Letters to the Editor

1637

A novel somatic K-Ras mutation in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive clonal disorder of hematopoietic precursor cells of early childhood characterized by excessive proliferation of myeloid cells inltrating both hematopoietic and non-hematopoietic tissues.1 Mutations constitutively activating the Ras-signaling pathway in genes such as KRAS, NRAS, PTPN11 or NF1 play a substantial factor in pathogenesis of this myeloproliferative disorder (reviewed in Lauchle et al.2 and Niemeyer and Kratz3). Ras controls the activation of pivotal downstream effector pathways by switching between active guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound conformations. Highly conserved regions within Ras that are crucial for the function of Ras as a molecular switch include the phosphate-binding (P) loop (codons 1116), the switch I (codons 3337) and switch II regions (codons 5966).4 Cancer-associated RAS mutations typically introduce amino-acid substitutions at positions Gly12, Gly13 or Gln61. Mutant Ras proteins are locked in the active state due to a defective intrinsic GTPase activity and resistance to GTPase activating proteins (GAPs). We report on two patients with JMML in whom we have detected a novel recurrent in frame insertion of a GGT-triplet resulting in an additional glycine residue in the P loop of K-Ras. We obtained tissue samples (bone marrow, peripheral blood and buccal swab) from two JMML patients. JMML was diagnosed based on clinical, hematological and cytogenetic criteria.5 Informed consent was obtained from the patients parents. DNA was extracted and mutation analysis of the KRAS gene was performed by denaturating high-performance liquid chromatography (DHPLC) of puried PCR products as recently described.6 Specimens showing abnormal DHPLC proles were amplied and sequenced using standard methods.6 Patient one was a 2-month-old infant who presented with a perianal abscess and splenomegaly. The peripheral blood count showed leukocytosis of 100 G/l, with 11% blasts and 31%

monocytes, hemoglobin 100 g/l and platelet count 110 G/l. Bone marrow aspirate was hypercellular with 7% blast forms. Cytogenetics uncovered loss of chromosome 7 in 96% of the metaphase cells. Currently, the patient is awaiting hematopoietic stem cell transplantation (HSCT). Patient two, a 3-year-old boy, presented with malaise, fever and respiratory distress. He had lymphadenopathy, mild splenomegaly and hepatomegaly. Peripheral blood counts demonstrated leukocytosis with 18 G/l, 3% blast forms, hemoglobin 50 g/l and platelets 82 G/l. Bone marrow aspirates showed increased cellularity with 8% blast forms. Cytogenetic analysis revealed 45, XY, 7 in 96% of bone marrow cells. The patient received a nonidentical related transplant with a single mismatch in the DRB1 locus from his mother. He remains in clinical and hematological remission 5 years later. Mutation analysis revealed a novel heterozygous in-frame insertion in KRAS, c.36_37insGGT, in the patients leukaemia cells but not in buccal cells (Figure 1). This insertion predicts a p.G12_G13insG mutation with an additional glycine between Gly12 and Gly13 of the P loop. Others have previously described and biochemically characterized another insertion mutation in the P loop of K-Ras, p.G10_G11insG, identied in a child with acute myeloid leukemia.7 These investigators elegantly showed that expression of the mutant protein in NIH 3T3 cells caused cellular transformation, and expression in COS cells activated the Ras-mitogen-activated protein kinase signaling pathway. In COS cells this mutant accumulated in the active GTP state. Biochemical analysis showed an impaired intrinsic GTP hydrolysis and resistance to GAPs.7 Similar insertion mutations of Ras with three extra amino acids inserted into the P loop have been studied by Klockow et al.8 These mutants show a strongly attenuated binding afnity for nucleotides, leading to a preference for GTP binding. Both the intrinsic as well as the GAP-mediated GTP hydrolysis were severely impaired. Microinjection of these mutants into PC12 cells induced neurite outgrowth. Interestingly, their ability to stimulate the MAP kinase pathway as measured by a reporter gene assay in RK13 cells was much higher than that of the

Figure 1 Heterozygous GGT triplet insertion between codon 12 and 13 of KRAS. (a) KRAS mutation analysis of DNA from both patients JMML bone marrow (BM) cells showed an abnormal DHPLC pattern (left) while analysis of DNA from buccal cells (BC) showed a normal wild-type pattern (right). (b) Sequence analysis revealed a heterozygous insertion of three bases GGT predicting an extra glycine between residues Gly12 and Gly13 of the protein.
Leukemia

Letters to the Editor

1638
normal oncogenic mutant p.G12V. This characteristic was attributed to an increased stimulation of c-Raf1 kinase activity by the insertional Ras mutant. We did not functionally characterize the novel mutation described in this report. However, the observation of these mutants in two JMML samples together with biochemical experiments described above to characterize similar mutations suggests that K-Ras p.G12_G13insG acts as an activating mutation contributing to malignant transformation. Notably, both patients presented here had a monosomy 7 in their leukemic cells. Possibly, this novel lesion cooperates with monosomy 7 during leukemogenesis.

