Key words: minimal residual disease (MRD), flow cytometry (FCM), childhood leukemia Fig. 1 Schematic diagram of numbers of leukemic cells in acute leu

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1 Detection of Minimal Residual Disease and Its Clinical Application for Childhood Leukemia Shohei YOKOTA,*1 Tomomi OKAMOTO*1 and Masahito TSURUSAWA*2,3 Department of Hematology, Kyoto Prefectural University of Medicine Department of Pediatrics, Aichi Medical University Chairman of Japanese Children's Cancer and Leukemia Study Group Abstract More than 1012 tumor cells are present in a leukemia patient at diagnosis. They decrease in number along with the chemotherapy and become undetectable by a microscope. Since the 80's several methods have been invented to visualize the existence of minimal residual disease (MRD). The most useful MRD assays currently available are polymerase chain reaction (PCR) amplification of fusion transcripts and rearranged T-cell receptor and immunoglobulin genes, and flow cytometric (FCM) detection of aberrant immunophenotypes. Both PCR and FCM methods allow detection of 1 leukemic cell in 10,000 normal cells in at least 90% of patients with acute lymphoblastic leukemia. A number of clinical studies have elucidated that MRD assays are increasingly important in the clinical management of patients with acute leukemia. Several studies in children and adult patients with acute lymphoblastic leukemia and acute myeloid leukemia have shown a strong association between MRD and risk of relapse, irrespective of the methodology used to detect residual disease. Those who are positive for bone marrow MRD have a bad prognosis either after a standard protocol or the salvage therapy and stem cell transplantation for relapsed patients. These findings lead to the idea of modifying the therapeutic regimen according to the amount of MRD. The international BFM group in Europe and Japanese Children's Cancer and Leukemia Study Group (CCLSG) have started MRD-based protocol for ALL children. Reprint requests to Shohei Yokota, Department of Hematology, Kyoto Prefectural University of Medicine, 465, Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, Japan

2 Key words: minimal residual disease (MRD), flow cytometry (FCM), childhood leukemia Fig. 1 Schematic diagram of numbers of leukemic cells in acute leukemia patients during and after chemotherapy and during development of relapse Approximately 1012 leukemic cells are present at diagnosis. Two or three log reduction is achieved by induction therapy leaving at least 109 cells residual. The remission which is defined by light-microscopy allows the existence of at least 109 cells. The detection limit of morphologic, as well as molecular genetic methods is indicated. Using molecular techniques, residual cells as many as 106 cells can be detected.

3 Table 1 Incidence of aberrant phenotypes in prec B- ALL

4 Fig. 2 Detection of MRD by PCR amplification

5 Table 2 Chromosome aberrations as major PCR targets for MRD detection in ALL Table 3 in AML Chromosome aberrations as major PCR targets for MRD detection

6 Fig. 3 Structure of Ig H, K, TCR 6, y chain gene

7 Fig. 4 Real time quantitative PCR A: Intercalating PCR. DNA-binding dyes such as SYBR Green (circles) are intercalated in double strand of PCR products along with the DNA polymerization. B: TaqMan PCR. Quantitative PCR is preformed with appropriate VH consensus primer, patient tumor specific (ASO) primer, and VH gene family-specific consensus TaqMan probe. The probes are labeled at the 5' end with reporter dye (R) and the 3' end with quencher activity (Q).

8 Fig. 5 Bone marrow sampling points of prospective MRD study by BFM group

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Minimal residual disease levels in bone marrow and peripheral blood are comparable in children with T cell acute lymphoblastic leukemia (ALL), but not in precursor-b-all.

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Detection methods of minimal residual disease and their prognostic value in patients with malignant hematological diseases はじめに (m

92 124 3 92 96 2015 Detection methods of minimal residual disease and their prognostic value in patients with malignant hematological diseases はじめに (minimal residual disease: MRD) MRD MRD ( 1) MRD PCR