Correspondence
1142 6 Meeus P, Demuynck H, Martiat P, Michaux L, Wouters E, Hagemeijer A. Sustained, clonal karyotype abnormalities in the Philadelphia chromosome negative cells of CML patients successfully treated with imatinib. Leukemia 2003; 17: 465–467. 7 Marktel S, Marin D, Foot N, Szydlo R, Bua M, Karadimitris A et al. Chronic myeloid leukemia in chronic phase responding to imatinib: the occurrence of additional cytogenetic abnormalities predicts disease progression. Haematologica 2003; 88: 260–267. 8 Braziel RM, Launder TM, Druker BJ, Olson SB, Magenis RE, Mauro MJ et al. Hematopathologic and cytogenetic findings in imatinib mesylate-treated chronic myelogenous leukemia patients: 14 months’ experience. Blood 2002; 100: 435–441. 9 Andersen MK, Pedersen-Bjergaard J, Kjeldsen L, Dufva IH, Brondum-Nielsen K. Clonal Ph-negative hematopoiesis in CML after therapy with imatinib mesylate is frequently characterized by trisomy 8. Leukemia 2002; 16: 1390–1393.
10 Feldman E, Najfeld V, Schuster M, Roboz G, Chadburn A, Silver RT. The emergence of Ph, trisomy-8+ cells in patients with chronic myeloid leukemia treated with imatinib mesylate. Exp Hematol 2003; 31: 702–707. 11 Bumm T, Muller C, Al-Ali HK, Krohn K, Shepherd P, Schmidt E et al. Emergence of clonal cytogenetic abnormalities in Phnegative cells in some CML patients in cytogenetic remission to Imatinib but restoration of polyclonal hematopoiesis in the majority. Blood 2003; 101: 1941–1949. 12 Gozzetti A, Tozzuoli D, Crupi R, Gentili S, Bocchia M, Raspadori D et al. Emergence of Ph negative clones in chronic myeloid leukemia (CML) patients in complete cytogenetic remission after therapy with imatinib mesylate. Eur J Haematol 2003; 71: 313–314. 13 Morel F, Ka C, Le Bris MJ, Herry A, Morice P, Bourquard P et al. Deletion of the 5’abl region in Philadelphia-positive chronic myeloid leukemia: frequency, origin and prognosis. Leukemia Lymphoma 2003; 44: 1333–1338.
Somatic PTPN11 mutation with a heterogeneous clonal origin in children with juvenile myelomonocytic leukemia
Leukemia (2004) 18, 1142–1144. doi:10.1038/sj.leu.2403374 Published online 15 April 2004 TO THE EDITOR
Juvenile myelomonocytic leukemia (JMML) is a clonal myeloproliferative disorder seen in young children and is characterized by leukocytosis with monocytosis, thrombocytopenia, hepatosplenomegaly, and elevated fetal hemoglobin caused by a reversion to fetal-like erythropoiesis.1 The Ras signaling pathway is known to be deregulated by mutations in the RAS gene or the neurofibromatosis type 1 gene (NF1) in approximately 40% of JMML cases. The PTPN11 gene, which encodes the nonreceptor protein tyrosine phosphatase SHP-2, has recently been shown to play a crucial role in the pathogenesis of JMML: SHP-2 acts as an upstream regulator of RAS, and somatic PTPN11 mutations were found in 34% of JMML patients without Noonan syndrome.2 Germline PTPN11 mutations are known to lead to Noonan syndrome, a multiple malformation syndrome characterized by unique facial features, neck webbing, and pulmonic stenosis. A subset of patients with Noonan syndrome also develop JMML. Currently, the level of hematopoietic differentiation at which somatic PTPN11 mutations arise in JMML patients remains unknown. In the present study, we performed a PTPN11 mutation analysis of leukemic marrow cells, Epstein–Barr virus (EBV)-transformed B lymphocytes, IL-2-stimulated peripheral blood (PB) cells, and buccal smear cells from two JMML patients without Noonan syndrome to document possible hematopoietic lineage-dependent genomic alterations at the PTPN11 locus. Both patients reported here had neither the features nor a family history of neurofibromatosis type 1 or Noonan syndrome. The study protocol was approved by the Ethics Committee of Keio University’s School of Medicine and the National Cancer Center. After obtaining informed consent from the patients’ Correspondence: Dr T Mori, Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; E-mail:
[email protected] Received 13 December 2003; accepted 11 March 2004; Published online 15 April 2004 Leukemia
