| Part I:Develope a novel method for detecting the chimerism:next-generation sequencing(NGS)analysis based on single nucleotide polymorphism(SNP)[objective]To develope a novel method for detecting the chimerism of donor cells after hematopoietic stem cell transplantation:next-generation sequencing(NGS)analysis based on single nucleotide polymorphism(SNP).Furthermore,we evaluated the feasibility,sensitivity and accuracy in detecting chimera using this method.[Methods]1.Screening of SNP loci:We downloaded SNP loci with a distribution frequency of 45%-55%in the East Asian population from the GenBank and performed linkage disequilibrium analysis to screen 48 SNP loci from 22 different autosomes.It was expected that the resolution of these loci to distinguish between donors and recipients was 99.99%.2.Preparation of artificial chimera samples:Total peripheral blood was collected from two volunteer donors(V1-V2).All peripheral blood samples were separated into mononuclear cells by Ficoll density gradient centrifugation.Mononuclear cell samples from V1(female)and V2(male)were paired as "donor" and "recipient,"respectively.A total of eight artificial chimeric mononuclear cell mixtures,as donor/recipient chimeras,were mixed by counting at eight percentages(10%,5%,1%,0.5%,0.1%,0.05%,0.01%,0.005%)for each artificial mixture.3.Three methods,including SNP-NGS technology,STR(short fragment tandem repeat)-PCR combined with capillary electrophoresis fragment analysis,and XY-FISH(fluorescence in situ hybridization),were used to detect these artificial chimeras.In addition,SNP-NGS and STR-PCR methods were used to detect the sample before mixing to determine the donor/recipient genotype.Sensitivity of these various detection methods was compared and analyzed.Finally,correlation analysis was performed by calculating the Pearson correlation coefficient to evaluate the effectiveness and feasibility of the SNP-NGS technology.[Results]1.When using untagged multiple PCR primers to amplify the target gene,the SNP-NGS analysis result of the artificial chimera showed that 19 different sites of donor and recipient SNP were screened,and the detection range of donor cell mosaic rate was 0.05%to 10%,but its detection limit was 0.1%(that is,the detection limit for the proportion of small populations of cells is 0.1%).Pearson correlation analysis showed that when the chimeric rate of small population cells was between 0.1%and 10%,the correlation coefficient between the measured value of SNP-NGS and the theoretical value was 0.961,indicating that there was a good positive correlation.2.When tagged multiple PCR primers was uesed to amplify the SNP target gene during SNP-NGS detection process(referred to as"optimized SNP-NGS"),the optimized SNP-NGS analysis results of artificial chimera showed that 19 different sites of donor-recipient SNP were also screened.And the detection range of donor cell mosaic rate was 0.005%to 10%,but the detection limit was close to 0.01%(ie,the detection limit for the proportion of small populations of cells was close to 0.01%).Pearson correlation analysis showed that when the chimeric rate of small population cells was between 0.01%and 10%,the correlation coefficient between the measured value of the optimized SNP-NGS and the theoretical value was 0.963,indicating that there was a good positive correlation.When the mosaic rate of the small population was between 1%and 10%,the correlation coefficient between the measured value of the optimized SNP-NGS and the theoretical value was 0.910.When the mosaic rate of the small population was between 0.01%and 1%,the correlation coefficient between the measured value of the optimized SNP-NGS and the theoretical value was 0.994,indicating that there was nearly a complete positive correlation.3.When STR-PCR combined with capillary electrophoresis analysis was used to detect artificial chimera,the results showed that 12 different sites between donor and recipient STR were screened,and the detection limit of chimerism was close to 1%.(That is,the detection limit for the proportion of small populations of cells is close to 1%).Pearson correlation analysis showed that when the mosaic rate of small population cells was between 1%and 10%,the correlation coefficient between the measured value of STR-PCR fragment analysis and the theoretical value was 0.925.When XY-FISH was used to detect a group of artificial chimera samples,the analysis results showed that the detection range was 0.05%to 10%,but the detection limit of chimeric rate was close to 0.1%(That is,the detection limit for the proportion of small populations of cells is close to 0.1%).4.When the theoretical mosaic rate of small population cells was between 1%and 10%,the Pearson correlation analysis between the measured value of optimized SNP-NGS and the measured value of STR-PCR analysis showed that the correlation coefficient was 0.99,indicating that there was nearly a complete positive correlation.