| Allogeneic stem cell transplantation(allo-SCT) remains an important therapeutic modality for many patients with hematological malignancies and some benign hematological disorders and solid tumors, at the same time, it is also one of the most effective curative method. The major obstacles for achieving long term disease-free survival after allo-SCT are the severe complications graft-verse-host-disease(GVHD) and relapse. Several previous works suggest that an accurate quantitative analysis of chimerism kinetics would permit early detection of patients with a high risk of GVHD or those liable to relapse. So Detection of mixed chimerism(MC) is pre-requisite in order to assess the graft status and decide later therapeutic strategy. The classical methods of chimeric detection includes:(1) red cell phenotypes; (2) RBC isozyme;(3) karyotype; (4)HLA antigens; (5) Restriction fragment length polymorphism(RFLP); (6) variable number tandem repeats(VNTR); (7) Short tandem repeats(STR),et al. Most cases can get the proof of implantation using methods above, but they do have their own inherent drawbacks. We established a new chimerism detection method in China based on real-time quantitative PCR(RQ-PCR) detecting single nucleotide polymorphism(SNP) locis in the first part of our study, trying to make it more economical, simple and accurate and applying more useful prognosis markers; the second part we study the relationship between early chimerism(+7d,+14d) of the CD4+T cell,NK cell and Treg cell subtype and the clinical outcomes, especially the chimerism on day 7. We tried to find some new valuable prognosis markers before reaching complete chimerism(CC) and early events after SCT.Part One:the establish of the chimerism detection method based on RQ-PCR detecting SNP locis after allo-SCT.Motheds:1.650 Peripheral blood or/and bone marrow samples from 65 patients were collected from the donor and recipient before SCT and from the recipient at different intervals after SCT(+7d,+14d,+21d,+30d,+60d,+90d,+6m,+1y,+2y).2.Genomic DNA was extracted using a salting-out method or by DNAzol or Chelex-100 resin. The concentration and purity of DNA were measured by optical density at 260nm and 280nm with a spectrophotometer and preservated at -20℃; 3.18 SNP locis from 9 different chromosomes were selected, donor and recipient samples before SCT were then screened for informative SNP loci using RQ-PCR method (Taqman probe).4. Gene glyceraldehyde-3-phosphate-dehydrogenase(GAPDH) was amplified simultaneously as an internal control for normalizing input DNA. A standard amplification curve was generated from serial dilutions of the GAPDH plasmids; SNP-PCR was performed in an ABi 7500 real-time PCR device on the informative SNP loci and the chimerism were analyzed by the formula. 5. Known artificial serial dilutions (100%～0.01%) of host cells diluted in donor cells were quantified to assess the accuracy and sensitivity of the method. And conventional STR-PCR, FISH assays and special fusion genes detected by the RQ-PCR method were used to validate the SNP-PCR results.6. Statistical analysis:SPSS 11.0 package was used and a p value of 0.05 was considered significant. Results were denoted with mean±standardized deviate and linear regression analysis for the related test, single sample t test for the intra-and interassay variability.Results:1. The average slope of the 17 times amplification of the GAPDH plasmid was-3.39 and the average intercept was 39.97, correlation coefficients were all more than 0.995, which was close to the desired level. The intra-and interassay variability was 0.50% and 1.1 %, respectively, which were both in control; 2. The amplification efficiency of GAPDH is almost the same as that of the 18 selected SNP locis, so one standard curve is sufficient for quantification of both the SNP loci and the internal control; 3. Over 95.4% can find informative markers in 65 donor/recipient pairs; The S1b, S2, S7b and Sgst SNP loci were most informative in our cases.4. The linear correlation with artificial mixed chimerism is >0.99 and a sensitivity of 0.01%; these values have proven to be reproducible; Comparisons with STR-PCR and