The Significance Of Chimerism Monitor In Lineage-specific Immune Cells After Allogenei Hematopoietic Stem Cell Transplantation

Objective:To investigate the significance of chimerism monitor in lineage-specific immune cells in judging engraftment,acute graft versus host disease(aGVHD)and relapse/graft rejection after allogeneic hematopoietic stem cell transplantation,and analysing the relationship between CMV/EBV infection and chimerism evolution.Methods:Sixty-eight patients underwent allogeneic hematopoietic stem cell transplantation between June 2016 to December 2018 in our department were enrolled in this research,including Myelodysplastic syndrome(n=19),acute myeloid leukemia(n=15),acute lymphoblastic leukemia(n=9),sever aplastic anemia(n=6),Non-Hodgkin lymphoma(n=6),chronic myeloid leukemia(n=3),mixed phenotype acute leukemia(n=3),paroxysmal nocturnal hemoglobinuria(n=2),AA-PNH syndrome(n=1),multiple myeloma(n=2)and Thalassemia(n=2).All the patients were tested chimerism of specific immune cells and complete blood cell by short tandem repeats-Polymerase chain reaction(STR-PCR)per month,with a minimun follow-up of one year.Meanwhile,we collected and extracted clinical datas of each patient,including engraftment time,the status and grade of aGVHD,relapse time and the imformation of CMV/EBV infection.All datas obtained were analyzed by rectospective statistical analysis.Results:1.Myeloablative stem cell transplantation(MTS)group with 57 patients and reduced intensity conditioning/non-myeloablative stem cell transplantation(RIC/NST)group with 11 patients.In MST group,the average time of absolute neutrophil count(ANC)>0.5×10^9/L was 15.41±4.38d(10-30d),and the average time of plantelet count(PLT)>20×10^9/L was 23.60±6.78d(8-40d);in RIC/NST group,the results came as15.51±2.26(11-20d)and 23.43±7.31(11-40d),respectively.The differences of ANC and PLT engraftment time between MST and RIC/NST group were not statistically significant(PANC=0.632,PPLT=0.823).2.Relationship between chimerism and engraftment:14 days after transplantation,the chimerism of NK cell,B cell,complete blood cell and T cell were:98.79±2.71%(84.06-99.99%)?98.77±3.01%(79.51-100%)?98.45±3.33%(81.28-100%)and 96.54±3.72%(78.42-99.88%),separately,in which T cell chimerism was the lowest,and it was statistically significant(PT vs NK<0.001,PT vs B<0.001,PT vs complete blood cell=0.002).The same results were found in the chimerism between NK cell,B cell,T cell and complete blood cell in 28 days after transplantation(PT vs NK=0.005,PT vs B=0.01,PT vs complete blood cell=0.004).60 days after transplantation,T cell chimerism was lower than NK and B cell,which was statistically significant(PT ? NK<0.001,PT ?B=0.009).28 days after transplantation,the full donor chimerism(FDC)rates of each immune cell population were:NK cell 94.92%(56/59),T cell 81.67%(49/60),B cell 93.22%(55/59)and complete blood cell 95.52%(64/67).FDC rate of T cell was the lowest,which was statistically significant(P=0.037).3.Reltionship between chimerism and aGVHD:14 days after transplantation,T cell chimerism rate among aGVHD group and non-aGVHD group were 95.68±5.54%and 97.12±1.50%,respectively,P=0.142;and the values were 96.49±5.25%and 96.00±7.43%in 28 days after transplantation,P=0.778.While the differences were not statistically significant.60 days after transplantation,T cell chimerism of aGVHD group was higher than that of non-aGVHD group(98.96±1.85%vs 95.98±7.72%),P=0.043.At 14 days after transplantation:12 cases(22.22%)suffered from ?-?°o aGVHD and 9 cases(16.67%)suffered from ?-?° in T cell FDC group,2 cases(50%)suffered from ?-?° aGVHD and 1 case(20%)suffered from ?-?° in T cell MC group,the difference was not statistically significant(P=0.550).And the severity of aGVHD between T cell FDC and MC group in 28 days and 60 days after transplantation were not statistically different(P28d=0.088,P60d=0.245).Meanwhile,14,28 and 60 days after transplantation,the cumulative incidence of aGVHD in Tcell FDC and MC group were not statistically significant(P14d=0.423,P28d=0.501,P60d=0.148).4.Relationship between chimerism and relapse:Among the 55/68 cases of stable full donor chimerism(SFDC)group,3 cases(5.45%)occurred relapse,the 1 year overall survival(OS)was 69.05%(29/42);4/68 cases of increased mixed chimerism(IMC)group,2 cases(50.0%)occurred relapse,the 1 year OS was 50.0%(2/4);8/68 cases of decreased mixed chimerism(DMC)group,no patient occurred relapse,the 1 year OS was 100%(4/4);1/68 case of never full donor chimerism(NFDC)group,this patient died of relapse 8 months after transplantation.The relapse rate(RR)in IMC and NFDC groups were higher than that in SFDC and DMC group,the difference was statistically