Immunophenotypic Patterns of Childhood Acute Leukemias in Indonesia RESEARCH COMMUNICATION Immunophenotypic Patterns of Childhood Acute Leukemias in Indonesia Eddy Supriyadi1*, Pudjo H Widjajanto1, Anjo JP Veerman1,2, Ignatius Purwanto1, Yetty M Nency4, Stefanus Gunawan5, Selvi Nafianti6, Dewajani Purnomosari7, Umi S Intansari8, Guus Westra3, Sutaryo1, Jacqueline Cloos2,3 Abstract Background: Immunophenotyping, as suggested by WHO, may improve diagnosis of childhood leukemia since it offers a better classification of the hematopoietic lineage of malignant cells as compared to morphology. Therefore, we aimed to determine the proportion of the immunophenotypic subtypes of acute leukemia in Indonesian children. Methods. Samples were obtained from patients (0-14 years of age) in 4 hospitals in Indonesia. We analyzed 541 suspected leukemia samples presented over a 4-year period (March 2006 - July 2010) by flow cytometry. Immunophenotyping allowed classification into acute myeloid leukemia (AML) and ALL (B-lineage and T-lineage ALL). Results. Of 541 samples, 136 were tested using a single color method and 405 with a three-color method. Concordance with morphology was very good (κ=0.82) using the three-color method with a panel of 15 monoclonal antibodies (n=387). A relatively high percentage of acute leukemia was classified as AML (23%). Of the ALL samples 83% were B-lineage ALL and 17% T- lineage ALL. Nine out of 239 morphological ALL were labeled AML, and 12/79 morphological AML were in fact ALL. Conclusion. Immunophenotyping in a multi-center study proved feasible and appears particularly important for prognostic assessment of childhood leukemia in low income countries such as Indonesia. Key words: Childhood leukemia - AML - ALL - immunophenotyping - Indonesia Asian Pacific J Cancer Prev, 12, 3381-3387 Introduction and stage of differentiation (Behm and Campana, 1999; Pui et al., 2008; Van Dongen, 2003). This technique The diagnosis of acute leukemia is based on improves both accuracy and reproducibility of acute morphological and cytochemical investigations of bone leukemia classification. It is considered particularly marrow samples and/or peripheral blood smears. The useful for identifying acute myeloid leukemia (AML) French-American-British (FAB) group established with lymphoid marker expression and, conversely, for criteria of acute leukemia based on morphologic ALL with myeloid marker expressions. Immunological characteristics of the malignant clones. It defines three studies of leukemic blasts are essential for identifying subtypes of acute lymphoblastic leukemia (ALL): L1, L2 biphenotypic and bilineage leukemias. Approximately and L3 and 8 subtypes of acute myeloblastic leukemia 6% of ALL cases express two or more myeloid markers. AML (Lilleyman et al., 1986; Pui et al., 1993; 2008; This subtype of acute leukemia is known to respond Bhatia et al., 1999; McNally et al., 2002). well to intensive therapy and should therefore be treated Refinement in classification of acute leukemias is according to appropriate risk-based protocols (Pui et al., accomplished by immunophenotyping. Differences 1990a; Huh and Ibrahim, 2000). Immunophenotypic in expression of surface membrane antigens or analysis is essential for accurately determining the cytoplasmic components are used to identify and lineage of the malignant clone of leukemic blasts besides classify lymphoproliferative disorders by cell of origin the light microscopic diagnosis of childhood leukemia 1Pediatric Hematology Oncology Division, Department of Pediatrics, Dr Sardjito Hospital, Medical Faculty, Universitas Gadjah Mada, Yogyakarta, Indonesia, 2Pediatric Oncology/Hematology Division, Department of Pediatrics, 3Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands, 4Pediatric Hematology Oncology, Kariadi Hospi- tal, Medical Faculty, Diponegoro University, Semarang, 5Pediatric Hematology Oncology, Prof Kandou Hospital, Medical Faculty, Sam Ratulangi University, 6Pediatric Hematology Oncology, Adam Malik Hospital, Medical Faculty, University of Sumatera Utara, Medan, 7Department of Histology and Cell Biology, 8Department of Clinical Pathology, Medical Faculty, Universitas Gadjah Mada, Yogyakarta, Indonesia *For correspondence: [email protected] Asian Pacific Journal of Cancer Prevention, Vol 12, 2011 3381 Eddy Supriyadi et al Table 1. Reported Incidence of B- and T-lineage ALL stratification into Standard Risk (SR) and High Risk in Various Countries (HR) ALL, as well as for determining mature B-ALL, B-lineage T-lineage which needs a different treatment schedule. In addition, ALL (%) ALL (%) a specific immunophenotype identified at diagnosis might be useful for evaluating microscopic residual Europe disease by flowcytometry during