parents, the patients’ leukemic marrow cells were screened for PTPN11 mutations. Genomic DNA was isolated using a desalting column (Qiagen, Chattsworth, CA, USA). Exon 3 and exon 8 in PTPN11, known to be mutation hot spots,2 were analyzed by direct sequencing of the PCR products amplified from the genomic DNA using a previously described method.3 To obtain T lymphocytes for the mutation analysis, PB cells were cultured and expanded using a TLY Culture kit25 (Lymphotec, Tokyo, Japan), which comprises plastic flasks coated with antiCD3 monoclonal antibody and RPMI-1640 medium containing 10% fetal calf serum and IL-2, as described previously.4 Almost all the IL-2-stimulated PB cells cultured by this method have been shown to be CD3-positive lymphocytes.4 To prevent contamination of DNA derived from myeloid cells, high molecular weight DNA was isolated from the IL-2-stimulated PB cells after culture for 3 weeks according to standard protocols. Patient 1: A 2-year-old boy presented with marked hepatosplenomegaly, skin lesions, thrombocytopenia (25.0 109/l), and leukocytosis (26.8 109/l) with myeloid precursors and a monocyte percentage of 16.5%. A bone marrow aspirate revealed granulocytic hyperplasia consisting of less than 5% blasts and a normal karyotype (46,XY). An elevated fetal hemoglobin level (50%) and a decreased neutrophil alkaline phosphatase (NAP) score of 29 were consistent with a diagnosis of JMML. Analysis of the genomic DNA derived from the leukemic bone marrow aspirate revealed an A4C transition in exon 3 at nucleotide 227, leading to an E76A substitution. The E76A substitution mutation was not present in an ethnically matched healthy control population of 100 subjects. Furthermore, the E76A substitution mutation was not found in the patient’s EBVtransformed B lymphocytes (Table 1). The patient was treated with 6-mercaptopurine (6-MP). At 4 months after the diagnosis of JMML, the patient underwent allogeneic bone marrow transplantation (allo-BMT) from an HLA-identical sibling. Patient 2: A 4-month-old boy presented with progressive hepatosplenomegaly, thrombocytopenia (71.0 109/l), and leukocytosis (49.8 109/l) with myeloid precursors and a monocyte percentage of 25.5%. A bone marrow aspirate revealed marked granulocytic hyperplasia consisting of less than 5% blasts and a normal karyotype (46,XY). A mildly elevated fetal hemoglobin level (3.6%) and a decreased NAP score of 75 were
Correspondence
1143 Table 1
Molecular findings in hematopoietic and buccal cells from two patients with PTPN11 mutation Mutation Nucleotide
Patient 1 Patient 2
227A4C 227A4T
BM
Lymphoid
Amino acid
EBV B cells a
E76A E76V
Mutant Mutanta
Buccal scrape
IL-2-stimulated PB
a
Wild-type Mutanta
ND Mutanta
ND Wild type
a
Each sample was harvested at the time of diagnosis. BM, bone marrow; EBV, Epstein–Barr virus; PB, peripheral blood; ND, not determined.
Table 2 Cytogenetic findings and HbF level in patient 2 with the PTPN11 mutation Patient 2
Karyotype BM
HbF EBV B cells
At diagnosis 46,XY [20] ND 3.6% 14 months after 46,XY [1] 46,XY [20] 32.4% diagnosis 46,XY,der(7)t(3;7)(q21;q22) [18] 46,XY,del(7)(q22) [1] BM, bone marrow; EBV, Epstein–Barr virus; HbF, fetal hemoglobin; ND, not determined.
consistent with a diagnosis of JMML. Analysis of the genomic DNA derived from the leukemic bone marrow aspirate obtained at the time of diagnosis revealed an A4T transition in exon 3 at nucleotide 227, leading to an E76V substitution; this mutation was not present in an ethnically matched healthy control population of 100 subjects. In this patient, the E76V substitution was also present in the EBV-transformed B lymphocytes and the IL-2-stimulated PB cells, but not in the buccal cells (Table 1). The patient was treated with 6-MP and maintained in a chronic phase. A cytogenetic analysis of a bone marrow aspirate obtained 14 months after diagnosis revealed the advent of an abnormal karyotype: 46,XY,der(7)t(3;7)(q21;q22)[18]/46,XY, del(7)(q22)[1]/46,XY[1]. However, the EBV-transformed lymphocytes established from the patient’s PB at the same time showed a normal karyotype (46,XY). The patient’s fetal hemoglobin level had increased to 32.4% at that time (Table 2). The patient underwent an allo-BMT from a 5/6 HLA-matched unrelated donor 18 months after diagnosis. We identified mis-sense mutations E76A and E76V in the PTPN11 gene in leukemic myelomonocytic marrow cells from two JMML patients who did not exhibit any features of Noonan syndrome. These