Essential thrombocythemias without V617F JAK2 ...

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Apr 6, 2006 - patients.5,6 However, in those studies, only HUMARA assay was performed to assess clonality, a methylation-based test that is not completely ...
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patients, through the binding of any IgG isotypes contained in immune complexes of autoimmune disorders associated with WM. This hypothesis may explain that FcgRIIa-131HH patients had a shorter survival than FcgRIIa-131RH patients. A similar effect might be observed in FcgRIIa-131RR patients, through the binding with CRP, because FcgRIIa-131R is a receptor for CRP, and CRP works similarly to IgG in causing events downstream to the binding of several FcgRIIa receptors.5 The expression of the FcgRIIa on some B cells and mast cells8 may contribute also to the effects of its ligands on WM cells. In conclusion, the present study indicated that AIHA occurred more frequently in WM patients with the FcgRIIa-131HH genotype. In addition, FcgRIIa-131R/H polymorphism might influence the outcome of the patients.

Acknowledgements We thank Audrey Gravey, Vale´rie Grandie`res and Catherine Vanicatte for their excellent technical assistance and Marc Daeron (U255, Institut Pasteur, Paris) for his thoughtful comment. This work was supported by the Comite´ de la Somme de la Ligue contre le Cancer.

S Poulain1, I Dervite2, X Leleu3, J Fernandes4, L Stalnikiewicz2, A-S Moreau3, V Coiteux3, S de Botton3, P Duthilleul1 and P Morel2 1 Service d’He´matologie -Immunologie-Cytoge´ne´tique, Centre Hospitalier, Valenciennes, France; 2 Service d’He´matologie Clinique, Centre Hospitalier Schaffner, Lens, France; 3 Service des Maladies du Sang, Centre Hospitalier Regional Universitaire, Lille, France and

4

Service d’He´matologie Clinique, Centre Hospitalier, Valenciennes, France E-mail: [email protected]

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References 1 Jonsson V, Kierkegaard A, Salling S, Molander S, Andersen LP, Christensen M et al. Autoimmunity in Waldenstro¨m’s Macroglobulinemia. Leuk Lymph 1999; 34: 373–379. 2 Dimopoulos M, Kyle R, Anagnostopoulos A, Treon S. Diagnosis and management of Waldenstrom’s macroglobulinemia. J Clin Oncol 2005; 23: 1564–1577. 3 Rosse WF, Hillmen P, Schreiber AD. Immune-mediated haemolytic anemia. Hematology (Am Soc Hematol Educ Program) 2004, 48–62. 4 Van Royen-Kerkhof A, Sanders E, Walraven V, Voorhorst-Ogink M, Saeland E, Teeling J et al. A novel human CD32 mAb blocks experimental immune haemolytic anaemia in FcgRIIa transgenic mice. Br J Hematol 2005; 130: 130–137. 5 Marnell L, Mold C, Du Clos TW. C-reactive protein: ligands, receptors and role in inflammation. Clin Immunol 2005; 117: 104–111. 6 Mathsson L, Tejde A, Carlson K, Ho¨glund M, Nilsson B, NilssonEkdahl K et al. Cryoglobulin-induced cytokine production via FcgRIIa: inverse effects of complement blockade on the production of TNFa and IL-10. Implications for the growth of malignant B-cell clones. Br J Hematol 2005; 129: 830–838. 7 Fabijanska-Mitek J, Lopienska H, Zupanska B. Gel test application for IgG subclass detection in auto-immune haemolytic anaemia. Vox Sanguinis 1996; 72: 233–237. 8 Okayama Y, Hagaman D, Woolhiser M, Metcalfe D. Further characterization of Fc gammaRIIa and Fc gammaRIII expression by cultured human masts cells. Int Arch Allergy Immunol 2001; 124: 155–157.