[Conclusions]The sensitivity of the optimized SNP-NGS method to detect artificial chimera was close to 0.01%.Pearson correlation coefficient analysis showed that the measured value of the optimized SNP-NGS was highly positively correlated with the theoretical value of chimeric rate and the measured value of STR-PCR.Moreover,when the optimized SNP-NGS was used to detect microchimerism(the proportion of small population cells<1%),there was nearly a complete positive correlation between the measured value of SNP-NGS and the theoretical value.Therefore,the optimized SNP-NGS technology is a reliable method for detecting chimerism after hematopoietic stem cell transplantation,and it can effectively evaluate the microchimerism.Part II:Application of optimized SNP-NGS in HLA-mismatched micro-transplantation as consolidation treatment for acute myeloid leukemia[objective]To evaluate the value of optimized SNP-NGS in monitoring donor cell engraftment,predicting leukemia recurrence and survival prognosis after HLA-mismatched micro-transplantation for acute myeloid leukemia(AML).[Methods]1.A retrospective analysis of 72 AML patients diagnosed and treated in our hospital from November 2012 to November 2015,aged between 12 and 73 years old.These patients have completed the HLA-mismatched micro-transplantation.The eligibility criteria included:1)Before micro-transplantation,the patients achieved CR1 after one or two induction therapy cycles;2)ECOG performance status score was ≤2 before micro-transplantation;3)All patients underwent consolidation therapy with micro-transplantation after CR1.Risk stratification refers to the 2017 European Leukemia Network(ELN)recommendation.We evaluated the efficacy and safety of HLA-mismatched micro-transplantation as consolidation therapy for patients with AML in the low-risk group,intermediate-risk group and high-risk group.2.Collect cryopreserved cells-or DNA-samples of micro-transplant patients and donors in clinical laboratories.In order to ensure the homogeneity of the study population,the samples from 48 young patients with AML after HLA-mismatched micro-transplantation and donors/recipients before micro-transplantation were successfully collected.Two methods,optimized SNP-NGS technology and STR-PCR combined with capillary electrophoresis analysis,were used to detect the chimerism of these samples,and the effectiveness of optimized SNP-NGS in the detection of micro-chimerism of donor cells in clinical micro-transplantation was evaluated.To further explore the value of donor cell microchimerism in micro-transplantation,our center retrospectively analyzed the clinical data of AML patients who underwent HLA-mismatched micro-transplantation as consolidation regimen after the first complete remission and used optimized SNP-NGS to detect the post-transplantation microchimerism rate and assess the impact of microchimerism on the clinical prognosis of patients.[Results]1.The median LFS of 72 patients was 34 months(range 3-84 months),the median OS was 50 months(range 4-84 months),and the 5-year LFS and OS were 44.0%and 55.6%,respectively.The 5-year LFS in the low-risk group,intermediate-risk group,and high-risk group were 67.0%,34.5%,and 8.3%,respectively(P=0.000).The 5-year OS were 80.6%,48.3%,and 8.3%,respectively,in the low-risk,intermediate-risk,and high-risk group(P=0.000).2.None of the 48 young patients with AML formed a complete or mixed donor chimerism state after micro-transplantation.In our study,microchimerism of donor cells was detected in all 48 patients by using the optimized SNP-NGS detection method,and 46 patients received long-term monitoring.Only two patients tested for donor chimerism within two months after the treatment.In these 46 patients who received long-term chimerism monitoring,the longest persistence of donor microchimerism was 30 months.The median was 10.5 months(2-30 months).The median duration of donor microchimerism in patients with relapsed disease was 6 months(2-18 months),and the median in patients with non-relapsed was 12 months(2-30 months),P=0.001.Thirteen