Multiparameter flow cytometry and minimal residual disease in patients with acute leukemia

Abstract

The investigation of minimal residual disease (MRD) with multiparameter flow cytometry (FC) immunophenotyping has proven to be a powerful approach for disease monitoring in patients with acute leukemia (AL). This technique mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis.

The immunophenotypic profiles exhibited by B- and T-cells in normal bone marrow (BM) and CD34+ cells in hernatopoietic stem cells from blood collections (HSC-BC) have been established. Well-defined triple monoclonal antibody combinations showed patterns of antigen expression that corresponded to various maturation stages. Aberrant phenotypes were found in 98 % of B-precursor ALL and in all studied T-ALL.

The identification of B- and T-cell subsets in BM allowed definition of "empty spaces". Areas of flow cytometric dot plots where no normal cell populations are located. Discrimination between normal and malignant cells can be done and used for MRD detection.

The immunophenotypic evaluation of MRD during therapy provides crucial information about the response to treatment and the risk of relapse. In 97 % of 70 uniformly-treated children with ALL, the FC technique could be used for follow-up and detection of MRD. Patients with MRD levels greater or equal to 0.01 % after Induction and the following timepoint showed a significantly higher risk of relapse than patients with MRD values under 0.01 % (p<0.05).

Experience from the BIOMED-1, a European Concerted Action, contributed to a MRD evaluation in a standardized way, obtained with optimal quality and sensitivity.

Two main methods of MRD detection were compared: FC with 'live-gate" analysis and allelespecific oligonucleotide (ASO)-PCR, detecting patient-specific T-cells receptor gamma/delta gene-rearrangements. The comparison showed significantly consistent results in 78 % of the samples. BM samples taken during the first phase of treatment showed lower consistency when compared to samples taken during the later phase of treatment.

An investigation of MRD levels in HSC-BC and BM samples before collection and transplantation of hernatopoietic stem cells was done. Low levels of MRD were found in HSCBC from 24 % of AL patients. MRD levels in HSC-BC correlated to the presence of MRD in the BM samples taken before collections. However, we could not find any correlation between the MRD levels in HSC-BC and outcome after hernatopoietic stem cells from blood transplantation.

The investigation of levels and the dynamics of MRD by sensitive and quantitative FC technique can provide a basis for further clinical studies and decisions concerning patient therapy.