Quantitative analysis of chimerism after allogeneic stem cell transplantation by real‐time polymerase chain reaction with single nucleotide polymorphisms, standard tandem repeats,and Y‐chromosome‐specific sequences
Authors: Koldehoff Michael , Steckel Nina K. , Hlinka Michal , Beelen Dietrich W. , Elmaagacli Ahmet H. 
Published: July 2006

AbstractWe compared the results of chimerism analyses with real‐time SNP‐PCR to those obtained by the classical STR‐PCR method in 135 hematopoietic stem cell transplantation recipients. Using 10 different SNP gene loci, the SNP‐PCR method was able to discriminate patient from donor cells in 125 of 135 cases (93%), whereas the use of 11 different STR gene loci with the STR‐PCR analysis using agarose or polyacrylamide gel resolution resulted in accurate donor‐host discrimination in all patients. Of the 470 analyzed samples we found in 74% concordant results for both chimerism methods. In all 26% discordant cases the SNP‐chimerism method showed mixed chimerism (MC), whereas the STR‐method found complete chimerism (CC). As a consequence, the SNP‐PCR chimerism analysis method detected a MC prior to the occurrence of relapse significantly earlier than the STR‐PCR chimerism method (120 vs. 30 days, P < 0.007). The probability of relapses was significantly higher in patients with increasing MC (70%) compared to 30% in patients with CC (P < 0.00001) associated with a significantly shorter overall survival in patients with increasing MC. The multivariate Cox model showed that chimerism analsis with SNP‐PCR was the only significant risk factor predicting relapse (RR 6.08, P < 0.0001).Furthermore, we analyzed the chimerism status in male recipients with a female donor in 580 samples of 134 patients using quantitative real‐time PCR of Y‐chromosome‐specific sequences and compared the results with interphase XY‐fluorescent in situ hybridization (FISH). MC without signs of relapse was detected in 35% of samples using quantitative real‐time PCR of Y‐chromosome‐specific sequences. The detected Y‐DNA amounts were low compared to the amounts detected in 104 samples of 42 patients with leukemic relapse at the time of analysis (P < 0.0001). Quantitative real‐time PCR of Y‐chromosome‐specific sequences detected therefore an increasing MC with high residual host DNA amounts approximately 143 days (mean) prior to the occurrence of relapse. By comparing the results of Y‐chromosome PCR with the XY‐FISH analysis we found concordant results in 73% in patients with myeloablative regimens. The XY‐FISH could detect 12 relapses, whereas the Y‐chromosome PCR detect 36 relapses by MC (P < 0.005). Residual host cells gradually decreased during the posttransplant period from a mean of 5.4 ng (first months) to 0.5 ng (above 5 years) without evidence of relapses. The probability of relapses was significantly higher in patients with increasing MC (100%) compared to 8% in patients with CC (P < 0.00001) associated with a significantly shorter overall survival in patients with increasing MC. The multivariate Cox model showed that chimerism analysis of Y‐chromosome‐specific sequences is an important risk factor for relapse (RR 17.0, P < 0.0001).We conclude that the use of real‐time SNP or Y‐PCR may be superior to the STR‐PCR or interphase XY‐FISH methods in detecting patients who are at high risk for relapse after transplant. Am. J. Hematol., 2006. © 2006 Wiley‐Liss, Inc.