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1903 age-dependent incidences and the stability during disease evolution. Cancer Res 2006; 66: 3310–3316. 5 Lin LI, Chen CY, Lin DT, Tsay W, Tang JL, Yeh YC et al. Characterization of CEBPA mutations in acute myeloid leukemia: most patients with CEBPA mutations have biallelic mutations and show a distinct immunophenotype of the leukemic cells. Clin Cancer Res 2005; 11: 1372–1379. 6 Boissel N, Renneville A, Biggio V, Philippe N, Thomas X, Cayuela JM et al. Prevalence, clinical profile, and prognosis of NPM mutations in AML with normal karyotype. Blood 2005; 106: 3618–3620. 7 Tiesmeier J, Czwalinna A, Muller-Tidow C, Krauter J, Serve H, Heil G et al. Evidence for allelic evolution of C/EBPalpha mutations in acute myeloid leukaemia. Br J Haematol 2003; 123: 413–419.
8 Suzuki T, Kiyoi H, Ozeki K, Tomita A, Yamaji S, Suzuki R et al. Clinical characteristics and prognostic implications of NPM1 mutations in acute myeloid leukemia. Blood 2005; 106: 2854–2861. 9 Smith LL, Pearce D, Smith ML, Jenner M, Lister TA, Bonnet D et al. Development of a quantitative real-time polymerase chain reaction method for monitoring CEBPA mutations in normal karyotype acute myeloid leukaemia. Br J Haematol 2006; 133: 103–105. 10 Gorello P, Cazzaniga G, Alberti F, Dell’oro MG, Gottardi E, Specchia G et al. Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations. Leukemia 2006; 20: 1103–1108.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
JAK2 V617F mutation analysis in different myeloid lineages (granulocytes, platelets, CFU-MK, BFU-E and CFU-GM) in essential thrombocythemia patients Leukemia (2006) 20, 1903–1905. doi:10.1038/sj.leu.2404341; published online 3 August 2006
Myeloproliferative disorders (MPD) are a heterogeneous group of clonal hematopoietic stem cell malignancies that classically include four related entities: chronic myelogenous leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IM). With the exception of IM, all of these disorders are characterized by increased hematopoiesis and overproduction of blood elements, with the predominance of one myeloid lineages. In CML, the granulocytic lineage is the most hyperplastic, whereas the erythroblastic and megakaryocytic lineage are predominantly involved in PV and ET, respectively. These diseases are characterized by a clonal hematopoiesis, which involve the hematopoietic stem cells, as all myeloid lineages are monoclonal. A hallmark of MPD is the independence or hypersensitivity of hematopoietic progenitors to numerous cytokines. In PV, one of the most relevant characteristics is endogenous erythroid colony formation (eBFU-E). Erythroid progenitor cells proliferate in semisolid cultures in the absence of exogenous erythropoietin (EPO). Similar growth pattern is observed in a significant proportion of patients with ET and IM, but never in healthy individuals. Some studies have revealed that erythroid colony formation (BFU-E) in PV are hypersensitive not only to EPO but also to several other hematopoietic growth factors, including interleukin (IL)-3, stem cell factor, granulocyte–macrophage colony-stimulating factor (GM-CSF), insulin-like growth factor and thrombopoietin (TPO). Several studies have revealed that megakaryocytic colony formation (CFU-Mk) is growth factorsindependent in the majority of ET patients. This endogenous megakaryocytic colony formation (eCFU-MK) has also been shown, but in a lesser degree, in some PV, IM and even in CML, but not in reactive thrombocytosis. Recently, the existence of an activating mutation of Janus kinase 2 (JAK2) tyrosine kinase in granulocytes has been reported in a high proportion of patients with MPD1–4 (80% of PV, 50% of ET and 50% of IM). JAK2 is a cytoplasmatic tyrosine kinase with a key role in signal transduction from diverse cytokines and growth factors (extracellular ligands), including those for IL-3, IL-5, EPO, GM-CSF, granulocyte colonystimulating-factor receptor (G-CSF) and TPO, all of which play a major role in myeloid development. The JAK2 V617F is located in the pseudokinase domain of the JAK2 gene, a region
that inhibits JAK2 kinase activity. This mutation in the pseudokinase autoinhibitory domain results in a constitutive kinase activity and induces cytokine hypersensitivity or independence of factor-dependent cell lines.1–4 On the other hand, Lu et al.5 have shown that coexpression of the JAK2 V617F mutant kinase with a homodimeric type I cytokine receptor (such as the EPO receptor, the TPO receptor or the G-CSF receptor) is necessary for transformation of hematopoietic cells to growth factor independence and for hormone-independent activation of JAK-STAT signaling pathway. This phenomenon reveals the molecular basis for the prevalence of JAK2 V617F in diseases of myeloid lineage cells that express these type I cytokine receptors, but not in lymphoid lineage cells that do not. The incidence of JAK2 mutation in ET patients ranges from 25 to 57% depending