Flow cytometry with anti HLA-antibodies: a simple but highly sensitive method for monitoring chimerism and minimal residual disease after HLA-mismatched stem cell transplantation.

Transplantation of HLA-mismatched stem cells may allow determination of chimerism status of single cells by differential expression of HLA molecules. Monoclonal antibodies against HLA antigens can be used to determine the HLA type of sub-populations by standard flow cytometry. Blood samples from 23 patients transplanted from HLA-mismatched family donors were monitored using HLA-specific antibodies. Suitable antibodies could be found for all donor recipient pairs by using differences in HLA Bw4 and Bw6 groups or other serological antigens. Pretransplant controls of donor and recipient were used to correct for variable fluorescence intensities of the antibodies and sub-populations. Owing to the high sensitivity, cell populations with a minimum frequency of 0.1% were detectable. Flow-cytometric analysis was confirmed by chimerism analysis of immunomagnetically isolated T cells by standard PCR technique. In addition to chimerism evaluation, HLA antibodies improved the detection of leukemic cells after transplantation with aberrant phenotype. In conclusion, flow cytometry using antibodies against HLA antigens is an interesting tool for determination of chimerism and minimal residual disease after HLA-mismatched transplantation. Information about the chimerism status is given on a single-cell level and allows fast and convenient analysis of sub-populations.

Increasing mixed chimerism defines a high-risk group of childhood acute myelogenous leukemia patients after allogeneic stem cell transplantation where pre-emptive immunotherapy may be effective.

Children with leukemias and increasing mixed chimerism (increasing MC) after allogeneic stem cell transplantation have the highest risk to relapse. Early immunological intervention was found to be effective in these cases. To substantiate this on a defined group of pediatric acute myelogenous leukemia (AML) patients, we performed serial analysis of post transplant chimerism and pre-emptive immunotherapy in patients with increasing MC. In total, 81 children were monitored, 62 patients revealed complete chimerism (CC), low-level MC or decreasing MC. Increasing MC was detected in 19 cases. Despite early immunological intervention relapse was still significantly more frequent in patients with increasing MC (9/19) than in patients with CC, low-level or decreasing MC (8/62, P<0.005). The probability of 3-year event-free survival (EFS) was 52% for all patients (n=81), 59% for patients with CC, low-level MC, 60% for patients with decreasing MC (n=62), and 28% for patients with increasing MC (n=19, P<0.005). Patients with increasing MC who received early immunological intervention showed a significantly enhanced probability for event-free survival (pEFS 36%, n=15) compared to patients with increasing MC without intervention (pEFS 0%, n=4, P<0.05). These results prove that pediatric AML patients with increasing MC are at highest risk for relapse and that early immunological intervention can prevent relapse in these patients.

Clinical relevance of serial quantitative analysis of hematopoietic chimerism after allogeneic stem cell transplantation in children for severe aplastic anemia.

Allogeneic stem cell transplantation (allo-SCT) is a well-established treatment modality for children with severe aplastic anemia (SAA). Treatment failures are rare and mostly caused by graft rejection. Increasing mixed chimerism represents a stage at the very beginning of graft rejection, where immunological intervention might be an effective prophylactic approach. To substantiate this, we: (1) monitored peripheral blood cells from children with SAA after allo-SCT and performed pre-emptive immunotherapy in patients with increasing MC. In all, 23/34 courses of 32 children with SAA after allo-SCT showed a complete chimerism (CC) throughout and 10/34 developed different types of mixed chimerism (MC). Altogether, 4/10 with MC spontaneously developed decreasing MC, 2/10 courses persisted with low proportions of autologous cells below 30% (stable-MC), 4/10 developed increasing MC and one patient showed an autologous recovery. All patients with CC, decreasing MC or stable MC remained in continuous complete remission (CCR). In all, 2/4 patients with increasing MC developed graft rejection. Based on these observations, 2/4 new patients with increasing MC received low-dose DLIs prophylactically, and remained in CCR without any GVHD. These results substantiate that low-dose DLI in children with SAA and increasing MC can prevent graft rejection with a calculable risk to induce severe GVHD.