Lineage-Specific Chimerism after HSCT in Patients with High Risk Haematological Malignancies.

Monitoring of chimerism levels after hemopoietic stem cell transplantation (HSCT) has become a standard test for documentation of important clinical events such as engraftment, graft rejection or leukemic relapse. Sensitive and informative molecular techniques (PCR for STR/VNTR markers) are preferable methods for many laboratories for assessment of donor- or recipient-derived hemopoiesis. However, the sensitivity and specificity of this method may be improved using lineage-specific analysis. Separation of cell population allows not only to detect the kinetics of lineage engraftment but, in some cases, to reveal residual potentially malignant cells. We performed chimerism analysis in 10 pts with high risk haematological malignancies underwent alloHSCT in 2002–2004 ( 4pts - ALL, 3 of them - in 2-nd CR, 2pts - acute non-differentiated leukemia in 2-nd CR, 2 - AML in 2-nd CR, 1 pt with MDS and 1 pt - CML). 6 pts were transplanted after conventional conditioning (Busulfan 16 mg/kg and Cytoxan 120 mg/kg) and 4 - after reduced-intensity conditioning (Busulfan 8 mg/kg, Fludarabin 150 mg/m² and ATG 40 mg/kg or Cytoxan 900 mg/m2). GVHD was prophylacted by Cyclosporin A (CSA) and Methotrexate(MTX) in 5 pts and Cy +MTX +Prednisone in 5 pts. The median follow-up was 339 days (90–600), median age was 32 yrs. The evaluation of chimerism was performed on bone marrow (BM) and following subpopulations of peripheral blood (PB): CD3+, CD19+ and granulocytes (GR) on +30, +60,+90, +180, +270, +360 days after HSCT using PCR-based method for VNTR/STR analysis. The analysed subpopulations have been obtained using immunomagnetic separation technique (Dynal). We also investigated the mononuclear fraction (MN) left after immunomagnetic separation for revealing of possible residual malignant cells. We revealed differencies in chimerism pattern in BM and PB cell populations. Only 4 pts showed complete chimerism (CC) in PB cell subsets while 7 patients had CC in BM on day+30. Four pts showed stable CC in all cell populations during all time of observation. Six pts demonstrated mixed chimerism (MC) in PB subpopulations (CD3+ - 6 pts, CD19+ - 4 pts, MN - 4 pts, GR - 2pts). Four of them showed stable or increasing donor signal (> 80%) and 2 pts expressed decreased donor chimerism (< 80%). In 4 pts with stable or decreasing MC CSA was cesated and 3 of them became CC to +180 d. 3 pts relapsed, 2 of them - with decreased donor chimerism in MN and CD3 subsets and one patient relapsed on day +90 despite CC in all cell populations. GVHD developed in 6 pts and always was predicted by CC in CD3+ subset. Pts with MC never demonstrated any GVHD. Conditioning regimen had no impact on chimerism status. In conclusion, investigation of chimerism in PB cell subsets permitted detection of minimal persistent recipient cell populations with high sensitivity than in BM. Close monitoring of chimerism allowed to identificate the patients with risk of relapse and convert them to CC using cessation of immunosupression.