Essential thrombocythemias without V617F JAK2 mutation are clonal hematopoietic stem cell disorders Leukemia (2006) 20, 1181–1183. doi:10.1038/sj.leu.2404214; published online 6 April 2006

Diagnosis of essential thrombocythemia (ET) relies on a set of criteria defined by the Polycythemia Vera Study Group and recently updated by WHO classification. Most of those criteria allow the exclusion of other myeloproliferative disorders (MPD) including polycythemia vera (PV), chronic myelogenous leukemia and myelofibrosis with myeloid metaplasia. Differentiating primary from secondary causes of thrombocytosis has important therapeutic implications but can sometimes be difficult. A key evidence for MPD is the demonstration of clonal proliferation of hematopoietic stem cells. In the absence of a molecular marker and due to the rarity of chromosomal abnormalities in ET, assays based on X-linked polymorphic markers in female patients were developed to investigate clonality.1–3 We have previously shown that the simultaneous study of several marker genes using methylation and expressionbased assays associated to the study of both granulocytes and platelets provides the finest evaluation of clonality status of hematopoiesis.1,2 Using X-chromosome inactivation patterns (XCIPs) analysis, several groups reported that circulating granulocytes and platelets derived from ET patients displayed a monoclonal pattern. However, the persistence of polyclonal patterns in a subset of patients was also reported, possibly associated with a lower risk of thrombosis.3 In the absence of

specific molecular markers, demonstration of clonal myeloproliferation was impossible in those cases. The discovery of an activating V617F JAK2 mutation in most patients with PV4 not only helps understand disease pathophysiology but represents also a clear molecular marker for this disease. In ET, the role of V617F JAK2 mutation is far less evident. Indeed, only half of ET patients harbor the mutation and its role in growth factor-independent in vitro proliferation of megakaryocytes has not yet been demonstrated. If diagnosis of PV will certainly rely on the presence of the mutation, in ET a positive marker is still necessary in almost half of patients, in whom V617F JAK2 is not present. It was recently reported that a similar proportion of ET patients with monoclonal XCIPs patterns could be found in JAK2 mutated and non-mutated patients.5,6 However, in those studies, only HUMARA assay was performed to assess clonality, a methylation-based test that is not completely reliable for patients’ samples analysis because of possible abnormal DNA methylation in malignancies.7 We studied a series of female ET patients in whom XCIPs had previously been analyzed by our group using both gene methylation and expression assays in all hematopoietic cell lineages1,2 for the presence of mutant JAK2 and tested if the mutation correlated with monoclonal phenotype that would be expected if JAK2 mutation plays a role in disease development. The studied population (n ¼ 44) and XCIPs analysis based on DNA and RNA assays were previously reported.1,2 In summary, Leukemia

Letters to the Editor

1182 Table 1 Patients

JAK2 mutation, XCIPs analyses and patients characteristics V617F JAK2 DNA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

 +      +    + + + +    + +  + +    + + + +       + +    +  

XCIPs analysis

Platelet RNA Unfractionated blood Failed +      +    + + +  +   + +  + NA    + +  +  +  NA  Failed NA + NA     

MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO PO PO PO PO PO PO PO MO MO MO MO MO MO MO MO MO MO MO PO PO

Patients characteristics at diagnosis

Granulocytes

Platelets

T Lymph.

Age

MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO PO PO PO PO PO PO PO MO MO MO MO MO MO MO MO MO MO MO PO PO

MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO PO PO PO PO PO PO PO NI NI NI NI NI NI NI NI NI NI NI NI NI

MO MO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO PO MO MO PO PO PO PO PO PO PO PO PO PO PO

36 34 37 80 21 50 33 65 55 75 57 71 69 69 39 77 69 65 48 63 59 38 63 71 24 25 25 43 30 37 76 57 68 70 46 16 43 68 42 63 47 61 37 16

Follow-up Treatment (months) 53 148 77 18 1 57 34 49 100 52 98 1 2 12 1 1 48 38 24 123 120 1 1 1 124 59 336 132 85 108 50 7 56 48 12 70 19 1 2 36 42 84 1 2

HU HU IFNa HU 0 HU HU HU HU HU HU 0 0 HU 0 0 0 HU HU HU HU 0 0 0 0 0 0 0 HU IFNa HU HU HU 0 HU HU 0 0 0 HU HU HU 0 0

Clinical symptoms

Platelet count (109/l)

+ +  +  +   +     +  +       +      +  +   + +  +  +  +   

1100 889 2160 812 1886 1700 1750 803 800 710 800 1150 900 2000 1400 650 787 745 1069 950 768 674 1200 751 950 751 780 600 648 1098 660 1300 962 693 1060 907 1500 843 990 1025 1067 631 880 900

Abbreviations: HU, hydroxyurea; IFNa, interferon alfa; JAK, Janus kinase; MO, monoclonal; NA, sample not available; ; NI, not informative for RNA markers; PO, polyclonal; T Lymph., T lymphocytes; XCIPs, X-chromosome inactivation patterns.

