Minimal residual disease in acute myeloid leukemia in children - standardization and evaluation of immunophenotyping in the AML-BFM-98 study
Abstract
Background: Minimal residual disease is a prognostic factor in AML. However, the impact on treatment stratification is not established.
The AML-BFM 98 MRD study started in 1/2000 in order to evaluate, standardize and establish immunophenotyping in AML in children. Methods: In a first phase the participating laboratories in Muenster, Goettingen, Vienna and Prague agreed on identical antibody-panels and standardized procedures of sample processing, analysis and data management.
The consensus panel was evaluated and adapted to 3- and 4-color flowcytometry. The complete panel was applied to each follow-up sample in orderto minimize the risk offalse negative results due to the loss or shift of antigens during treatment, a known phenomenon in myeloid blasts.
Between 1/2000 and 9/2001 165 of 198 protocol patients were analysed at diagnosis, in 149 children at least two follow-up samples were available. Results: Three kinds of immunophenotypes could be defined [1].
Asynchronous expression of stem cell and myeloid antigens i.e. CD34/CD117 combined with CD13/CD15 had a low specificity because precursors in regenerating or normal bone marrow expressed this pattern in 0.47% (0.1 to 1.5%) [2].
The aberrant co-expression of stem cell antigens and lymphatic antigens such as CD7 or CD2 showed a median level of specificity (0.07% (0.04 to 0.19%) [3]. Aberrant expression of stem cell antigens combined with B-lymphatic (CD19, CD10) or NK-cell antigen (CD56) showed the best specificity.
The maximal level in normal bone marrow was 0.05%. Sensitivity of different immunophenotypes was evaluated by diluting known leukemic blasts in regenerating bone marrow.
Minimal level of sensitivity was found to be at 10(-3) to 5 x 10(-4). According to these data highiy specific immunophenotypes could be detected in 33%, median specificity was seen in 71% and low specificity was seen in 88% of the protocol patients.
Two laboratories analyzed simultaneously 17 samples of children with AML from diagnosis and during therapy. A high correlation of blast quantification could be demonstrated (correlation r(2) = 0.98; blasts < 5% r(2) = 0.91).
In addition, two independent explorers quantified the raw data of 16 samples. All results correlated well (r(2) = 0.97; blasts < 5% r(2) = 90.94).
Conclusion: The prospective study phase, started 1/2002, aims to test the impact of MRD diagnostics as an independent prognostic factor in AML in children. This might facilitate future treatment stratification and consequently optimize outcome.