FISH had no statistical significance(P>0.05); the quantitative results of special fusion gene transcripts of complete chimerism samples were negative, and positive in mixed chimerism samples.Part Two:Prognosis values of the immune cell subsets'early chimerism(day 7 and 14) after allo-SCT.Motheds:1. A total of 650 peripheral blood (PB) or/and bone marrow (BM) samples were collected from donors and recipients before SCT and from recipients at different intervals after SCT(+7d,+14d,+21d,+30d,+60d,+90d,+6m,+1y,+2y, et al). General information were collected, including sex, age, disease category, the number of chemotherapy treatments before SCT, disease stage (CR1/CP1 or not) at SCT, stem cell source, HLA typing, type of regime, ABO type, implantation rate of PB cell, complication, outcome and survival time. DNA were extracted with the mothed in the first part; 2. Chimerism of the 650 samples were detected with the SNP-PCR method descriped in the first part and relationships between outcome after allo-SCT and day 7,14 chimerism level were analyzed.3. A total of 150 bone marrow (BM) samples were collected from recipients at different intervals after SCT(+14d,+30d,+60d,+90d,+6m,+1y, et al). General information was collected(which was the same as part one).4. CD3+,CD56+,CD4+CD25+ cell subsets were achieved through MACS sorting, DNA were extracted with the mothed in the first part; 5. Chimerism of the sorted samples were detected with the SNP-PCR method and relationships between outcome after allo-SCT and day 14 chimerism level were analyzed.6. Statistical analysis:SPSS 11.0 package was used and a p value of 0.05 was considered significant. Results were denoted with mean±standardized deviate; Fisher's exact analysis for count data, t test for measurement data, Kaplan-Meier method for survival analysis.Results:1. Both RIC and standard regimes were well tolerated. Only 2 patients failed to undergo engraftment and 2 rejected their grafts; all the others achieved engraftment successfully.2. The median periods required for recovery of absolute neutrophil(NRT) and platelet count(PRT) were 14 and 16 days, respectively. Patients whose NRT(PRT) were<14d had higher day 7 chimerism than those with NRT(PRT)>14d, but the difference is not statistically significant,77.3% vs 69.6% and 78.4% vs 69.5%(P>0.05). Counts of white blood cells, hemoglobin and platelets in PB on day 7 were similar in patients whose chimerism was less than and greater than 65%, which were 0.38±0.75 vs 0.33±0.65×109/L(P=0.819), 83.6±11.8 vs 77.4±17.8 g/l(P=0.207) and 22.9±26.9 vs 25.5±37.6×109/L (P=0.801) respectively(P>0.05), the level of chimerism on day 7 had no direct relationship with the count of peripheral blood.3. Of the 65 patients in the study,13 experienced leukemia relapse. The media chimerism level on day 7 and day 14 of relapse patients were much lower than non-relapse patients,51.86% vs 83.02% and 82.89% vs 95.37%(P<0.01); 24 developed varying degrees of GVHD,14 aGVHD and 13 cGVHD (10 localized and 3 extensive). Grades II to IV aGVHD were seen in 8 patients; 4. With increasing chimerism on day 7, the probability of developing GVHD increased(P=0.001) and repalse rate decreased (P=0.0001), the incidence of extramedullary relapse was not significantly different; Day 7 chimerism <65% was strongly associated with increased risk of relapse or rejection (68.8% vs 8.7%,P=0.0001) and>85% was distinctly associated with increased risk of both whole GVHD (aGVHD+cGVHD) and cGVHD(46.43% vs 9.52%, P<0.01), no significant difference was found in the incidence of aGVHD(P=0.091); 5. With increasing chimerism on day 14, repalse rate decreased (P=0.002), but the probability of developing GVHD not increased significantly(P>0.05); When we further devided the day 14 chimerism by 85% and 95%, relapse rate obviously increased in the<85% group(P<0.01), whole GVHD and CGVHD appeared more in the>95% patients(P<0.01).6.21 of the 65 patients in the study died during the two and a half years of follow-up, the 24-month probability of OS for the entire group was 56%. OS of the group whose day 7 chimerism were between 65-85%(which we called "optimal survival chimerism level" on