IDS, G6PD and P55 gene polymorphisms were used for XCIPs analysis in both DNA (extracted from unfractionated blood and purified granulocytes) and RNA (prepared from the same cell fractions and from purified platelets). DNA samples were also screened for heterozygosity of the HUMARA gene, and clonality analyses were performed using HUMARA trinucleotide repeat polymorphism. Cell fractions containing T lymphocytes (as control for skewed lyonization), granulocytes and platelets were studied using RNA markers when at least one of them was informative (n ¼ 31). When no RNA marker was informative, the same fractions except platelets were studied using the HUMARA DNA polymorphism (n ¼ 13). Two methods were used to detect JAK2 mutation: sequence analysis on stored whole blood genomic DNA (sensitivity 5–10% to detect the mutated allele) and platelets RNA, and singlenucleotide polymorphism genotyping assays performed in DNA Leukemia

samples using real-time PCR-based mutation detection (Taqmans ABI Prism 7700).8 Sensitivity of PCR assay reached 0.5–1% of HEL cell line DNA diluted in non-mutated cell line DNA and 2–4% of a homozygously mutated patient’s DNA diluted in normal DNA. This PCR assay also allowed semiquantitative evaluation of the proportion of mutant and wild-type JAK2 alleles, as described.8 V617F JAK2 mutation was found in 19 of the 44 patients (43%) (Table 1), a proportion in agreement with published series of ET patients.4 In five cases, DNA sequencing showed a small peak of mutated nucleotide, but platelet RNA sequencing and allele-specific PCR clearly demonstrated the presence of the mutation. Semiquantitative evaluation of mutated JAK2 in PCR assays was available in 16 of the 19 positive patients and showed variable mutated allele content from one patient to another: 60% of total JAK2 in one (6%), 40% in three (19%),

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1183 30% in nine (56%) and 10% in three (19%) patients, respectively. No differences were found between JAK2-mutated and non-mutated patients for age (52716 years versus 49720, P ¼ 0.58), clinical symptoms at presentation (31 versus 36%, P ¼ 1), platelet count (1004  109/l7362 versus 1024  109/ l7347, P ¼ 0.86), follow-up duration (60780 months versus 48736, P ¼ 0.65) or utilization of cytoreductive treatment (9/19 versus 15/25, P ¼ 0.42). Of the nine patients with polyclonal pattern in granulocytes, four (44%) had the V617F JAK2 mutation. Moreover, those four mutated cases (patients no. 27–30, Table 1) belonged to the group of seven patients with polyclonal patterns observed in both granulocytes and platelets. Presence of the mutation despite a polyclonal pattern can probably be explained by the higher sensitivity of mutation detection by PCR (that can detect 2–4% of mutated cells), compared to XCIPs assays that require at least 25% of clonal cells in the tested population to show a monoclonal pattern.3 Those results show that many ET with an apparent polyclonal XCIPs are clearly clonal MPD. In this series, combined analysis of four clonality marker genes in both DNA and RNA assays demonstrated a high proportion of monoclonal patterns in myeloid cells (31 of the 40 cases with random XCIPs in T lymphocytes, 77%), a proportion higher than in studies based on the HUMARA DNA assay only.5,6 Of the 31 patients with monoclonal hematopoiesis demonstrated by XCIPs analysis, 13 (42%) had the mutation. Twenty-two patients were informative for RNA markers with a monoclonal pattern in platelets as well as in granulocytes (patients 3–24, Table 1), and 10 (45%) had the JAK2 mutation. Of note, the three patients with a ratio of mutant JAK2 determined by real-time PCR of only 10% belonged to this group of monoclonal patients (patients no. 12, 15 and 23). The absence of JAK2 mutation in 55% of patients with clear monoclonal hematopoiesis (as demonstrated by XCIPs analysis, including in platelets) suggests that molecular events other than V617F JAK2 mutation can lead to clonal megakaryocytic proliferation in a large proportion of ET patients. In four patients, a nonrandom X-chromosome inactivation pattern was found (i.e. monoclonal pattern in all cell fractions including T lymphocytes, patients 1, 2, 32 and 33, Table 1). In such situation, clonality cannot be established by XCIPs assays. V617F JAK2 mutation was present in two of them. An important issue for an accurate detection of JAK2 mutation is the type of cells used for analysis. To date, most published studies used DNA prepared from whole blood, granulocytes or mononuclear cells. Very recently, JAK2 mutation was also found in platelets of five ET patients.9 In the same study, Campbell et al. did not found the mutation in the platelets of five patients with wild-type JAK2 in granulocytes. In the present study, we could compare JAK2 mutation detection between whole blood DNA (mainly representative of granulocytes) and platelet-derived RNA. Overall, JAK2 mutation was detected in 17 of the 43 (39%) DNA samples available, and in 13 of the 38 (34%) exploitable RNA samples, confirming that both cell fractions can be used for mutation detection in ET and that the mutation is present in cells of the megakaryocytic lineage. Contrary to the study by Campbell et al., V617F JAK2 was detected in platelets in two of the 24 patients (8%) with wildtype JAK2 in the DNA samples. These results suggest that larger series of ET patients are required to definitely determine usefulness of platelet studies in order to exclude presence of the mutation in patients with wild-type JAK2 in whole blood or granulocytes. In conclusion, in this series of 44 ET patients, V617F JAK2 mutation demonstrated clonal hematopoietic proliferation in

