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Debate Round-Table: Comments concerning chimerism studies

Leukemia volume 15, pages 1986–1988 (2001)Cite this article

Allogeneic stem cell transplantation (allo-SCT) is the treatment of choice for many patients with hematological malignancies, as well as some non-malignant hematological disorders such as β-thalasemia major and severe aplastic anemia. Successful allotransplantation is a complex process that requires the engraftment of transplanted pluripotent hematopoietic stem cells which re-establish normal hematological and immune functions. Thus, following allo-SCT, it is important to be able to distinguish between host and donor origin of bone marrow and blood cells in order to monitor the engraftment process. Consequently, the monitoring of chimerism has become a routine instrument at centers performing allo-SCT. Successful engraftment has been associated with stable complete donor hematopoietic chimera (CC) in which only hematopoietic cells of donor origin are detectable. The detection of recipient cells coexisting with donor cells (mixed chimerism, MC) has been associated with both graft rejection and disease relapse. However, other reports produced discrepant results, thus, the incidence and significance of the detection of MC remains unclear.1,2 Some studies considered MC as a sign of poor prognosis, while others showed that survival was identical in patients with MC or complete donor hematopoiesis.1,2 These conflicting results in the literature might be explained by a number of factors. Several of these factors – related to either transplant procedures (less intense conditioning regimens or graft-versus-host disease prophylaxis, fewer stem infused cells, the use of T cell depletion, the CD34+ cells selected, or bone marrow vs peripheral blood stem cells) or patient features (lower age, underlying disease, β-thalasemia major vs acute leukemia) – have been shown to be associated with an increase in mixed chimerism. In our series of 119 adult sibling unmanipulated allogeneic transplants (88 and 31 patients using myeloablative and non-myeloablative regimens, respectively) a chimerism analysis by PCR-VNTR assay using six minisatellite regions (Table 1) was carried out. MCT 118 (50%), YNZ (30%) and ST14 (15%) minisatellites proved to be the most useful, enough to monitor 95% of the patients. The chimerism analysis performed in the early post-transplant period (on days +21 to +56) showed that the presence of MC correlated with the underlying disease, with a higher incidence in CML (35%) and MDS (33%) than in other diseases (AML, ALL, NHL, MM) (P = 0.04), which was probably due to the less intensive chemotherapy received before allo-transplant. The MC was also higher in patients who had undergone non-myeloablative all-SCT (18 vs 10%, P = 0.1) but without reaching statistical significance, probably due to the number of patients studied. The clinical significance of MC was different: its persistence was associated with a trend to relapse in ALL or AML, while in patients with anemia (in all four patients with aplastic or sideroblastic anemia) MC could be detected over a long period (up to +220 days) without graft failure.3 Moreover, in patients with non-myeloablative conditioning, the clinical relevance of MC in the early post-transplant period was not associated with a bad prognosis. Thus seven out of 10 patients with early MC became full donor chimeras later on, and, interestingly, none of these seven patients relapsed.

Table 1 VNTRs primers characteristicsa
Full size table

The other important factors that influence the detection of MC are related to technical reasons. Thus, the sensitivity of the method used, the post-transplant time at which it is detected as well as whether the study was carried out in whole peripheral blood or bone marrow or in a specific hematological subpopulation (eg lymphoid vs myeloid chimerism), can largely influence the detection of MC. Several methods have been successfully applied to evaluate chimerism after allo-SCT. These included cytogenetic, Y body detection, protein polymorphism and red cell phenotyping. More recently, DNA-based methodology has provided more sensitive techniques. The use of polymerase chain reaction (PCR) amplification of variable numbers of tandem repeats (VNTR) or the short tandem repeats (STR) loci, has become the most widely used approach. Numerous assays have been published to date, usually reporting a sensitivity detection limit of between 0.5 and 1.5%. However, in practice, these methods have some limitations. First, the real sensitivity is usually lower, mainly when the size of PCR-VNTR fragments are different, and could condition a preferential amplification of one of the two alleles. The methods are non-automated, time-consuming and they require extensive pre-testing of combined donor/recipient VNTR samples to find an informative marker. Moreover, these methods only give qualitative or semiquantitative information on the degree of chimerism. Additional limitations are that most laboratories use different microsatellite regions and/or ‘in house’ techniques (different primers, PCR-conditions, etc) that hamper comparisons and standardization of results between different institutions. To overcome these problems, a quantitative technique using a commercially multiplex STR PCR Kit (Amp FSTR profiler PCR amplification Kit) with subsequent fluorescence-based detection by automated DNA-sequencer with Genescan and Genotyper software, has recently been described.4 In the 31 patients who had undergone a non-ablative allo-SCT from a related donor, comparative molecular analyses of chimerism status at day +28 after transplantation, were carried our in our laboratory. Chimerism studies were performed by both a PCR-VNTR analysis of six minisatellite regions (YNZ22, VNTR 33.6, MCT118, HVR-Globin, H-RAS and ST-14)5,6,7 and the commercial PCR assay with multiplex amplification of 10 STR loci (D3S1358, vWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, THO, FGA) plus the gender marker Amelogenin and fluorescence detection (ampFlSTR SGM Plus PCR Amplification Kit; Applied Biosystems, Norwalk, CT, USA). All patients had several informative STR (median 5, range 3–8), while one patient did not have an informative VNTR loci. The incidence of MC was higher with STR than it was with PCR-VNTR analysis (35% vs 18%). The percentages of host DNA in the cases with MC by STR and CC by PCR-VNTR analysis ranged between 0.8% and 5.5%. Thus, the discrepancy in the percentage of patients with MC was due to the higher sensitivity of the STR approach. Consequently, according to our results, the STR-PCR analysis using a commercial multiplex STR-PCR Kit is a more sensitive, reproductive and accurate method for monitoring hematopoietic chimerism. However, this assay has some limitations (eg stutter peaks) and it requires specific equipment (an automated DNA sequencer). A detailed description of this procedure for quantitative chimerism analysis has recently been reported.8

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  1. Department of Haematology, University Hospital of Salamanca, Salamanca, Spain

    M González, R López-Pérez, R García-Sanz, JA Pérez-Simón & JF San Miguel

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González, M., López-Pérez, R., García-Sanz, R. et al. Debate Round-Table: Comments concerning chimerism studies. Leukemia 15, 1986–1988 (2001). https://doi.org/10.1038/sj.leu.2402310

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