Leukemia (1999) 13 , 32–37  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http:/ / www.stockton-press.co.uk/ leu RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML) C Flotho 1 , S Valcamonica 2 , S Mach-Pascual 3 , G Schmahl 1 , L Corral 2 , J Ritterbach 4 , H Hasle 5 , M Arico ` 6 , A Biondi 2 and CM Niemeyer 1 1 Children’s Hospital, University of Freiburg, Germany; 2 Pediatric Clinic University of Milan, Ospedale San Gerardo, Monza, Italy; 3 Brigham and Women’s Hospital, Harvard Institutes of Medicine, Boston, MA, USA; 4 Children’s Hospital, University of Giessen, Germany; 5 Department of Pediatrics, University Hospital of Aarhus, Denmark; and 6 Clinica Pediatrica, Universita ` Pavia, Italy Juvenile myelomonocytic leukemia (JMML) is a malignant hem- atopoietic disorder of early childhood with excessive prolifer- ation of the myeloid and monocytic lineage. Deregulation of the RAS signal transduction pathway is thought to play a key role in its pathogenesis. We examined peripheral blood or bone marrow cells of 36 children with JMML for activating point mutations in codons 12, 13 and 61 of the NRAS and KRAS proto-oncogenes by allele-specific restriction assay, single- strand conformation polymorphism and/or direct sequencing. Codons 12, 13 and 61 of HRAS were examined in 26 of these patients. We detected RAS mutations in six cases (17%) located at N12 (n = 2), N13 (n = 3) and K13 (n = 1). In addition, we perfor- med clonality studies on different cell lineages in four of these patients applying the RAS mutation, the karyotype and X-chro- mosome inactivation patterns as clonal markers. Erythroid cells carried mutant RAS, indicating clonal origin. In EBV B cell lines, one of three patients studied harbored a RAS mutation, while the other two patients had polyclonal B cells with wild- type RAS. T lymphocytes were examined in one patient; they were polyclonal and had wild-type RAS. It is likely that JMML is a heterogeneous disease with respect to clonal involvement of different lineages. Keywords: juvenile myelomonocytic leukemia; juvenile chronic myelogenous leukemia; RAS mutations; clonality; childhood Introduction Juvenile myelomonocytic leukemia (JMML) is a hematopoietic malignancy of childhood characterized by prominent hepato- splenomegaly, frequent skin involvement, leukocytosis, mon- ocytosis and the presence of immature precursors in periph- eral blood (PB). 1,2 Its annual incidence is about 1.3/million children. 3 The disorder has also been referred to as ‘juvenile chronic myelogenous leukemia’ (JCML) or ‘chronic myelo- monocytic leukemia in childhood’ (CMML). 1 JMML is a clonal disorder as assessed by X-chromosome inactivation studies. 4 Hematopoiesis in JMML features an excessive proliferation of myeloid progenitor cells and a specific hypersensitivity for GM-CSF in vitro. 5 Because stimulation with GM-CSF is known to be associated with RAS activation, 6 mutations resulting in a deregulated RAS pathway are of particular interest in JMML. p21RAS is a family of guanine nucleotide binding proteins located at the inner cell membrane which forward stimuli for cell proliferation and differentiation to the nucleus through a kinase cascade. 7 The RAS proteins cycle between an active, GTP-bound conformation and an inactive, GDP-bound form. 8 GTPase-activating proteins (GAPs) negatively regulate the sig- nal output of RAS proteins by accelerating GTP hydrolysis. 9 Several GAPs connected with RAS proteins have been ident- ified, including neurofibromin, the product of the neurofib- romatosis type 1 (NF 1) tumor suppressor gene (NF1). NF1 is Correspondence: C Flotho, Universita ¨ts-Kinderklinik, Mathilden- strasse 1, 79106 Freiburg, Germany; Fax: 49 761 270 4518 Received 16 April 1998; accepted 14 September 1998 clinically diagnosed in about 15% of children with JMML. 1 In addition, truncating NF1 mutations can be shown in a pro- portion of children with JMML without clinical evidence of NF1. 10 While NF1 -/- knock-out mice die in utero at an embry- onic stage, cultured fetal NF1 -/- hematopoietic cells demon- strate a pattern of selective hypersensitivity to GM-CSF. 11,12 Like decreased GAP activity, specific point mutations of the RAS genes can result in persistent activation of the RAS sig- naling pathway. 13 In this study, we wanted to determine the frequency of point mutations at codons 12, 13 or 61 of the NRAS, KRAS or HRAS genes in a large cohort of children with JMML. In addition, mutant RAS genes, karyotype analysis and, in female patients, X-chromosome inactivation clonality assays were used as markers for clonal involvement of different cell lineages in JMML patients. Materials and methods Patients Thirty-six children with JMML were studied. Clinical data are given in Table 1. NF1 was known in three patients. Chromo- somal analyses in leukemic cells were available in 32 of the 36 children and showed a normal karyotype in 23 children, monosomy 7 in four children and complex aberrations in five children. Cell cultures Adherence depleted mononuclear cells (MNC) from PB were cultured in a serum-deprived semi-solid methylcellulose sys- tem 14 at 5 × 10 4 cells/ml in the presence of erythropoietin (2 U/ml). After 14 days of culture at 37°C in a 4% CO 2 atmos- phere, colonies derived from erythroid burst-forming units (BFU-E) were harvested. EBV transformed B lymphocytes were grown in RPMI 1640 (Gibco, Eggenstein, Germany) sup- plemented with 10% fetal calf serum (FCS), 2 mml -glutamine, 100 U/ml penicillin/streptomycin. 15 Fluorescence-activated cell sorting (FACS) After lysis of erythrocytes, PB cells were maintained in RPMI 1640/10% FCS. Per 10 6 cells, 10 l phycoerythrin-conjugated monoclonal anti-CD3 (Laboserv, Giessen, Germany) was added. Cells were incubated for 15 min, washed twice and sorted on a FACStar Plus (Becton Dickinson, Heidelberg, Germany). The purity of CD3 + cells was greater than 90%.