43% of cases. Combining results of clonality assays and JAK2 sequencing allowed demonstration of clonal proliferation in 40 of 44 patients (90%). Of the 25 patients without detectable JAK2 mutation, 18 (72%) had a monoclonal pattern showing that XCIPs analysis can still be helpful to identify a clonal hematopoietic disorder in female patients with unexplained thrombocytosis. Furthermore, these results confirm that the majority of ET patients without V617F JAK2 mutation have indeed a clonal hematopoietic stem cell disease if comprehensive XCIPs assays are used. Identification of such patterns will improve selection of ET patients in whom molecular defects other than V617F JAK2 could be found.

Acknowledgements We thank William Vainchenker for helpful discussions and JeanPierre Le Coue´dic for technical assistance.

J-J Kiladjian1, N Elkassar2, B Cassinat3, G Hetet4, S Giraudier5, N Balitrand3, C Conejero5, J Briere1, P Fenaux1, C Chomienne1 and B Grandchamp4 1 AP-HP, Hopital Avicenne, Service d’Hematologie Clinique, and Paris 13 University, Bobigny, Paris, France; 2 Etablissement Francais du Sang PL and UPRES EA3863, Faculte de Medecine, Angers, France; 3 AP-HP, Cellular Biology Unit, Hopital Saint-Louis, Paris, France; 4 INSERM U656 and Service de Biochimie Hormonale et Genetique, Hopital Bichat and Paris 7 University, Paris, France and 5 AP-HP, Laboratory of Hematology, Hopital Henri Mondor, Creteil, France E-mail: [email protected] References 1 El-Kassar N, Hetet G, Briere J, Grandchamp B. Clonality analysis of hematopoiesis in essential thrombocythemia: advantages of studying T lymphocytes and platelets. Blood 1997; 89: 128–134. 2 El-Kassar N, Hetet G, Briere J, Grandchamp B. X-chromosome inactivation in normal females: incidence of excessive lyonization with age and comparison of assays using DNA methylation and those using analysis of transcript polymorphisms. Clin Chem 1998; 44: 61–67. 3 Harrison CN, Gale RE, Machin SJ, Linch DC. A large proportion of patients with a diagnosis of essential thrombocythemia do not have a clonal disorder and may be at lower risk of thrombotic complications. Blood 1999; 93: 417–424. 4 Kaushansky K. On the molecular origin of the chronic myeloproliferative disorders: it all makes sense. Blood 2005; 105: 4187–4190. 5 Antonioli E, Guglielmelli P, Pancrazzi A, Bogani C, Verrucci M, Ponziani V et al. Clinical implications of the JAK2 V617F mutation in essential thrombocythemia. Leukemia 2005; 19: 1847–1849. 6 Levine R, Belisle C, Wadleigh M, Zahrieh D, Lee S, Chagnon P et al. X-Inactivation based clonality analyis and quantitative assessment reveals a strong association between clonality and JAK2V617F in PV but not ET/MMM, and identifies a subset of JAK2V617F negative ET and MMM patients with clonal hematopoiesis. Blood, , prepublished online January 24, 2006; doi 10.1182/blood-2005-09-3900. 7 Vogelstein B, Fearon E, Hamilton S, Feinberg A. Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors. Science 1985; 227: 642–645. 8 James C, Delhommeau F, Marzac C, Teyssandier I, Couedic JP, Giraudier S et al. Detection of JAK2 V617F as first intention diagnostic test for erythrocytosis. Leukemia 2006; 20: 350–353. 9 Campbell P, Scott L, 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. Leukemia