day 7) was significantly higher than that of the other group, with the 24-month probabilities of OS 82.6% vs.47.0%(P=0.04). However, similar results were not reached considering the golden survival chimerism space 85-95% for day 14 samples (P>0.05).7. Disease category, the number of chemotherapy treatments before SCT, disease stage (CR1 or not) at SCT, type of donor, age, HLA typing, ABO type, and stem cell source showed no statistically significant differences between patients whose day 7 chimerism was 65-85% and those out of this region.8.32 cases all got successful T, NK cell sorting with the purity of 75-95%; There were 32 cases ready to sorting CD4+CD25+ cell.2 didn't have the informative SNP loci; we didn't acquire samples from 2 patients and 3 failed to get successful cell sorting, finally 25 cases got cell sorted with the purity 70-80%; 9. Day 14 T cell chimerism<80% had the tendency to relapse(40% vs 4.5%, P=0.024), but had no significant relationship with developing GVHD(P>0.05), neither for the NK cell.10. Day 14 chimerism of the CD4+CD25+ cell subset had no significant relationship with relapse and GVHD, but the margin between CD4+T and CD4+CD25+ cell do have close relationship, which means if the chimerism of day 14 CD4+CD25+ is higher than CD4+ T cell, the incidence of GVHD reduced, or reverse, the incidence of GVHD was 6.7% vs 50%(P=0.014).Conclusion:1. SNP-PCR method detecting chimerism after SCT is easy, accurate and reliable; Over 95.4% can find informative markers in 65 donor/recipient pairs, this system is suitable for popularization and application in Chinese people; the linear correlation with artificial mixed chimerism is>0.99 and a sensitivity of 0.01% and proved to be reproducible; Comparisons with STR-PCR and FISH had no statistical significance(P>0.05); and the results were consistent with the quantitative results of special fusion gene transcripts in AL patients.2. Patients whose NRT(PRT) were<14d had higher day 7 chimerism than those with NRT(PRT)>14d, but the difference was not statistically significant (P>0.05). Counts of white blood cells, hemoglobin and platelets in PB on day 7 were similar in patients whose chimerism was less than and greater than 65%, respectively(P>0.05), the level of chimerism on day 7 had no direct relationship with the count of peripheral blood;3. Under strong conditioning regimen, the median chimerism level on day 7 could reach 76.36%, and over 50% patients acquired CC on day 14, the implant rate was significant faster than the non-myeloablative stem cell transplantation(NST) reported; the implantation rate of donor T cells was significantly slower than NK cells;4. With increased chimerism on day 7, the probability of developing GVHD increased and repalse rate decreased; Day 7 chimerism<65% was strongly associated with increased risk of relapse or rejection and>85% was distinctly associated with increased risk of both whole GVHD(aGVHD+cGVHD) and cGVHD; When we devided day 14 chimerism by 85% and 95%, relapse rate obviously increased in the<85% group, whole GVHD and cGVHD appeared more in the>95% patients;Day 7 chimerism<65% and day 14 chimerism<85% can be used as rejection or recurrence early-warning markers; Day 7 chimerism>85% and day 14 chimerism>95% can be applied as GVHD early-warning markers;5. Day 14 T cell chimerism<80% had the tendency to relapse(P=0.024), but had no significant relationship with GVHD, neither for the NK cell;6. We can use magnetic beads to sort CD4+CD25+ cell subsets, about 90% achieved successful cell sorting; if the chimerism level of day 14 CD4+CD25+ cell is higher than CD4+ T cell, the incidence of GVHD reduced, or reverse. It can be used as an additional prognostic marker for patients who achieved high level chimerism on day 14;7.24-month probability of OS of patients in our study was 56%. OS of the group whose day 7 chimerism were between 65-85%(which we called "optimal survival chimerism leve"on day 7) was significantly higher than those out of the region. We believe that day 7 chimerism between 65% and 85% is an important indicator for the long-term survival.
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