Molecular evidence of persisting or reappearing recipient cells may be a reflection of either survival of leukemic cells or of survival of normal host hematopoietic cells or a combination of both. Surviving host hematopoietic cells may in turn facilitate the re-emergence of a malignant cell clone by inhibiting immunocompetent donor effector cells. For patients with CML, it could be clearly demonstrated that reappearance of host hematopoietic cells in the mononuclear cell fraction preceded hematological relapse87,88,89,90 Therefore, mixed chimerism has been considered to reduce the graft-versus-leukemia effect (GVL) in this particular group of patients.87,88,91

In patients with acute leukemias and MDS, several early studies have left the question unanswered whether patients with mixed chimerism do have an increased risk of relapse.37,39,92,93,94,95 In the middle of the 90s, it was realized that evolution of chimerism is a dynamic process and chimerism analysis should be carried out serially in short time intervals. Using STR–PCR-based serial analysis of microsatellite regions in short time intervals, it could be shown that patients with rapidly increasing mixed chimerism have the highest risk of relapse.13,14,15 These reports could be confirmed by others,96,97,98,99 whereas some studies did not find a correlation between chimerism and relapse.22,38,100,101 These discrepancies may partly be explained with sampling protocols used in the studies. Investigations of subpopulations in patients with acute leukemias showed that there might also be a difference between adult and pediatric patients. Guimond et al could demonstrate that mixed chimerism in T- and NK-cell subpopulations can frequently be found in pediatric patients with leukemia relapse, but not in children in remission. In contrast, mixed chimerism in these subsets was not found in adult patients with relapse.102 In our own study, we could show that persistent mixed chimerism in the early post-transplant period is caused predominantly by normal recipient hematopoietic cells.103 Its increase precedes the reappearance of the underlying disease. These findings therefore support the hypothesis that a state of mixed hematopoietic chimerism may reduce the clinical GVL effect of alloreactive donor-derived effector cells also in patients with acute leukemias and MDS, and thus facilitate the proliferation of residual malignant cells that may have survived the preparative regimen. Barrios et al104 could prove in 133 patients with acute leukemias that patients with increasing mixed chimerism have a significantly elevated risk to develop relapse. Based on these studies, several consecutive trials were initiated, evaluating the possibility to prevent relapse by pre-emptive immunotherapy on the basis of chimerism analysis in patients with acute leukemias.96,99,105,106,107,108 Most recently, our group could show in 163 children with ALL that STR-based chimerism analysis in short time interval is able to define a great cohort of children with impending relapse and also that overt relapse, in principle, could be prevented by pre-emptive immunotherapy on the basis of increasing mixed chimerism.109 However, these analysis showed also that (i) it was not possible to realize impending relapse in all patients and (ii) the time interval between the conversion of chimerism and relapse can be very short.

Chimerism analysis does provide information about the alloreactivity and/or tolerance induction of the graft, and thereby serves more likely as a ‘prognostic factor’ than as an indirect marker for minimal residual disease (MRD). It is moreover, important to stress that, due to its low sensitivity of about 1%, chimerism analysis is not a reliable procedure for the detection of MRD.

It could be shown in children transplanted for ALL that the level of MRD prior to transplant has a significant impact on post-transplant outcome.110,111,112 Patients with high level MRD at the time of transplant (>10−3 malignant cells in the background of nonmalignant cells) could be rarely cured. Most likely, neither the conditioning nor the alloreactive potential of the graft could clear the disease. In this group, relapse is also occurring although patients are complete chimeras throughout the follow-up. On the other hand, in patients who have a low MRD burden (<10−3 malignant cells in the background of nonmalignant cells), residual disease can be controlled by a conversion of mixed chimerism to complete chimerism, for example, by pre-emptive immunotherapy. This is illustrated in Figures 2 and 3.

Figure 2 The course of post transplant follow-up is given of a 5-year-old boy with c-ALL, who received an haploidentical transplantation in CR2. This patient has had a high level (>10−3) of MRD prior to transplant. The conditioning regimen could not substantially reduce the leukemia burden and such high leukemia load could not be controlled by the alloreactive potential of the graft. The patient relapsed on day 168 post transplant. Full size image

Figure 3 The course of a 16-year-old girl with c-ALL, who was transplanted in CR2 from an HLA-identical unrelated donor. This patient received a low-dose DLI 1 × 105/kg body weight (BW) when increasing mixed chimerism was developed. This immunotherapy led to a conversion of mixed to complete chimerism and by then MRD was cleared. Full size image

MRD should be monitored using disease-specific PCR techniques as, for example, TCR- or IG- gene rearrangements for ALL and BCR/ABL fusion mRNA transcripts for CML (for reviews, see van Dongen et al113, Gabert et al114 and van der Velden et al115). When a disease-specific marker is not available, for example, regularly in patients with AML, chimerism analysis in cell subpopulations may serve as a surrogate marker for MRD. A very elegant approach was presented by Thiede et al,116,117 who could show that mixed chimerism in CD34-positive cells is predictive for relapse in patients with AML and ALL in peripheral blood. They could show that increasing autologous cells within this subset precedes relapse with a median interval of 52 days (range 12–97).118 To enrich this rare subpopulation in the periphery, however, 50 ml of blood is needed, which limits the applicability of this procedure to adult patients only. Mattsson and coworkers performed a prospective analysis in 30 patients with AML and MDS. They have investigated chimerism in CD33-, CD7- or CD45-positive cells and found significantly more relapses in patients whose subpopulation was mixed, compared to patients with complete chimerism.119 In ALL patients, several studies have been performed evaluating the impact of mixed chimerism after enrichment of the cell population carrying the leukemic phenotype (possible targets could be: CD10, CD19, CD34 for precursor B-ALL, CD3, CD4, CD5 and CD8 for T-lineage ALL).28,120,121,122,123 These studies showed a remarkable correlation between minimal residual disease and mixed chimerism in the respective subset. However, large studies in ALL patients, indicating the predictive value of mixed chimerism in different subsets for the individual patient with regard to disease recurrence, are yet missing.

Taken together, serial and quantitative analysis of chimerism in the whole peripheral blood by STR–PCR allows the identification of patients at the highest risk for relapse. However, not all patients can be highlighted and the time interval between onset of mixed chimerism and relapse can be very short. Therefore, it is essential to perform these analyses weekly during the first 100, better during the first 200, days since the majority of relapses occurs during this time. Performing chimerism analysis in subpopulations increases the sensitivity of the approach enormously. In this setting, chimerism analysis can be considered as a surrogate marker for minimal residual disease. Combining chimerism and MRD analysis does allow accurate documentation of engraftment and surveillance of post-transplant remission status, thus providing a rational basis for individual pre-emptive immunotherapy strategies to prevent recurrence of the underlying disease.