Erasmus MC-Sophia Childrens Hospital, Rotterdam, The Netherlands and 5 Department of Pediatric Oncology, Skejby Hospital, Aarhus University Hospital, Aarhus N, Denmark E-mail: [email protected] References
1 Niemeyer CM, Arico M, Basso G, Biondi A, Cantu Rajnoldi A, Creutzig U et al. Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases. European Working Group on Myelodysplastic Syndromes in Childhood (EWOG-MDS). Blood 1997; 89: 35343543. 2 Lauchle JO, Braun BS, Loh ML, Shannon K. Inherited predispositions and hyperactive Ras in myeloid leukemogenesis. Pediatr Blood Cancer 2006; 46: 579585. 3 Niemeyer CM, Kratz C. Juvenile myelomonocytic leukemia. Curr Oncol Rep 2003; 5: 510515. 4 Vetter IR, Wittinghofer A. The guanine nucleotide-binding switch in three dimensions. Science 2001; 294: 12991304. 5 Hasle H, Niemeyer CM, Chessells JM, Baumann I, Bennett J M, Kerndrup G et al. A pediatric approach to the WHO classication of myelodysplastic and myeloproliferative diseases. Leukemia 2003; 17: 277282. 6 Schubbert S, Zenker M, Rowe SL, Boll S, Klein C, Bollag G et al. Germline KRAS mutations cause Noonan syndrome. Nat Genet 2006; 38: 331336. 7 Bollag G, Adler F, elMasry N, McCabe PC, Conner Jr E, Thompson P et al. Biochemical characterization of a novel KRAS insertion mutation from a human leukemia. J Biol Chem 1996; 271: 3249132494. 8 Klockow B, Ahmadian MR, Block C, Wittinghofer A. Oncogenic insertional mutations in the P-loop of Ras are overactive in MAP kinase signaling. Oncogene 2000; 19: 53675376.

Note added in proof

C Reimann1, M Arola2, M Bierings3, A Karow1, MM van den Heuvel-Eibrink4, H Hasle5, CM Niemeyer1 and CP Kratz1 1 Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg, Germany; 2 Division of Pediatric Hematology and Oncology, Tampere University Hospital, Unit of Pediatric Hematology and Oncology, Tampere, Finland; 3 Department of Haematology and Stem Cell Transplantation, University Medical Centre, Utrecht, The Netherlands; 4 Department of Pediatric Oncology/Hematology,

A novel translocation t(3;21)(p21;q22) in acute myelogenous leukemia preceding a late-appearing Philadelphia chromosome
Leukemia (2006) 20, 16381640. doi:10.1038/sj.leu.2404283; published online 22 June 2006

The Philadelphia (Ph) chromosome is present in more than 95% cases of chronic myeloid leukemia (CML) and encodes the Bcr-Abl oncoprotein with constitutive tyrosine kinase activity, which is believed to be crucial in cell transformation. Only rare cases of CML are truly negative for both the Ph chromosome and BCR-ABL1 rearrangement.1 The presence of a late-appearing Ph chromosome in patients with acute myelogenous leukemia (AML) represents a very infrequent occurrence.2 Therapy with imatinib mesylate (Gleevec) renders sustained hematologic responses in 95% of patients in early chronic phase CML and 3169% in advanced phase CML.3 AMN107 is a phenylamino-pyrimidine derivative 20 to 30-fold more potent as an Abl inhibitor than imatinib, which renders hematologic response rates ranging from 44 to 89% and cytogenetic responses ranging from 22% in blast phase to 29% in accelerated phase and 50% in chronic phase (Kantarjian et al. Blood 2005; 106: 15a, abstract). We report a patient with AML who developed a novel t(3;21)(p21;q22) during the course of therapy. Shortly thereafter, he exhibited a late-appearing Ph chromosome. Therapy with the tyrosine kinase inhibitors imatinib mesylate and AMN107 was completely unsuccessful.
Leukemia

A 62-year-old man with history of leukopoenia since 1990 underwent a bone marrow aspiration and biopsy in February 2002 owing to worsening pancytopenia. A diagnosis of myelodysplastic syndrome (MDS) with a normal diploid male karyotype was made and the patient was initially observed. In August 2002, he presented to an outside hospital with a white blood cell (WBC) count of 75 109/l and 80% myeloid blasts in the bone marrow. A diagnosis of AML M2 in the FrenchAmericanBritish (FAB) classication was established. Cytogenetic study showed again a normal diploid male karyotype. He received induction therapy with cytarabine hydrochloride (ara-C) and idarubicin on a 7 3 regimen (ara-C and idarubicin), and achieved a complete remission. Consolidation treatment with one course of high-dose ara-C (HDAC) was given, after which the patient remained pancytopenic and no further therapy was provided. A bone marrow aspirate 2 months later showed tri-lineage dysplasia with 3% blasts, and a normal diploid male karyotype. The patient presented to the University of Texas M D Anderson Cancer Center (MDACC) in April 2003 seeking treatment options for persistent pancytopenia. Observation was recommended because he was not transfusion dependent and had no signs and symptoms of disease. In August 2004, the patient was seen again at MDACC and was found to have relapsed disease. A bone marrow aspirate demonstrated 28% blasts and a normal diploid male karyotype. Imunophenotypic