Debate Round-Table: Comments concerning chimerism studies

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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

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

Sequential molecular analysis of chimerism after allo-SCT has been shown to be useful for predicting engraftment, graft failure, rejection or impending relapse.9,10,11 Furthermore, more detailed analysis within a specific leukocyte subset (granulocytes, monocytes, NK cells, T cells, B cells, etc) could provide more information in order to distinguish patients with different clinical situations (eg differentiate patients with delayed engraftment from those with irreversible rejection and predict the occurrence of GVHD).12 In addition, some reports show that chimerism studies can be useful for monitoring therapy and detection of minimal residual disease (MRD). In our series of 31 patients who had undergone non-ablative allo-SCT, 21 cases could be monitored for MRD by flow cytometry and/or RT-PCR techniques.13,14,15 In eight of them (two CML, one AML Ph+, one AML, one MDS, three MM), in whom there was persistence of MRD and/or MC, an immunotherapy strategy was initiated (faster taper off immunosuppression and/or donor lymphocyte to infusion). In all eight patients, a CC was reached and MRD became negative in five of them. In the remaining three patients (all MM patients) a slow decrease of monoclonal component and abnormal BM plasma cells is currently being observed. MRD clearance was preceded by full donor chimera and GVHD in all cases. Interestingly, none of these patients have relapsed to date, suggesting that a combination of both techniques can be used to guide an immunotherapy strategy. Regarding their utility as a technique for MRD detection, in contrast to the immunophenotype and/or RT-PCR analyses that are restricted to certain types of leukemia, the chimerism studies can be used in all cases of allo-SCT. However, their low sensitivity does not allow the detection of minor host cell fraction if they represent less than 1% of the whole leukocyte sample. In this regard, five out of 12 patients from our series of non-ablative allo-SCT with CC presented MRD positive by cytometry and/or RT-PCR analysis. However, a very attractive alternative could be to carry out the chimerism studies on FACS-sorted material (eg cells with immunophenotype of the original leukemia clone or CD34+ cells) which allows the detection of residual, potentially malignant cells within a 103–104-fold excess of donor-derived cells.16,17 This possibility makes the assay an alternative for the sensitive detection of MRD.

In conclusion, we view chimerism studies to be a powerful diagnostic tool for numerous clinical situations. However, due to the many variables that can influence their final result and the introduction of new therapeutic strategies, the data in the literature are still controversial and open to question. Only large prospective trials in different transplant settings with a standardized technique will allow for scheduled therapeutic interventions in the future. In this sense, international efforts to standardize the technical approach, such as the Biomed I13 or EAC projects18 are worthwhile in order to clarify the role of chimerism studies on allo-transplantation outcome.

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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) doi:10.1038/sj.leu.2402310

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