Clinical Observation And Pathogenetic Study On The Sclerodermatous Chronic Graft-versus-host Disease After Hematopoietic Stem Cell Transplantation

Background & ObjectiveChronic graft-versus-host disease (cGVHD) is a complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) which clinical manifestation resembling autoimmune and other immunologic diseases. Acording to with or not with the clinical manifestations of aGVHD, cGVHD divided into classic cGVHD (without features or characteristics of aGVHD) and overlapping cGVHD (diagnositic or distinctive features of cGVHD and aGVHD appear together). The main organs involved include skin, oral cavity, lung, liver, kidney, gastrointestinal tract, eye, genital tract, muscle and bone, blood and immune system. Reported incidence rates of cGVHD after allo-HSCT range from 30% to 70%. Skin is the most common target organ, and sclerodermatous chronic graft-versus-host disease (ScGVHD) is one of which rare occurred, and characterized by the manifestations of cutaneous sclerosis, fasciitis, or joint contracture. ScGVHD is characterized by cutaneous and its appendages features such as sclerosis, telangiectasia, atrophy, hyper-or hypopigmentation, contractures, erythema, ulceration, hair loss and nail changes. According to the lesions range, ScGVHD distributed into generalized type and localized type. Generalized type involve more than two anatomic sites, and localized type affect one or two anatomic site.ScGVHD affect allo-HSCT patient’s survival time and quality of life, although it show reduced relapse due to an enhanced graft versus leukemia effect, the mortality may be approximately 20 to 40%owing to extracutaneous involvement. While ScGVHD has some clinicalmanifestation and histopathological similarities with SSc, the pathogenic mechanisms of the 2diseases are thought to differ. Differ from SSc, ScGVHD rarely involve visceral organs with pulmonary, renal, cardiac and so on; SSc begins in deeper layers of the skin and then extends to superficial layers, whereas ScGVHD usually begins in superficial layers of the skin and then extends to deeper layers. In recent years, there has been a growing attention at ScGVHD, some large sample researches were reports, but there are only a few case reports in China at present. Moreover, the domestic and foreign researchers are concerned about if the increasing proportion of HLA-mismatched donor may lead to the change of incidence rate and clinical characteristics of ScGVHD.GVHD has a very complex pathological mechanism, but ultimately is the result of donor T cells recognizing recipient’s disparate histocopatibility antigens which leads to the imbalance of differentiation of Th cells and cytokine storm cascade, and causing injures to recipient’s normal tissues. T cell activatation, proliferation and differentiation are considered important factors for the development of GVHD, the CD4+T cells will differentiate into Thl, Th2, Thl7 and Tregs after activation. Newly reported other subsets of Th cells such as Th9, Th22 and Tfh, these founding enriched our understanding about T cell differentiation. Thl cells distinctively produce IFN-y, IL-2, and TNF-a. In contrast, Th2 cells produce IL-4, IL-5, and IL-13. Th17 cells produce their signature cytokines:IL-17, TGF-β, IL-21, IL-23, and IL-6. Tregs produce IL-10 and TGF-β. Th-cell subsets and cytokines secreted by them plays a vital and complex role in the pathogenesis of GVHD. Now we considere that the increasing proportion of Th1 and/or Th17 cell may promote the occurrence of GVHD, Tregs can decrease the incidence of GVHD due to the biological function of regulating immunity, and Th2 cell may be associated with fibrosis disease. At present, there is no report about the Th-cell subsets and cytokines in the pathogenesis of ScGVHD, exploring the mechanisms will provide further understanding for the long-term GVHD in allo-HSCT and explore more effective treatment methods to improve the prognosis.Methodâ… . Analysis of clinial characteristics and risk factors of sclerodermatous chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation1.Case information:Two hundred and fifty-nine patients who underwent allo-HSCT from January 2012 to December 