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We recently investigated samples of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and normal bone marrow (BM). We found that leukemic blasts, compared to their physiologic counterpart cells, frequently display aberrant phenotypes with respect to levels of expression of certain antigens. Using multiparameter flow cytometry, these differences enabled us to trace leukemic cells admixed to normal BM, which suggested that this approach might be a useful strategy for minimal residual disease detection. In the present study, we used the same multiparameter approach ("comparative phenotype mapping") to prove that such quantitative phenotypic differences really exist between malignant and normal BCP when simultaneously present in the BM. We demonstrate this by five exemplary follow-up BM samples from patients with BCP-ALL, all of which showed phenotypically aberrant cells according to levels of expression of CD10, CD11a, CD19, CD34, CD44, or CD45RA, as well as according to altered orthogonal light scattering properties. We confirmed the leukemic nature of these cells by polymerase chain reaction-based detection of bcr1/abl transcripts, and of leukemia clone-specific immunoglobulin heavy chain rearrangements in only the suspicious cells when sorted by flow cytometry, but not in normal BCP or non-B cells. Comparative phenotype mapping thus allows one to distinguish between normal and leukemic cells, and we show that it may enable rapid, specific, and quantitative detection of residual/resurgent leukemia in BCP-ALL.

Leukemic cells of B-lineage acute lymphoblastic leukemia (ALL) are regarded as the malignant counterparts of immature, physiologic B cell precursors (BCPs). To determine whether phenotypic differences exist between these corresponding cell types, we investigated samples of normal pediatric bone marrow (n=30) as well as of B-precursor ALL at diagnosis (n=53; common and pre-B subtype). Using three-color multiparameter flow cytometric analysis, we compared the leukemic populations with the physiologic BCPs of corresponding maturity with respect to the intensity with which they expressed a series of antigens. In some of these antigens, leukemia-associated aberrations were frequently observed. In particular, overexpression of CD10 was displayed by 65% of ALL samples, whereas 58% of leukemic cases aberrantly exhibited very low or no CD45RA expression. Regarding CD11a and CD44, 47% and 35% of ALL populations were aberrant as defined by either the absence or significant overexpression of the antigen. In contrast, antigen densities of CD49d, CD49e, and CD99 on leukemic cells were in the normal range of values for BCPs. Combining the patterns of frequently aberrant markers in a comprehensive analysis, we were able to identify individual phenotypic leukemic cell aberrations in up to 98% of investigated cases. CD10 and/or CD45RA were aberrant in 86% of cases overall, emphasizing the high discriminative potential of these two markers. Using comparative phenotype mapping based on quantitatively aberrant, leukemia-associated antigenic patterns, we were able to detect leukemic blasts among normal bone marrow cells at frequencies as low as 10(-5). We speculate that our approach may have a profound impact on the development of new strategies for minimal residual disease investigations in patients with BCP-ALL.