mutations have been previously identified in JMML patients without Noonan syndrome and are considered pathogenic, thus lending further support to the notion that somatic PTPN11 mutations can cause JMML. The absence of the E76A change in the EBV-transformed B lymphocytes in patient 1 suggests that the mutation occurred at the level of a myeloidcommitted precursor cell. In patient 2, however, the presence of the E76V change in both the EBV-transformed B lymphocytes and the IL-2-stimulated PB cells, but not in the buccal cell germ line, indicates that the PTPN11 mutation occurred at the level of a pluripotential hematopoietic stem cell before it committed to a myeloid or lymphoid lineage. Somatic mutations with a heterogeneous clonal origin have also been observed in JMML patients with RAS or NF1 mutations.5,6 Thus, we believe that causative mutations in RAS, NF1, or PTPN11, all of which result in the deregulation of the RAS pathway, can occur in both primitive myeloid precursors and pluripotential hematopoietic stem cells. Patient 2, who harbored the PTPN11 mutation in the EBVtransformed B lymphocytes, acquired 7q monosomy in the bone
marrow cells 14 months after diagnosis. The normal karyotype in the EBV-transformed B lymphocytes, established at the same time, demonstrates the serial acquisition of two ‘hits’: the first hit being the PTPN11 somatic mutation in a pluripotential hematopoietic stem cell and the second one being 7q monosomy in a subclone with a myeloid origin. Furthermore, the concurrent increase in fetal hemoglobin suggests clonal evolution and proliferation of JMML cells that acquired 7q monosomy at the level of a primitive myeloid precursor common to a granulocyte–macrophage and erythroid lineage. In this case, not only 7q monosomy but also a partial trisomy of 3q may have also contributed to the clonal evolution of JMML cells, considering that 3q trisomy has been occasionally reported in JMML.7,8 In conclusion, PTPN11 mutant clones can originate from either a pluripotential hematopoietic cell or a cell with a primitive myeloid lineage.
Acknowledgements This work was supported by Pfizer Fund for Growth & Development Research, the Program for the Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion, and Product Review of Japan, and the Grant for Clinical Research for Evidence Based Medicine from the Ministry of Health, Labour, and Welfare of Japan. We thank C Hatanaka for her technical assistance.
H Shimada1 T Mori1 N Shimasaki1 K Shimizu2 T Takahashi1 K Kosaki1
1
Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan; and Molecular Oncology Division, National Cancer Center Research Institute, Tokyo, Japan
2
References 1 Arico M, Biondi A, Pui CH. Juvenile myelomonocytic leukemia. Blood 1997; 90: 479–488. 2 Tartaglia M, Niemeyer CM, Fragale A, Song X, Buechner J, Jung A et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 2003; 34: 148–150. 3 Kosaki K, Suzuki T, Muroya K, Hasegawa T, Sato S, Matsuo N et al. PTPN11 (protein-tyrosine phosphatase, nonreceptor-type 11) mutations in seven Japanese patients with Noonan syndrome. J Clin Endocrinol Metab 2002; 87: 3529–3533. 4 Sekine T, Shiraiwa H, Yamazaki T, Tobisu K, Kakizoe T. A feasible method for expansion of peripheral blood lymphocytes by culture with immobilized anti-CD3 monoclonal antibody and interleukin-2 for use in adoptive immunotherapy of cancer patients. Biomed Pharmacother 1993; 47: 73–78. 5 Flotho C, Valcamonica S, Mach-Pascual S, Schmahl G, Corral L, Ritterbach J et al. RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML). Leukemia 1999; 13: 32–37. Leukemia
Correspondence
1144 6 Miles DK, Freedman MH, Stephens K, Pallavicini M, Sievers EL, Weaver M et al. Patterns of hematopoietic lineage involvement in children with neurofibromatosis type 1 and malignant myeloid disorders. Blood 1996; 88: 4314–4320. 7 Michalova K, Bartsch O, Starvy J, Jelinek J, Wiegant J, Bubanska E. Partial trisomy of 3q detected by chromosome painting in a case
of juvenile chronic myelomonocytic leukemia. Cancer Genet Cytogenet 1993; 71: 67–70. 8 Tosi S, Mosna G, Cazzaniga G, Giudici G, Kearney L, Biondi A et al. Unbalanced t(3;12) in a case of juvenile myelomonocytic leukemia (JMML) results in partial trisomy of 3q as defined by FISH. Leukemia 1997; 11: 1465–1468.