2014 at Nanfang hospital, Southern Medical University, and survived at least 100 days after allo-HSCT were enrolled in this retrospective analysis. There were 169 male and 90 female among them, and the median age at allo-HSCT was 29 (10-59) years. In these patients, there were 99 cases were diagnosed with acute myeloid leukemia (AML),96 cases with acute lymphoblastic leukemia (ALL),12 cases with acute unclassified leukemia (AUL), 21 cases with chronic myeloid leukemia (CML),15 cases with non Hodgkin’s lymphoma (NHL),7 cases with myelodysplastic syndromes (MDS),1 case with multiple myeloma (MM),7 cases with aplastic anemia (AA),1 case with paroxysmal nocturnal hemoglobinuria (PNH). The disease risk at transplantation was high in 137 cases and standard in 122 cases.2.Method:The incidence, clinical manifestations, laboratory examination of ScGVHD were analyzed statistically. The 259 patients were divided into ScGVHD group (n=22) and non ScGVHD group (n=237) according to the ScGVHD occurrence. A number of factors affected the occurrence of ScGVHD, including disease state, age and sex of donors and recipients, donor type, HLA type, ABO blood type, stem cell source, conditioning regimen, GVHD prophylaxis, whether occurred aGVHD and cGVHD, DLI and so on. Follow-up study to October 2015, the median follow-up time is 29 (3～44) months.3.Statistical analysis:Statistical analyses were performed using the SPSS for Windows (version 20.0) with P<0.05 considered statistically significant. Univariate analysis using the Pearson chi 2 test, Fisher’s exact probability test and independent sample t-test. Multivariate analysis using binary logistic regression.â…¡. The research on the role of Th1/Th2/Th17/Treg cells and cytokines in the pathogenesis of sclerodermatous chronic graft-versus-host disease1.Case information:10 patients(6 male and 4 female) who were diagnosed with ScGVHD at Nanfang hospital, Southern Medical University from July 2015 to March 2016. The average age of ScGVHD patients was 28.8 (18-48) years old. And 14 patients(9 male and 5 female) who gained engraftment after allo-HSCT and free from GVHD as control, the average age of control group was 28.6(19～42) years old.2.Specimen collection:collect 7～8ml peripheral blood of ScGVHD patients and control patients by sterile heparin sodium anticoagulation tubes, place at room temperature, and handling within 4 hours.3.Separation of peripheral blood mononuclear cells(PBMC):ficoll density gradient centrifugation was used to separate the mononuclear cells by lymphocyte separation medium.4.Detection of IFN-y+Th1, IL-4+Th2, IL-17A+Th17 cells by flow cytometry: using intracecellular flow cytometry staining method, resuspend the separated PBMC with culture medium, cuture cells for 5-6 hours after adding cell stimulation and protein transport inhibitors, harvest of the cells and then staining with cell surface antigensâ†'fixing and permeabilizing the cellsâ†'staining with intracecllular antigens, flow cytometry analysis after washing.5.Detection of CD4+ CD25+ Foxp3+ Treg cells by flow cytometry:using intranuclear flow cytometry staining method, collect the separated PBMC and then staining with cell surface antigensâ†' fixing and permeabilizing the cellsâ†'blocking the nonspecific binding siteâ†'staining with intranuclear antigens, flow cytometry analysis after washing.6.Detection of levels of plasma IFN-y, IL-4, IL-10, IL-17A, TGF-β, TNF-a by ELISA assay.7.Statistical analysis:Statistical analyses were performed using the SPSS for Windows (version 20.0) with P<0.05 considered statistically significant. All continuous data were shown as mean values±standard deviations(x±s). The homogeneity of variance should be test at first, if meet, using two independent sample t-test, and using the rank transformation test if not dissatisfaction. Using Pearson correlation test to analyze the correlation between the variables.Results1.In this study,134 (51.7%) of the 259 patients developed cGVHD including localized in 98 (73.1%) cases and generalized in 36 (26.9%) cases.22 patients presented with sclerodermatous cGVHD, that is the incidence of ScGVHD was 8.49%in transplant patients (survival at least 100 days) and 16.4% in cGVHD