Leukemia (2002) 16, 2213–2221 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Expression of myeloid-specific genes in childhood acute lymphoblastic leukemia – a cDNA array study T Niini1, K Vettenranta2, J Hollme´n3, ML Larramendy1, Y Aalto1, H Wikman1, B Nagy1, JK Seppa¨nen3, A Ferrer Salvador1, H Mannila3, UM Saarinen-Pihkala2 and S Knuutila1 1Departments of Pathology and Medical Genetics, Haartman Institute and Helsinki University Central Hospital, University of Helsinki, Finland; 2Division of Hematology-Oncology and Stem Cell Transplantation, Hospital for Children and Adolescents, Helsinki University Central Hospital, Helsinki, Finland; and 3Laboratory of Computer and Information Science, Helsinki University of Technology, Espoo, Finland Several specific cytogenetic changes are known to be associa- of hundreds to thousands of genes to be evaluated in a single ted with childhood acute lymphoblastic leukemia (ALL), and experiment, and the screening of genes with over- or under- many of them are important prognostic factors for the disease. Little is known, however, about the changes in gene expression expression. The method can be used to search for novel mol- in ALL. Recently, the development of cDNA array technology ecular changes that are associated with the development has enabled the study of expression of hundreds to thousands and/or prognosis of leukemia. In this study we used cDNA of genes in a single experiment. We used the cDNA array arrays to study gene expression profiles in the leukemic blast method to study the gene expression profiles of 17 children cells of 17 children with precursor-B ALL. with precursor-B ALL. Normal B cells from adenoids were used as reference material. We discuss the 25 genes that were most over-expressed compared to the reference. These included four genes that are normally expressed only in the myeloid lin- Materials and methods eages of the hematopoietic cells: RNASE2, GCSFR, PRTN3 and CLC. We also detected over-expression of S100A12, expressed Patients in nerve cells but also in myeloid cells. In addition to the myeloid-specific genes, other over-expressed genes included A total of 17 patients (6/11, M/F) with ALL were included in AML1, LCP2 and FGF6. In conclusion, our study revealed novel information about gene expression in childhood ALL. The data the study. Fourteen patients were analyzed at diagnosis and obtained may contribute to further studies of the pathogenesis three at relapse. Table 1 shows the key clinical data and the and prognosis of childhood ALL. karyotypes of the patients. The mean age of the children was Leukemia (2002) 16, 2213–2221. doi:10.1038/sj.leu.2402685 5.0 years (range 1.2–12.7 years) and the mean WBC was 38.6 Keywords: acute lymphoblastic leukemia; gene expression; × 109/l (range 1.2–272.4 × 109/l). All patients had an early myeloid-specific genes; cDNA array precursor-B phenotype, except patient 8, who had a precur- sor-B disease. In 16 of the 17 cases the blast population expressed the CD34 antigen, in 10 cases in conjunction with Introduction the expression of the myeloid antigens CD13 and CD33. Only in patient 8 was the blast cell population devoid of the Cytogenetic changes are important prognostic markers in expression of both CD34 and CD13/33. The patients were div- childhood acute lymphoblastic leukemia (ALL). For example, ided into standard, intermediate, and high-risk categories and the translocation t(12;21)/ETV6-AML1 is a marker of favorable treated according to the protocols of the Nordic Society of prognosis and most patients with this abnormality are best Pediatric Hematology and Oncology (NOPHO).3 Two of the treated with conventional chemotherapy. In contrast, patients patients analyzed at primary diagnosis have relapsed, but 12 with t(4;11)/MLL-MLLT2 and t(9;22)/BCR-ABL have a poor continue in first remission, one after allogeneic bone marrow prognosis, and high-dose chemotherapy with bone marrow transplantation from an unrelated donor. All three patients transplantation is advocated (for review see Ma et al1). Despite studied at relapse have received a bone marrow transplant, the extensive knowledge about chromosomal abnormalities in two from an unrelated donor and one from a matched sibling. ALL, little is known about the changes in gene expression in One of the three has died following a post-transplant relapse, the disease. but two remain in second complete remission. In addition to genetic markers, the prognostic tools in child- hood ALL include white blood cell