commoner cytopenia beyond day28post-transplant (CB-28PT), those exact development mechanism is not well known.Etiologies possibly include myelotoxic agents, viral infections and transplant-related factors such as donor’s age, HLA Typing, patients’ CMV status, the source of the stem cells and chronic GVHD (cGVHD). The CB-28PT in TM-HSCT was rarely reported so far, regardless of both its causes and its relation to KIR ligands. Herein, we focused and analyzed our data of CB-28PT in TM-HSCT,ObjectiveThis study is aim to explore reasons for cytopenia which occurred on+28days after Allogeneic hematopoietic stem cell transplantation. By analyzing risk factors of cytopenia to find out related factors of cytopenia, so as to provide a guideline for clinical treament.MethodWe retrospectively analyzed187consecutive TM patients underwent HSCT between January1,2009and October31,2012in our center. The median age at transplantation was6years(range2.0～16years),including117boys and70girls.The median follow-up time was19(r:1-44) months. Of them,120received alternative donor (109UD plus11parents donor) transplant (ADT) and67received MST. Of187TM patients,a total of39patients were identified with cytopenia,,39patients with cytopenia and148patients without cytopenia were compared.According patients’ age,hepatomegaly and ferritin level,we graded native pediatric thalassemia major patients into grades Ⅰ,Ⅱ,Ⅲ. Cytopenia-related risk factors included recipient/donor age,sex and CMV serostatus,HLA Typing,ABO match,stem cell source,transplant type,acute GVHD and chronic GVHD.Conditioning regimen and prevention of GVHDThe NF-08-TM protocol was used for all the patients, which was consisted of cyclophosphamide (Cy;day-10to day-9); fludarabine (Flu; day-8to day-4); thiotepa (TT; day-5); and intravenous busulfan (Busulfex; day-8to day-6) at a dose that was dependent on the age of the patient. The average plasma steady-state concentrations (Css) of busulfan were tested on day-8and targeted to range between300-600μg/L. The dose of busulfan was increased or decreased by1.2mg/kg when Css was less than300μg/L or more than600μg/L, and was given in divided doses on day-6. All patients received3mg/kg of azathioprine and30mg/kg of hydroxyurea daily beginning at day-45before transplantation.The prophylaxis of GVHD included Cyclosporine A (Cs-A, started at1.5mg/kg/day intravenously from day-10to day-2, before being increased to3mg/kg/day at day-1up to day25, and subsequently administered orally at targeted concentrations of200±50ng/ml); mycophenolate mofetil (MMF, administered on day1at15mg/kg bid and was discontinued on day30if there were no signs of≥grade Ⅱ aGVHD and short-term methotrexate (MTX, administered on days1,3and6at15,10and10mg/m2, respectively). In ADT, all the patients received PBSCT and most were UD-HSCT.Criterion of engraftmentThe blood type of peripheral blood should be testd in the28th day,first month,second month,third month,sixth month,and first year,even more as appropriate.Criterion of engraftment are divided into Indirect indicators and a direct indicator.A indirect index:patients was alive more than21days after hematopoietic stem cell transplantation; hematopoietic recover and values of erythrocyte,granulocyte,lymphocyte and megakaryocyte were normal,presentation of clinical GVHD.Hepatomegaly and splenomegaly shrink to normal;lactate dehydrogenase level reduced to normal.A direct index:red cell antigen(ABO and Rh type changed into donor’s type), genetic mark of cell(DNA of peripheral blood or bone marrow changed into donor’s),HLA antigen(mismatchcases,HLA antigen changed into donor’s),Molecular genetic evidence(PCR proliferation technology was used to analyze patient’s peripheral blood after HSCT,calculated donor’sgenetic mark’s ration). Definitions of cytopeniasThe one of the following conditions was defined as CB-28PT in current study:1) WBC counts less than3.0×103/mm3for four weeks or longer.2) Neutrophil counts less than0.5X103/mm3for four weeks or longer.3) Hgb lees than9.0g/dL for four weeks or longer.4) Platelet less than20.0×103/mm3for four weeks or longer. Autoimmune haemolytic anaemia (AIHA) was identified with documented significant haemolysis and a positive direct antiglobulin test (Coombs test).Statistical analysisUnivariate and multivariate logistic regression models were used to estimate odds ratios and confidence intervals (95%CI) for risk factors associated with cytopenia as defined above. Covariates included recipient/donor age, sex and CMV serostatus, HLA Typing, ABO match, stem cell source, transplant type, acute GVHD and chronic GVHD. Varibles with a significance were analyzed level of less than0.05in the univariate models were candidates for the multivariates models. All P values are two-sided and unadjusted for multiple comparisons. Mixed linear model was applied to analysis the relationships between WBC count and Cs-A blood concentration. Chi-square test was used to assess the morbidity of cytopenia between ADT-HSCT and MSD-HSCT. The SPSS13.0software package was used for the analyses.ResultsOf187TM patients underwent HSCT, a total of39patients were identified with CB-28PT, giving an overall cumulative incidence of20.9%at one year (95%CI15.0%-26.7%). The median time of CB-28PT occurring was day102(r:48-189) post-transplant. The time of median duration of CB-28PT was70days (r:28-308days). Of the39patients,24,9and3patients’ WBCs were less than3.0,2.0, and1.0X103/mm3, respectively. Thirty-four had leucopenia only, two had AIHA, one had thrombocytopenia, one had leucopenia with AIHA and one had leucopenia with thrombocytopenia. With comparing ADT with MST, there was higher incidence of CB-28PT in ADT group (25.8%vs.11.9%, p=0.025). There was furthermore higher CB-28PT (31/120,36.4%vs.8/67,19.7%, p=0.045) in HLA mismatched ADT group.In univariate analysis, the risk factors for CB-28PT included donors’s age, HLA-mismatched donor (HLA-A and HLA-DRB1mismatched) and stem cell source (MST vs. ADT, OR=0.389,P=0.028). The number of patients with HLA-B and HLA-C mismatch was too small to compare them with Match. In a multivariate model, we identified HLA-mismatched donor as only significant risk factor for cytopenia.Mixed linear model was well used to analysis the relations of WBC count and level of Cs-A blood concentration,Methylprednisolone dose,patients’ sex and patients’ age.The result showed a significant positive relation with Cs-A blood concentration(β=0.002037P<0.001), and Methylprednisolone dose(β=0.6053P<0.001),also have positive relation with patients’ age(《=0.1357P=0.0364),but there was interaction between methylprednisolone dose and patients’ age, no relation with patients’ sex(P=0.7241).We adjusted doses of Cs-A and MP, or changed Cs-A to tacrolimus with or without MTX to treat34patients with leucopenia only. WBC count of33patients recovered to normal in median time of70days (range:28-308). One patient died of complication of diabetics and lung infection. Additionally, two patients were given ATG (5mg/kg in total), one of them had the WBC up to normal and the another one is still about2.0×103/mm3; The remained three patients more than3months after transplantation still accompanied by cytopenia until end of follow-up.ConclusionThese facts (HLA mismatch, ADT, response to immunosuppressants, rash appearance with reducing doses of Cs-A and MP, WBC count linking Cs-A, significant high incidence in C1C1recipients and time of occurrence of CB-28PT) were usually related to cGVHD in HSCT experience of malignant diseases. For this reason, we propose that CB-28PT may be a primary manifestation of cGVHD in young pediatric patients undergoing allogeneic HSCT, despite CB-28PT could not diagnosed as cGVHD by current standard of Seattle’s or NIH’s criteria.