patients.2.2 (9.1%) cases of 22 ScGVHD patients shown joint sclerosis type with clinical manifestation of joint contracture and activity limitation without cutaneous sclerosis. The remaining 20 (90.9%) cases shown cutaneous sclerosis with localized ScGVHD in 7 (31.8%) cases and generalized ScGVHD in 13 (59.1%) cases. Cutaneous lesions manifested sclerosis, hyper-or hypopigmentation, scaling, ulceration, blistering and so on. There was no Reynolds phenomenon in all 22 patients. In 22 ScGVHD patients, there were 7 cases with oral GVHD,7 cases of liver GVHD,3 cases of lung GVHD,1 case of renal GVHD, and no digestive tract involvement. The median RS score was 11 (2-42) in 20 cases of cutaneous ScGVHD.15 cases of 22 SCGVHD patients had taken autoimmune antibody detection, which 11 cases of anti nuclear antibody (ANA) positive,2 cases of anti mitochondrial antibody (AMA) positive,1 case of anti centromere antibody (ACA) positive and anti ScL-70 antibody were negative in all. The basic therapy of ScGVHD is Cyclosporine A (CsA)/tacrolimus (FK506) combined with glucocorticoid hormone and other immunosuppressor such as cyclophosphamide, methotrexate, thalidomide and so on. Anti-CD20 monoclonal antibody rituximab was adding into the therapeutic regimen in 2 patients and tyrosine kinase inhibitor imatinib mesylate imatinib in 1 patient.1～14 times MSC infusion was accepted in 9 patients because of disease progression or conventional treatment invalid.77.3%(17/22) patients was benefit from treatment and 1 patient dead of obliterative bronchiolitis complicated with severe pulmonary infection, the remaining cases survived.3.The univariate analysis showed condition regimen (TBI/without TBI, P=0.031), GVHD prophylaxis (MMF/without MMF, P=0.046), occurred cGVHD(P=0.008), donor lymphocyte infusion (DLI) (P=0.001) were closely associated with occurrence of ScGVHD. By multivariate analysis, cGVHD (RR=3.512,95%CI=1.235-9.987, P=0.018) and DLI(RR=5.217,95% CI=1.698～16.029, P=0.004) were independent risk factors.4. The Thl cells proportion in CD4+T cell of ScGVHD patients was slightly higher (4.96±4.71 vs 3.86±3.69) when compared with control patients, Th2 cell proportion was slightly lower (1.29±1.62 vs 2.00±1.85) and Th17 cell proportion was slightly higher (1.44±2.69 vs 0.66±0.50), but they were no statistical significance. The plasma level of IL-4 in ScGVHD is significantly higher than control group(2.90±1.49 vs 1.54±0.97, P=0.013).5. The percentage of CD4+ CD25+ Foxp3+ Treg cells in ScGVHD patients was lower than that in non GVHD control group (1.73±1.69 vs 3.93±2.82, P=0.039) with statistical significance. The plasma TGF-P level is significantly higher (731.31±445.54 vs 328.06±222.81,P=0.008) in ScGVHD group.6.The plasma TGF-β level of ScGVHD patient was significantly positively correlated with skin involvement RS score (r=0.703, P=0.023).Conclusion1. ScGVHD is a complication after allogeneic hematopoietic stem cell transplantation at a median of 12.5 months after transplantation(range,4-28). The incidence of ScGVHD was 8.49%(22/259) in all transplanted patients and 16.4% (22/134) in those with cGVHD. While the result is consistent with those reported in the literature indicating a positive control of long-term complication after allo-HSCT in new era of haploidentical transplantation.2. The plasma TGF-P level of ScGVHD patient was positively correlated with skin involvement RS score, it suggested that plasma TGF-β level could be used as a monitoring index of disease severity.3. Factors associated with an increased risk of ScGVHD by univariate analysis included the use of TBI in conditioning regimen, the use of MMF in GVHD prophylaxis, cGVHD and DLI. By multivariate analysis, cGVHD and DLI were independent risk factors.4. Imbalance of CD4+T lymphocyte subsets(Th17/Treg) and cytokines(IL-4, TGF-β in ScGVHD patients may be one of the pathogenesis of this disease, which may contribute to early diagnosis and provide a new direction for the treatment of ScGVHD.5. The percentage of Tregs in ScGVHD patients’ peripheral blood CD4+T cell was significantly lower than that of the control group. It suggested that the adoptive infusion of Treg cells may be a effective treatment of ScGVHD.