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Approximately 40% of children with acute myeloid leukemia (AML) who respond to initial therapy subsequently relapse. Multidimensional flow cytometry employing a standardized panel of monoclonal antibodies enables the detection of small numbers of occult leukemic cells that persist during therapy using technology adaptable by most clinical laboratories. We performed a prospective, blinded evaluation of bone marrow specimens obtained from 252 pediatric patients with de novo AML to determine whether detection of occult leukemia defined as more than or equal to 0.5% blasts with aberrant surface antigen expression as determined by flow cytometry was predictive of subsequent relapse. Occult leukemia was detected in 41 (16%) of the 252 patients who responded to initial induction therapy. In time-dependent multivariate analyses that controlled for allogeneic marrow transplantation, variable intervals between sample submission, age, sex, white blood cell count at diagnosis, presence of splenomegaly or hepatomegaly, and presence of more than 15% blasts in the marrow after the first course of induction, patients harboring occult leukemia were 4.8 times more likely to relapse (95% confidence interval [CI] = 2.8 to 8.4, P <.0001) and 3.1 times more likely to die (95% CI; 1.9 to 5.1, P <.0001) than those lacking leukemia detectable by flow cytometry. In this analysis, flow cytometric evidence of leukemia after the initiation of therapy emerged as the most powerful independent prognostic factor associated with poor outcome. Among patients in whom a marrow sample was available for analysis at the end of consolidation therapy, overall survival at 3 years was 41% versus 69% for patients with and without occult leukemia, respectively (P =.0058).

Myeloid leukemia of Down syndrome (ML-DS) in young children is associated with distinct clinical and biological features and is typically initiated with oncogenic mutations in the X-linked megakaryocytic transcription factor GATA1. Here we present a 3-yr-old child with DS diagnosed with acute myeloid leukemia (AML), which lacks typical immunophenotypic and molecular characteristics of ML-DS, including GATA1 mutations. The leukemic blasts were found to have an MN1-ETV6 gene fusion, a high-risk oncofusion not previously described in DS patients. This report highlights the importance of immunophenotypic, cytogenetic, and molecular characterization of ML-DS for identification of rare cases with unique features that may benefit from treatment protocols that are more intensive than those developed for patients with typical GATA1 mutant ML-DS. [ABSTRACT FROM AUTHOR]

The RAM immunophenotype has been recently described as a subtype of acute myelogenous leukemia (AML) that is characterized clinically by extremely poor prognosis. We present a case of AML with RAM immunophenotype in a 5-year-old patient that resulted in poor outcome despite early hematopoietic cell transplant. We describe the unusual morphologic features that, along with the distinct immunophenotype, may provide initial diagnostic clues and further justify the classification of this AML variant as a rather distinct subtype. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Flow cytometry is becoming an important tool in the characterization of different hematological disorders. The aim of our study was to detect very rare populations in hematological malignancies using the comprehensive concept of immunophenotyping. Patients and Methods: Six patients, including three with acute myeloid leukemia, one with acute lymphoblastic leukemia, one with myelodysplastic syndrome, and one with B-cell lymphoma, were enrolled in this study. Serial bone marrow aspirates were analyzed by three-color multidimensional flow cytometry. Results: The core principles for the use of flow cytometric analysis are understanding deviations in abnormal antigen expression from normal cellular differentiating pathways and characterizing different patterns of antigenic aberrancy for each patient. Our results demonstrate that multidimensional flow cytometry can be used to: (1) monitor minimal residual disease after treatment; (2) aid in the differential diagnosis of cases, which are difficult to evaluate using conventional morphology; and (3) help in the staging of lymphoproliferative disorders. Conclusion: After selecting appropriate combinations of monoclonal antibodies, and applying advanced knowledge in immunophenotyping, flow cytometry is very sensitive and specific for the detection of rare populations in hematological disorders. [Copyright &y& Elsevier]

Highlights • DFS was higher in pediatric AML patients who had MRD detected by the "difference from normal" flow cytometry (ΔN) pre-HSCT. • MRD detected by ΔN was highly specific but did not identify most relapsing patients. • Quantitative assessment of WT1 gene expression pre HSCT was not predictive of DFS. Abstract We enrolled 150 patients in a prospective multicenter study of children with acute myeloid leukemia undergoing hematopoietic stem cell transplantation (HSCT) to compare the detection of measurable residual disease (MRD) by a "difference from normal" flow cytometry (ΔN) approach with assessment of Wilms tumor 1 (WT1) gene expression without access to the diagnostic specimen. Prospective analysis of the specimens using this approach showed that 23% of patients screened for HSCT had detectable residual disease by ΔN (.04% to 53%). Of those patients who proceeded to transplant as being in morphologic remission, 10 had detectable disease (.04% to 14%) by ΔN. The disease-free survival of this group was 10% (0 to 35%) compared with 55% (46% to 64%, P <.001) for those without disease. The ΔN assay was validated using the post-HSCT specimen by sorting abnormal or suspicious cells to confirm recipient or donor origin by chimerism studies. All 15 patients who had confirmation of tumor detection relapsed, whereas the 2 patients with suspicious phenotype cells lacking this confirmation did not. The phenotype of the relapse specimen was then used retrospectively to assess the pre-HSCT specimen, allowing identification of additional samples with low levels of MRD involvement that were previously undetected. Quantitative assessment of WT1 gene expression was not predictive of relapse or other outcomes in either pre- or post-transplant specimens. MRD detected by ΔN was highly specific, but did not identify most relapsing patients. The application of the assay was limited by poor quality among one-third of the specimens and lack of a diagnostic phenotype for comparison. [ABSTRACT FROM AUTHOR]