therapy. Conventional The Netherlands (Kamps et al., 2010) 88 12 United Kingdom (McNally et al., 2002) 87 13 immunophenotyping studies are performed on blood Germany (Moricke et al., 2009) 87 13 or bone marrow samples by flowcytometry, using an Bulgaria(Taskov et al., 1995) 70 30 array of monoclonal antibodies to identify cell surface North America (Pui et al., 2009) 84 16 and cytoplasmic antigens. Published guidelines for Central America (Howard et al., 2005) 90 10 compositions of monoclonal antibody panels for use South America with flowcytometry panels in the evaluations of acute Chile (Cabrera, 1996) 90 10 leukemias are available (LeBien et al., 1981; Braylan et Brazil (Rego et al., 1996) 71 29 al., 2001; Kebriaei et al., 2002). Australia (Milne et al., 2009) 90 10 The most common B-lineage ALL is the B lineage Africa Morocco (Dakka et al., 2007) 63 37 phenotype positive for the following: B cell markers Egypt (Kamel et al., 1989) 49 51 CD19, CD22, TdT, cytoplasmic CD79a, CD34 and Asia CD10. B-lineage ALL has been sub-classified according Malaysia-Singapore (Ariffin et al., 2007) 93 7 to maturation stage into: early pre B (pro-B), pre-B, Thailand (Tiensiwakul et al., 1999) 82 18 transitional (or late) pre-B and (mature) B-ALL (Behm Hong-Kong (Shing et al., 1999) 82 18 and Campana, 1999; Pui et al., 2002). In different Saudi Arabia (Roberts et al., 1990) 88 12 regions, various incidences of B-lineage ALL have Indonesia (this study) 83 17 been reported (Table 1). Burkitt’s leukemia displays an Table 2. Monoclonal Antibody Panel Used immunophenotype consisting of mature B cells (Pui et al., 1990b). T-lineage ALL can also be categorized into Old panel New panel phenotypic subgroups, correlating to differentiation MoAb Label Company MoAb Label Company stages of thymic T cells. T cell markers are cytoplasmic B CD10 FITC Dako CD10 PE Dako CD3 and CD7 plus CD2 or CD5. This lineage can be lineage CD19 FITC Dako CD19 PE Becton further subdivided into early, mid or late thymocyte Dickinson differentiation (Matutes et al., 1997; Van Dongen, 2003). CD22 FITC Sanguin CD22 FITC Sanguin The World Health Organization (WHO) classification IgM FITC Becton cCD79a PE Beckman divides ALL into two main groups only, i.e., B-lineage Dickinson Coulter and T-lineage ALL, without further categorization. T CD2 FITC Dako CD2 FITC Dako To our knowledge, there is no published data lineage CD7 FITC Becton cCD3 FITC Becton on immunological phenotyping of childhood acute Dickinson Dickinson CD7 PE Becton leukemias in Indonesia, where stratification for treatment Dickinson protocols until recently is based solely on clinical criteria MyeloidCD33 FITC Dako CD13 FITC Dako and morphology of lymphoblasts. Our study, the first Lineage CD33 PE Becton of its kind reported from Indonesia, was designed to Dickinson investigate the immunophenotyping profiles of childhood cMPO PE Dako acute leukemia and to determine the feasibility of Non TdT FITC Dako CD34 FITC Becton incorporating the results in further defining risk groups Lineage Dickinson for development of treatment protocols. IgG1 FITC Becton CD45 PerCP Becton Dickinson Dickinson cIgG1 FITC Becton Materials and Methods Dickinson cIgG1 PE Becton Origin of samples Dickinson Samples of blood and bone marrow aspirates from cTdT FITC Dako 541 patients, aged 0-14 years, suspected of leukemia MoAb, monoclonal antibody; FITC, fluorescein isothiocyanate; between March 2006 until July 2010 were received IgG1, Immunoglobulin G1; PBS, phosphate buffered saline; from Pediatric Cancer Units (PCU) of four Indonesian PE, phycoerythrin; PerCP, peridinin-chlorophyll proteins; teaching hospitals: Dr. Sardjito Hospital, Yogyakarta and TdT, terminal deoxynucleotidyl transferase Kariadi Hospital, Semarang, both are in Central Java, (Behm and Campana, 1999; Margolin et al., 2002). It is Adam Malik Hospital (Medan, North Sumatra) and Prof. in particular important to resolve an equivocal diagnosis Kandou General Hospital (Manado, North Sulawesi). of ALL versus AML, and to determine the lineage of The laboratory is in Yogyakarta thus samples from Dr. lymphoblasts along the lines of B lineage-ALL and Sardjito hospital, Yogyakarta, were analyzed fresh, and T-lineage ALL maturation. The latter is important for from the other PCU’s samples were sent by airmail and 3382 Asian Pacific Journal of Cancer Prevention, Vol 12, 2011 Immunophenotypic Patterns of Childhood Acute Leukemias in Indonesia analyzed within 24 hours. Table 3. Scoring System for Definition Acute Biphenotypic Leukemia Adapted from EGIL Single color vs. three-color method Scoring B-lymphoid T-lymphoid Myeloid Single color monoclonal antibody (MoAb) panel were used during the period March 2006 till November 2 cCD79a, CD22, cCD3 cMyelo cIgM Peroxidase 2007.