patients who received long-term monitoring of donor chimerism were classified as intermediate-risk,and the median duration of donor chimerism for non-relapsed and relapsed patients was 15 months(9-30 months)and 6 months(2-12 months),respectively(P=0.009).Twenty-seven patients in the low-risk group received long-term monitoring of donor chimerism after treatment,and the median duration of donor chimerism for non-relapsed and relapsed patients was 12 months(2-30 months)and 9 months(2-18 months),respectively(P=0.109).In patients whose duration of donor cell microchimerism was<10.5 months,the relapse rate was 69.6%(16/23).A lower relapse rate(26.1%,6/23)(χ2=8.712,P=0.003)was found in patients with a duration of microchimerism>10.5 months.The 13 cases with intermediate-risk AML were divided into two subgroups by a median time of 10.5 months,the relapse rate of the subgroup<10.5 months was 85.7%(6/7),and a lower relapse rate(16.7%,1/6)(x2=6.198,P=0.029)was seen in the other subgroup>10.5 months.In the low-risk group,the relapse rate of the subgroup<10.5 months was 45.5%(5/11),and a lower relapse rate(31.3%,5/16)(χ2=0.564,P=0.687)was seen in the other subgroup>10.5 months.The duration of donor microchimerism was not correlated with the infused mononuclear cells(MNCs)(R=0.121,P=0.144)or the number of CD34+cells(R=0.02,P=0.318).There was no statistically significant difference in the chimeric rate of donor cells between the patients with relapsed disease and those with non-relapsed on day 14 and day 60 after micro-transplantation.3.HLA-mismatched micro-transplantation as consolidation regimen in these patients with AML was well tolerated with mild non-hematological toxicity,while myelosuppression was common.Among 48 young patients,26 patients(54.2%)had non-infectious fever events after micro-transplantation.Moreover,the 5-year LFS(69.2%)and 5-year OS(76.9%)of patients with non-infectious fever events were significantly better than those of other patients(31.8%and 45.5%),P<0.05.Our further analysis found that the median duration of donor cell micro-chimerism in patients who developed non-infectious fever after micro-transplantation was 12 months(range,2-30 months),and that in patients who did not develop non-infectious fever was 9 months(range,2-18 months),P<0.05.4.Among 48 young patients with AML,the 5-year LFS and 5-year OS were 63.3%and 78.6%in the low-risk group,and the 5-year LFS and OS in the intermediate-risk group were 42.9%and 50.0%,respectively.However,the 5-year LFS and OS in the high-risk group were both 16.7%.Micro-transplantation as consolidation regimen for AML can improve the survival of patients in the low-and intermediate-risk group,and is not recommended for patients in the high-risk group.The 46 patients who received long-term monitoring of chimerism were divided into 2 groups based on the median duration of donor chimerism(10.5 months).Among them,patients with donor chimerism periods<10.5 months were group 1,and those with donor chimerism periods>10.5 months were group 2.The 5-year LFS and OS of group 1 were 30.4%and 43.5%,respectively,and the 5-year LFS and OS of group 2 were73.4%and 82.6%,respectively(P<0.05).For 27 patients in the low-risk group,the 5-year LFS and OS of group 1 were 54.5%and 72.7%,respectively,and the 5-year LFS and OS of group 2 were 68.2%and 81.3%,respectively(P>0.05).For 13 patients in the intermediate-risk group,both the 5-year LFS and OS of group 2 were 83.3%,and the 5-year LFS and OS of group 1 were 14.3%and 28.6%,respectively,P<0.05.[Conclusions]1.Optimized SNP-NGS method can effectively detect the micro-chimerism of donor cells,and the duration of donor chimerism is of great value for the survival and prognosis assessment of patients with AML,especially in the intermediate-risk group.Better LFS and OS were found in patients whose micro-chimerism of donor cells lasted longer.2.HLA-mismatched micro-transplantation as consolidation therapy for young patients with AML is safe and effective in low-and intermediate-risk group.Patients with non-infectious fever had longer microchimerism duration of donor cell and better survival.3.As a high-resolution,high-sensitivity,and high-throughput detection method,optimized SNP-NGS technology can effectively detect the chimerism of donor cells after micro-transplantation.It provides important supplementary means for relapsed-disease monitoring and survival assessment of patients after hematopoietic stem cell transplantation. |
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