on the study. These variation is likely owing to the sensitivity of the assay used to detect JAK2 V617F and the cell type analyzed (variabilty of the implication of different myeloid lineages in ET). In the majority of reports, the JAK2 V617F has been described in whole blood or isolated granulocytes, whereas limited information to referred to platelets and no data have been reported in isolated megakaryocytes.6 The aim of the present study was to analyze the presence of the JAK2 V617F in megakaryocytic cells obtained from peripheral blood in vitro cultures and in the remaining myeloid cells: peripheral blood granulocytes and platelets, as well as erythroid and granulomonocyte colonies obtained from peripheral blood in vitro cultures of patients with ET. We have evaluated six newly diagnosed ET patients according to the WHO’s diagnostic criteria. Written informed consent was obtained from every patient. All the assays were determined at diagnosis. In vitro cultures were performed as reported previously.7 Twenty milliliter of venous blood was collected in ethylendiaminetetraacetic acid and immediately processed. Neutrophils were isolated by Lymphoprep (1077 g/ml) density gradient, followed by dextran sedimentation. Total RNA was isolated from granulocytes or platelets using guanidinium thiocyanate method (Ultraspec; Biotecx Laboratories, Houston, TX, USA). cDNA was reverse transcribed from 1 mg of total RNA with Murine Moloney Leukemia Virus reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the standard procedures with random hexamers. Isolated colonies from in vitro cultures were carefully aspirated and washed with phosphate-buffered saline. RNA Leukemia
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1904 extraction was performed using the Genelute Mammalian Total RNA miniprep kit (Sigma-Aldrich, St Louis, MO, USA). cDNA was reverse transcribed using the high-capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). Analysis of JAK2 V617F was performed by direct sequencing using cDNA from granulocytes or purified colonies. Primers were designed using the Primer Express software and were as follows: forward: 50 -TTGGCCAAGGCACTTTTACAA-30 and reverse: 50 -TAGTGATCCAAATTTTACAAACTCCTGA-30 . Sequencing was performed with BigDye version 3.1 (Applied Biosystems) following the manufacturer’s instructions and analyzed on an ABI3100 Sequencer (Applied Biosystems). In non-conclusive cases, allele-specific polymerase chain reaction (PCR) was performed as described previously.1 All patients showed eBFU-E and/or eCFU-MK colony formation. These results are in agreement with others and with our previous experience in which we demonstrated eBFU-E and/or eCFU-MK in 85% of ET patients7,8 (unpublished data) and confirms the myeloproliferative nature of this entity. JAK2 mutation in granulocytes from peripheral blood by direct sequencing was detected in two out of six ET patients. When allele-specific PCR was used, the mutation was found in
Table 1 patients Patient
1 2 3 4 5 6
five out of six ET patients (no. 1–5). In patient no. 6, JAK2 mutation was not demonstrated by any of the two techniques. These results demonstrate the higher sensitivity of allele-specific assay and are in line with others and with our previous experience1–4,8 (Table 1). In five patients (no. 1–5), JAK2 V617F was demonstrated not only in granulocytes from peripheral blood but also in megakaryocytes and erythroblasts from endogenous colonies. In patient no. 6, JAK2 V617F was not found in erythroblasts from BFU-E, megakaryocytes from CFUMK, granulocytes from granulocyte–macrophage colony formation nor in granulocytes from peripheral blood. To further analyze the megakaryocytic lineage involvement in the patients included, we studied the JAK2 V617F in platelets from these patients, except for one of them from which no platelets were available. The mutation was detected in all patients in which the eCFU-MK were mutated and was negative in patient no. 6 who was also negative for eCFU-MK (Figure 1). ET is considered to be a clonal disease with the majority of myeloid lineages belonging to the same clone. Several reports have demonstrated JAK2 V617F in different myeloid lineages: erythroblasts, granulocytes and platelets in patients with MPD.
JAK2 V617F analysis in different myeloid (granulocytes, platelets, BFU-E, CFU-MK and CFU-GM) in essential thrombocythaemia
Diagnoses
ET ET ET ET ET ET
PB granulocytes sequencing/allele-specific PCR
M NM/M NM/M NM/M M NM/NM
In vitro cultures
PB platelets
BFU-E
eBFU-E
CFU-MK
eCFU-MK
CFU-GM
ND ND M ND M NM
M M M M M F
ND ND ND ND ND NM
M M M M M NM
ND ND ND ND ND NM
M M ND M M NM
Abbreviations: BFU-E, erythroid colony formation; CFU-GM, granulocyte-macrophage colony formation; CFU-MK, megakaryocytic colony formation; eBFU-E, endogenous erythroid colony formation; eCFU-MK, endogenous megakaryocytic colony formation; ET, essential thrombocythaemia; M, mutated; ND, not done; NM, non-mutated; PB, peripheral blood.