count (WBC) at diagnosis, age, gender, CNS/testicular involvement, response to primary Samples therapy and the phenotype of the blasts (mature B cell vs T cell vs precursor-B cell ALL). Groups of patients stratified using the Whole bone marrow specimens were diluted 1:10 in existing criteria remain, however, heterogeneous and result in RNA/DNA stabilization reagent for blood/bone marrow patients remaining in first complete remission and those (Boehringer Mannheim, Mannheim, Germany) for simul- relapsing either on or off therapy.2 A more comprehensive taneous cell lysis and stabilization of nucleic acids. The analysis of blast cell gene expression could provide us with samples were stored at −70°C until RNA isolation. novel prognostic tools as well as new insight into the patho- genesis of childhood ALL. The cDNA array methodology allows the expression levels Reference material Two separate pools of CD19-positive B cells from human Correspondence: S Knuutila, Department of Medical Genetics, Haart- man Institute, PO Box 21, FIN-00014 University of Helsinki, Helsinki, adenoid samples from healthy children were used as reference Finland; Fax: +358–9–191 26788 material. One pool was from six individuals and the other Received 19 February 2002; accepted 31 May 2002 from five. The B cells were purified using microbeads conju- Myeloid-specific genes in childhood ALL J Niini et al 2214 Table 1 Clinical characteristics and karyotype data of 17 children with ALL Patient Lab. code Sampling Sex Age Risk WBC Myeloid Myeloid Karyotype time dg (rel)b group dg (rel)c markers cells (%) 1 991506 Relapse F 1.8 (6.2) HR 99.4 (25.2) no 4 49,XX,+10,t(12;21)(p13;q22),+14,+21d 2 990663 Diagnosis F 4.2 HR 1.2 yes 5 64–66,XX,+X,+2,+3,+4,+5,+6,+8,+10,+11, +12,+14,+14,+16,+17,+18,+21,+21, +22,+2mar 3 981894 Diagnosis M 5.8 HR 4.0 yes 15 46,XY,t(12;21)(p13;q22)d 4 991170 Diagnosis M 3 IR 16.4 yes 10 46,XY,t(12;21)(p13;q22)d 5 GA99–17 Diagnosis F 8.9 SR 3.9 no 1 45–46,XX,-2,?t(2;14)(q?;q?),-12,-14,- 15,+mar,inc 6a 991477 Diagnosis F 4.7 HR 272.4 yes 2 47,XX,+21 7 GA00–10422 Diagnosis F 3.1 SR 4.9 no 15 46,XX 8 991843 Diagnosis M 12.7 IR 7.2 no 5 46,XY,del(9)(p11) 9 GA00–10526 Diagnosis F 3.2 IR 19.0 yes 5 55–57,XX,+X,+4,+6,+8,+8,+10,+14,+17, +18,+21,+21 10 981693 Diagnosis F 5.6 IR 26.3 no 10 48,XX,-20,+der21,+2mar 11a GA00–9977 Diagnosis F 3.8 HR 145.8 yes 2 44–45,X,-X,-9,-9,-11,-13,+3–4mar 12 GA00–9648 Relapse F 1.2 (3.5) IR 4.0 (14) no 15 46,XX,t(X;9)(q?;q11) 13 990710 Relapse M 4.7 (13.1) SR 7.6 (3.9) yes 10 55,XY,+X,1q+,+4,+6,+8,+14,+17,+18, +21,+21 14 GA00–9884 Diagnosis F 6.9 IR 10.6 yes 6 54–57,XX,+?X,+4,+6,+?14,+16,+21,+22 15 GA00–10058 Diagnosis F 3.1 SR 3.4 no 1 46,XX,-1,-1,+3mar,inc 16 GA00–10648 Diagnosis M 6.6 HR 7.0 no 3 54–55,XY,+X,+4,+6,+10,+14,+17,der(19) t(1;19)(q23;p13),+21,+21 17 GA00–10387 Diagnosis M 4.9 IR 22.5 yes 2 54,XY,+X,+Y,+6,+10,+14,+17,+21,+21 aThe patient has since relapsed. bAge at diagnosis (at relapse) in years. cWBC at diagnosis (at relapse) (×109/l). dThe t(12;21) was confirmed by fluoresence in situ hybridization. F, female; M, male; HR, high risk; IR, intermediate risk; SR, standard risk. gated to a monoclonal CD19 antibody (Miltenyi Biotec, cDNA array hybridization Bergisch Gladbach, Germany). CD19 is expressed from earl- iest recognizable B-lineage cells to activated B cell blasts, but Total RNA (3–4 ␮g) was converted into cDNA and labeled expression is lost on maturation to plasma cells. The pro- with 33P-dATP using the Atlas pure total RNA labeling system portion of T lymphocytes was analyzed in one of the pools, (Clontech) according to the manufacturer’s instructions. and it was less than 5%, indicating that 95% of the isolated Probes were purified and hybridized to the arrays according cells were B lymphocytes. to the manufacturer’s instructions. The arrays were exposed to an imaging plate (BAS-MP 2040S; Fuji, Kanagawa, Japan) for 3–7 days followed by scanning of the plate with a phos- Total RNA extraction phorimager (Bio-Imaging Analyzer, BAS-2500; Fuji). Two sep- arate reference hybridizations were performed from the two distinct pools of B cells. Nucleic acids were extracted using an mRNA isolation kit (Boehringer Mannheim) and following steps 1–6 in the manu- facturer’s instructions. The DNA was removed according to Quantitative real-time reverse transcriptase polymerase Clontech’s (Palo Alto, CA, USA) protocol for DNase treatment chain reaction (RT-PCR) of total RNA, with the exception that RNA precipitation was carried out overnight at −70°C.