Nucleolar localization of the carboxy-truncated form of the signal transducer and activator of transcription 5 (STAT5b) detected in CML and AML5
Leukemia (2004) 18, 1144–1145. doi:10.1038/sj.leu.2403371 Published online 15 April 2004 TO THE EDITOR
Leukemias are characterized by deregulation of hematopoietic cell differentiation and/or resistance to apoptosis. Among proteins involved in regulation of these pathways and suspected to play an important role in leukemogenesis are the Jak and STAT proteins. STAT (signal transducer and activator of transcription) 5a and STAT5b proteins are latent cytoplasmic transcription factors that translocate to the nucleus upon activation by cytoplasmic Jak tyrosine kinases. They participate in elemental cell biologic processes, including cellular growth, cell cycle progression and apoptosis. Based on several reports of STAT5 constitutive activation in leukemic cells, it has been hypothesized that STAT5 proteins may play an important role in leukemia. We previously reported on an original fusion protein between STAT5b and RARa in a patient with Acute Promyelocytic-Like phenotype,1 and we demonstrated that STAT5b gene rearrangement is rare in primary human hematologic malignancies.2 We decided on investigating the subcellular distribution of STAT5 proteins upon leukemia cells by immunochemistry experiments because the previous studies exclusively analyzed the STAT proteins in leukemias by Western blotting and electro mobility shift assay (EMSA) experiments on total nuclear protein extracts.3 Mononuclear cells from bone marrow or peripheral blood containing more than 80% blasts were isolated from 70 untreated patients with primary leukemias at initial diagnosis and STAT5 subcellular distribution was analyzed by indirect immunofluorescence with polyclonal antibodies directed against either STAT5a (L20), STAT5b (C17) or both STAT5a/b (N20) proteins. As summarized in Table 1, STAT5 nuclear distribution has been observed in 39/59 (67%) acute myeloblastic leukemia (AML) cases. Moreover, we have detected a nuclear localization of the STAT5 proteins in all chronic myelogenous leukemia (CML) and AML5 tested. Surprisingly, we pointed out a subnuclear localization for STAT5 proteins in AML5 (4/4), CML (4/5), AML2 (1/18) and in the K562 cell line expressing Bcr-Abl (Figure 1). In order to determine the precise subnuclear localization of the STAT5 proteins, we performed a double labelling experiment with both a polyclonal antibody to Correspondence: P Jonveaux, Laboratoire de Ge´ne´tique Me´dicale, EA 3441, CHU Nancy-Brabois, Avenue du Morvan, 54511 Vandoeuvre les Nancy, France; Fax: þ 33 3 83 15 37 72; E-mail:
[email protected] Received 10 November 2003; accepted 3 February 2004; Published online 15 April 2004 Leukemia
the a and b forms of STAT5a/b (N20) and a monoclonal antibody to Nucleolin/C23. A nucleolar colocalization of STAT5 with the nucleolin/C23 protein was observed (Figure 1). This is, to our knowledge, the first evidence of a nucleolar translocation of STAT transcription factors in hematological malignancies. Interestingly, we observed two different patterns of STAT5 subnuclear distribution in the K562 cells. Whereas STAT5 proteins revealed by an antibody directed against the highly homologous N-terminus domain of both STAT5a and STAT5b under a and b forms are located within the nuclei and the nucleoli, immunostaining with antibobies directed against the C-terminal domains of STAT5ba (C17) and STAT5aa (L20) revealed a distribution in the whole nucleus but not in the nucleoli as revealed by a total absence of co-localization with Nucleolin/C23. This pattern of nuclear distribution could be explained by a nucleolar localization of C-terminal truncated b forms of both STAT5a and STAT5b. Truncated STAT5 proteins lacking the transactivation domain have been observed in physiological conditions. These shorter b forms retain DNA binding ability but do not induce subsequent transcriptional activity.4 We thus hypothesized that the nucleolar STAT5 proteins might correspond to the truncated b forms of these transcription factors. We next tried to determine if nucleolar localization is a common event following activation of STAT5 proteins. We reported no evidence for STAT5 nucleolar distribution in activated peripheral blood mononuclear cells nor in lymphocytic U937, megacaryocytic MEG-01, Jurkat or in nonhematopoietic HeLa cell lines (data not shown). We demonstrated a nucleolar repartition of STAT5b isoforms only in K562 cells. This result raised the possibility that the nucleolar localization of STAT5 is part of the leukemogenic phenotype in CML. Several studies reported a STAT5 activation by BCR-ABL fusion protein in CML cells.5 We thus tested the contribution of the oncogenic tyrosine kinase BCR-ABL on the nucleolar localization of STAT5 proteins using a treatment with the kinase inhibitor STI571.6 K562 cells were treated with 1 mmol/l STI571, for 24–72 h. Western blot analysis of nuclear protein extracts from untreated or STI571 treated cells showed a dramatic decrease in STAT5ab phosphorylation but immunocytochemical analysis revealed that STI571 treatment of K562 cells did not affect the STAT5 nucleolar localization (data not shown). These results indicate that the oncogenic tyrosine kinase BCR-ABL is probably not responsible for STAT5 nucleolar localization. The nucleolus is a highly dynamic structure containing a large number of proteins and exhibiting a constant flow of RNA and proteins. Apart from its well-known involvement in the rRNA biogenesis, the nucleolus seems to be involved in the regulation of proteasome-dependent protein degradation. It has been proposed that trafficking through the nucleoli might be involved in protein turn-over. Recent data on STAT5 turn over analysis
