Reliable quantification of hematopoietic chimerism after allogeneic transplantation for acute leukemia using amplification by real-time PCR of null alleles and insertion/deletion polymorphisms

Jiménez-Velasco, Antonio; Barrios, Manuel; Román‐Gómez, José; Navarro, Germán; Buño, Ismael; Castillejo, Juan A.; Rodríguez, Antonia; García-Gemar, Gracia Mar; Torres, Antonio; Heiniger, Anabel

doi:10.1038/sj.leu.2403622

Cited by 70 publications

(60 citation statements)

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“…The detection of increasing MC preceded relapse by a median of 74 days in this study and was significantly related to the absence of chronic GVHD [26]. The same group published recently that real time PCR of null alleles and insertion/deletion polymorphisms predicted a significantly higher number of relapse (88.2% vs 44.4%) with a median anticipation period of 58 days [27].…”

Section: Discussionmentioning

confidence: 87%

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

et al. 2006

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We 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. 81:735-746, 2006. V V...

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Section: Discussionmentioning

confidence: 87%

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

et al. 2006

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“…Detection of 0.1% of target DNA has been reported for SNP 8,14,21 compared with 0.1% to 0.01% for bialellic short insertion/deletion polymorphisms. 6,14,22 The detection limit of our SNP-specific qPCR assay was evaluated with dilution experiments of allele-positive DNA in allele-negative DNA. A linear correlation of target DNA template fraction (r ϭ 99%) for all SNP markers was detected down to 0.1% of the test template (Figure 1).…”

Section: Discussionmentioning

confidence: 99%

Single Nucleotide Polymorphism-Based System Improves the Applicability of Quantitative PCR for Chimerism Monitoring

Gineikiene

Stoškus

Griškevičius

2009

The Journal of Molecular Diagnostics

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Recently , several studies demonstrated the feasibility of a real-time quantitative PCR (qPCR) approach for chimerism monitoring. qPCR offers a fast , sensitive, and elegant quantification of genotypes. However, before it becomes an established method for routine chimerism monitoring , a qPCR marker set for every transplant pair should be available. This requirement poses a major challenge since the genetic markers for qPCR-short insertions/deletions (Indels) and single nucleotide polymorphisms (SNPs)-published to-date do not guarantee applicability for every transplant pair. The aim of our study was to design and validate a new SNP allele-specific system to supplement an already existing Indel primer panel and improve applicability of the qPCR approach for chimerism status monitoring. Here , we present an approach for an economical in-house design of SNP allele-specific qPCR primers/probe sets with a locus-individualized reference system that allows for the accurate quantification of the respective informative locus using a simple ⌬⌬Ct method. We designed primers/probe sets specific for seven biallelic SNP loci and validated them in a population of 30 transplant pairs. Repeatability varied depending on the amount of quantifiable genotype. The combination of our SNPqPCR system and Indel primers increased recipient genotype identification from 86.6% to 96.6% when tested in a population of our transplant pairs. These results demonstrate the feasibility of our SNP-based qPCR approach to improve the applicability of a qPCR for chimerism monitoring.

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“…Quantitative PCR analysis using insertion/deletion polymorphism (indel qrtPCR) is a well-characterized and simple technique, which can be performed in peripheral blood with a better sensitivity compared with standard STR-PCR (0.01-0.1% vs 1-5%). 22,23 While this technique has been previously used by others, [24][25][26] its usefulness to predict relapse at the recipient's cell level o1% has never been clearly evaluated.…”

Section: Introductionmentioning

confidence: 99%

Chimerism analysis in peripheral blood using indel quantitative real-time PCR is a useful tool to predict post-transplant relapse in acute leukemia

Jacque

Nguyen

Golmard

et al. 2014

Bone Marrow Transplant

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Detection of increasing mixed chimerism (IMC) using standard PCR correlates with relapse after allo-SCT for acute leukemias (ALs). Quantitative real-time PCR of insertion/deletion polymorphism (indel qrtPCR) is a much more sensitive method, which can be performed on peripheral blood. We studied the significance of low increases of recipient cells (0.1%) detected by indel qrtPCR in a cohort of 89 transplants. We did not observe relapse among the 32 patients with no IMC. Fifty-seven patients presented a first IMC, which was followed by four different scenarios: a decreasing MC (26 cases, no relapse), a stable MC (1 case, 1 relapse), a second IMC (24 cases, 15 relapse) or no control of chimerism (6 cases, 5 relapses). In multivariate analysis, detection of two successive IMCs was strongly associated with relapse (hazard ratio (HR): 9.4, 95% confidence interval (CI): 3.8-23; Po0.0001). Among the 57 patients who presented at least one IMC, 27 underwent immunomodulation (tapering of immunosuppression or donor lymphocyte injection), leading to a 1-year relapse rate of 15.7% vs 57.6% in the 30 other patients (P = 0.0007). Altogether, these results indicate that chimerism analysis using indel qrtPCR in peripheral blood is a useful tool for detection of relapse in patients transplanted for AL.

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Reliable quantification of hematopoietic chimerism after allogeneic transplantation for acute leukemia using amplification by real-time PCR of null alleles and insertion/deletion polymorphisms

Cited by 70 publications

References 40 publications

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

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

Single Nucleotide Polymorphism-Based System Improves the Applicability of Quantitative PCR for Chimerism Monitoring

Chimerism analysis in peripheral blood using indel quantitative real-time PCR is a useful tool to predict post-transplant relapse in acute leukemia

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