Figure 1 JAK2V617 analysis by sequencing in cell populations (granulocytes, eBFU-E, eCFU-MK and platelets) of a representative mutated case (upper panels) and a non-mutated case (lower panels). Leukemia
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In the current study, JAK2 V617F has been simultaneously analyzed in granulocytes, erythroblasts, megakaryocytes and platelets from the same patient. The results found in the first five ET patients (no. 1–5) demonstrated that myelopoiesis in these patients is entirely clonal and is related to JAK2 V617F. This mutation was not found in one patient who showed eCFU-MK formation; nevertheless, this patient had monoclonal myelopoiesis (as determined by the human androgen receptor gene polymorphic marker clonality assay) (results not shown). These results support the hypothesis that some cases of true ET might be related to other molecular defects, as already has been suggested in the literature. In our experience, all ET patients with JAK2 V617F in peripheral blood granulocytes carry the mutation in the platelet-megakaryocytic lineage. This is the lineage predominantly involved in ET and indicates that in ET the mutation should be searched by more sensitive techniques and directly on platelets. In conclusion, our study describes the presence of JAK2 V617F in all myeloid cells including the megakaryocytic lineage. These results strongly support the implication of different myelopoietic cell lineages in ET and confirm the biological heterogeneity of this disease.
Acknowledgements This work was supported by the Grants FIS PI030345, C03/07 and C03/10 from the Spanish Ministry of Health.
L Florensa1,2, B Bellosillo2,3, C Besses2,4, E Puigdecanet1,2,3, B Espinet2,3, E Pe´rez-Vila1,2, R Longaro´n3, RM Vila`1, F Sole´2,3 and S Serrano1,2,3 1 Laboratori de Citologia Hematolo`gica, Departament de Patologia, Hospital del Mar, IMAS, Barcelona, Spain; 2 Unitat de Recerca en Neopla`sies Hematolo`giques-Parc Recerca Biome`dica Barcelona (URNHE-PRBB), Barcelona, Spain;
3
Laboratori de Citogene`tica i Biologia Molecular, Departament de Patologia, Hospital del Mar, IMAS, Barcelona, Spain and 4 Servei d’Hematologia Clı´nica, Hospital del Mar, IMAS, Barcelona, Spain E-mail:
[email protected]
1905
References 1 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–1061. 2 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387–397. 3 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005; 434: 1144–1148. 4 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779–1790. 5 Lu X, Levine R, Tong W, Wernig G, Pikman Y, Zarnegar S et al. Expression of a homodimeric type I cytokine receptor is required for JAK2V617F-mediated transformation. Proc Natl Acad Sci USA 2005; 102: 18962–18967. 6 Campbell PJ, Scott LM, Buck G, Wheatley K, East CL, Marsden JT et al. Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study. Lancet 2005; 366: 1945–1953. 7 Florensa L, Besses C, Woessner S, Sole´ F, Acı´n P, Pedro C et al. Endogenous megakaryocyte and erythroid colony formation from blood in essential thrombocythaemia. Leukemia 1995; 9: 271–273. 8 Bellosillo B, Besses C, Florensa L, Sole F, Serrano S. JAK2 V617F mutation, PRV-1 overexpression and endogenous erythroid colony formation show different coexpression patterns among Phnegative chronic myeloproliferative disorders. Leukemia 2006; 102: 18962–18967.
High ZAP-70 expression correlates with worse clinical outcome in mantle cell lymphoma Leukemia (2006) 20, 1905–1908. doi:10.1038/sj.leu.2404362; published online 17 August 2006
Zeta-associated protein 70 (ZAP-70) is a z-chain, CD3-receptorassociated protein tyrosine kinase (PTK) that is critical for initiating T-cell signaling. Relatively little is known with regard to its function in B cells, although there is evidence that ZAP-70 may enhance signal transduction via the B-cell receptor complex. In chronic lymphocytic leukemia (CLL), the presence of 420% of ZAP-70-positive CLL cells detectable by flow cytometry was found to correlate with rapid disease progression and shorter overall survival in stage Binet A patients.1 High level of ZAP-70 expression also has been associated with a relative lack of somatic mutations in the variable regions of the immunoglobulin heavy chain gene (VH), which is an established prognostic marker for CLL.2 Several recent studies have confirmed that ZAP-70 is expressed in a subset of mantle cell lymphoma (MCL).3–5 In this study, we aimed to determine if ZAP-70 carries any clinical significance in newly diagnosed MCL patients.
Immunohistochemistry was employed to assess ZAP-70 expression in 64 MCL tumors. The clinical characteristics of these patients are summarized in Table 1. All cases were newly diagnosed and collected between 1994 and 2003 at the Department of Laboratory Medicine and Pathology, Cross Cancer Institute. Treatment for each MCL patient was determined during our weekly lymphoma conference based on our institutional treatment protocol. For first-line treatment, 32 of the 64 patients received CHOP-based (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy, 15 had chlorambucil-based chemotherapy, and five received other treatments such as proteasome inhibitor PS341 and flavopirodol. The diagnosis of all MCL cases was based on the criteria described in the World Health Organization Classification Scheme; all cases were positive for CD5 and/or CD43 and cyclin D1, and negative for CD23. All tissues were routinely processed, formalin-fixed and paraffin-embedded. All but four cases had the small cell morphology. Three of the remaining four cases were blastoid MCL. The morphology of the other case was composed of diffuse areas of small lymphoma cells admixed with Leukemia