significant(P=0.006).The chimerism rate of each immune cell experienced varying degrees of decreasing before relapse.In addition,chimerism of three patients occurred extramedullary relapse all remained FDC,so chimerism monitoring lack the sensitivity of predicting extramedullary relapse.And 1 year OS between the four groups were not statistically different(P=0.174).5.Relationship between chimerism and virus infection:28 days after transplantation,T cell chimerism of cytomegalovirus(CMV)infection group and non-CMV infection group were 97.64±4.32%and 93.00±9.58%separately,P=0.012.While 14 and 60 days after transplantation,T cell chimerism between these two groups were not statistically different(P14d=0.362,P60d=0.118).Besides,28 days after transplantation,T cell chimerism of Epstein-Barr virus(EBV)infection group and non-EBV infection group were 99.241±1.03%and 94.62±7.64%,P=0.004.And 14 and 60 days after transplantation,T cell chimerism between these two groups were not statistically different(P14d=0.241,P60d=0.961).Otherwise,28 days after transplantation,the cumulative probability of CMV/EBV infection in T cell FDC group was higher than that in MC group.The differences were considered statistically significant(Pcmv=0.020,PEBV=0.047).6.Relationship between aGVHD and virus infection:26/29(89.66%)cases occurred CMV infection in aGVHD group,25/39(64.10%)cases occurred CMV infection in non-aGVHD group,the difference was statistically significant,(P=0.016).However,the incidence of EBV infection between aGVHD group and non-aGVHD group were 44.83%(13/29)and 35.90%(14/39),which was not statistically significant(P=0.457).Furthermore,CMV infection rate in ?-?°aGVHD group and ?-?° aGVHD group were 93.75%(15/16)and 84.62%(11/13),respectively(P=0.573).EBV infection rate in?-?° aGVHD group and ?-?° aGVHD group were 56.25%(9/16)and 30.77%(4/13),respectively(P=0.264).The differences were considered not statistically significant.7.Relationship between conditioning regimen and chimerism:28 days after allo-HSCT,the chiemerism of lineage immune cell subset in MST group were:98.91±2.30%(85.67-99.99%),96.48±6.46%(77.63-100%)and 98.78±3.64%(76.56-100%)in NK,T,B cell respectively;the result of RIC/NST group were 97.13±6.59%(78.54-99.81%),95.83±6.83%(81.68-99.88%)and 97.41±6.29%(79.57-99.87%)in NK,T,B cell.28 days after allo-HSCT,the difference of chimerism rate between MST group and RIC/NST group was not statistically significant(PNK=0.137,PT=0.775,PB=0.350).The same result was found in 60 days after transplantation(PNK=0.484,PT=0.410,PB=0.502).And 28 days after transplantation,the FDC rates of specific immune cell between the two groups were not significantly statistical different(PNK=O.313,PT=1.000,PB=0.433),it was not significantly statistical different in 60 days after transplantaion,either(PNK=0.522,PT=1.000,PB=0.515).8.Multivariate analyses of T cell chimerism:donor gender,donor age,conditioning regimen,HLA matching level,graft source,diagnosis,CD34+cell count of graft,mononuclear cell count of graft were enrolled to determine the relationship between T cell chimerism and these factors.The result indicted that donor gender(P14d=0.998,P28d=0.963,P60d=0.956),donor age(P14d=0.672,P28d=0.543,P6od=0.216),conditioning regimen(P14d=0.999,P28d=0.714,P60d=0.416),HLA matching level(P14d=0.580,P28d=0.220,P60d=0.392),graft source(P14d=0.280,P28d=0.719,P60d=0.401),diagnosis(P14d=0.934,P28d=0.659,P60d=0.351),CD34+ cell count(P14d=0.153,P28d=0.362,P60d=0.455),mononuclear cell count(P14d=1.000,P28d=1.000,P60d=1.000)were not significantly relative with T cell chimerism in 14 days,28 days and 60 days after transplantation.Conclusions:1.Relationship between chimerism and engraftment:we can consider the FDC of T cell as a mark of stable engraftment.Meanwhile,both MST and RIC/NST groups could achieve stable engraftment after transplantation.2.Relationship between chimerism and aGVHD:High chimerism rate of T cell in 60 days after allo-HSCT can be considered as a predictive factor of aGVHD.3.Relationship between chimerism and relapse:donor chimerism declining can be a prediction of disease relapse,while chimerism monitoring lack the sensitivity of predicting extramedullary relapse.Therefore,chimerism monitor,bone marrow morphology test combined with molecular biology study can provide strong evidences for estimating disease relapse as well as instructions for clinical treatment.4.Relationship between chimerism and virus infection:high chimerism rate of T cell during early post-transplantation period has predictive value for CMV and EBV infection.CMV infection can induce the forming of T cell FDC,which promote aGVHD.So CMV infection can be considered as a trigger of aGVHD.