PEDIATRIC PATIENTS WITH ESSENTIAL THROMBOCYTHEMIA ARE MOSTLY POLYCLONAL AND V617FJAK2 NEGATIVE

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1 Blood First Edition Paper, prepublished online July 18, 2006; DOI /blood PEDIATRIC PATIENTS WITH ESSENTIAL THROMBOCYTHEMIA ARE MOSTLY POLYCLONAL AND V617FJAK2 NEGATIVE Maria Luigia Randi 1, Maria Caterina Putti 2, Margherita Scapin 1, Enrica Pacquola 1, Fabio Tucci 3, Concetta Micalizzi 4, Luigi Zanesco 2, Fabrizio Fabris 1 1 Dept of Medical and Surgical Sciences, Internal Medicine, 2 Dept of Pediatrics, Pediatric Hematology Oncology, University of Padua Medical School, Padua, Italy 3 Pediatric Clinic, Meyer Hospital, Florence, Italy 4 Dept. of Pediatric Hematology-Oncology, G Gaslini Institute, Genova MLR and MCP contributed equally to this study Acknowledgment: We thank dr. C Consarino, Pediatric Hematological Service, Catanzaro Hospital, dr. P Pierani, Pediatric Clinic, Ancona, dr. K. Tettoni, Pediatric Clinic, Spedali Civili Brescia, dr. GM Fiori, Pediatric Clinic Cagliari for providing samples of some patients. Correspondence to: dr. ML Randi, Dept of Medical and Surgical Sciences, University of Padua, via Giustiniani 2, Padua, Italy Tel , Fax marialuigia.randi@unipd.it Copyright 2006 American Society of Hematology

2 ABSTRACT Essential thrombocythemia (ET) is rare in children and little or no information is available about clonality or JAK2 mutation. These analyses prove useful, however, for the diagnosis of adults myeloproliferative disorders (MPD). We evaluated the clonality status and V617FJAK2 mutation in 20 children affected by ET and compared them with 47 consecutive adult ET. Clonality was evaluated on the DNA of granulocytes and on the RNA of platelets. V617FJAK2 was analysed by sequencing tests, allele-specific PCR and digestion by BsaX I. A monoclonal pattern was found in 4 out of 14 children (28.5%) and 45% of informative adults. Heterozygous V617FJAK2 was found less frequently in children than in adults (p<0.009). Only two girls showed both the V617FJAK2 mutation and a monoclonal pattern; one of them was the only child presenting a major thrombotic complication. In contrast to adults, most children with ET do not show either a clonal disorder or the V617FJAK2 mutation. 152 words INTRODUCTION The diagnosis of essential thrombocythemia (ET) is usually made by exclusion criteria because no biological markers are known to differentiate it either from primitive myeloproliferative disorders (MPD) (1) or from reactive thrombocytosis (2). The primitively proliferative nature of ET was first documented by Fialkow (3) who analysed female patients for X chromosome inactivation patterns (XCIP) of G6PDH. It was found therein that 3 cases showed clonal expansion. However, further studies (4,5) demonstrated that only 20-35% of adult ET is monoclonal. Therefore, it is possible that monoclonal and polyclonal patterns define different disorders with a similar phenotype. It is relevant that recently a somatic mutation of janus kinasis 2 (JAK2) was found in about 35-50% of adult ET patients (6,7). ET in pediatric age is a rare disorder that might also embed a spectrum of different diseases (8). Unaware of previous comparative reports, we evaluated the clonal patterns and JAK2 gene mutations in pediatric ET patients. PATIENTS AND METHODS We studied 15 females and 5 males affected by ET in agreement with the Polycythemia Vera Study Group criteria (9) diagnosed in pediatric age, with a platelet count continuously over 800 x 10 9 /L (range x 10 9 /L) and without known causes of reactive/secondary thrombocytosis. None of the children had any family history of either MPD or thrombocytosis. None had a prothrombotic

3 condition. Moreover, in these patients serum erythropoietin (EPO) and thrombopoietin (TPO) contents, platelet function, standard cytogenetic analysis, bcr/abl rearrangement, sequences of TPO and c-mpl genes (10) were evaluated without finding any abnormality. The clinical findings and therapeutic options adopted are summarized in table 1. Approval was obtained from the Padua University Department of Medical and Surgical Sciences institutional review board for these studies and that informed consent was provided according to the Declaration of Helsinki. For comparison as adult controls, 36 consecutive adult females and 11 adult males, all younger than 50 years (mean age 43±7 years) affected by ET were studied in our Department, which is a reference Center for thrombo/hemorrhagic conditions. We also studied, as negative controls, 21 normal individuals (17 women and 4 males) with normal platelet count and no thrombotic complication nor pro-thrombotic state. Granulocytes, T-lymphocytes and platelets were isolated as described by Teofili et al. (11). DNA was extracted with the Miller method (12). RNA was extracted from platelets using TRIZOL reagent according to the manufacturer s protocol. The state of activation of the X-chromosome was determined by using a methylation-sensitive restrictive enzyme (HpaII) or Transcriptional Polymorphism on the active X-chromosome. XCIPs was assessed using: primers flanking the CAG repeats of HUMARA gene (13); nested primer pairs flanking the region of BstXI polymorphism of PGK gene (14); primers for allele-specific PCR of an exonic polymorphism of MPP1 gene (p55) on platelet RNA (15). To detect the V617FJAK2 mutation in peripheral blood granulocytes DNA, sequence analysis and allele- specific PCR was used. Digestion by BsaXI was used to determine homozygosis of JAK2 mutation (7). The comparison between variables was performed by chi-square statistics with Yates variables where p-values <0.05 were considered statistically significant. RESULTS AND DISCUSSION While reactive thrombocytosis is relatively common and some familial cases were reported (16), sporadic ET is extremely rare in childhood (17). These cases have clinical and laboratory characteristics different than those of adult ET (8). In the present study we found that pediatric ET differs from adult ET also with respect to the clonality pattern (table 2): indeed we show that 10 children exhibit a polyclonal pattern on granulocytes while only 4 are monoclonal (28.5%). Because a restriction of the clonal population to cells of the megakaryocytes lineage may exist (18), we also studied clonality in platelet mrna from 8 children (6 polyclonal and 2 monoclonal): the observed patterns were consistent. Clonality studies were interpretable for all but one child who had a

4 skewing pattern. On the other hand, adults, notwithstanding the relatively young age, were not informative in about half of the cases considered, as reported by others (4). Moreover, 45% of the informative patients were monoclonal, a percentage significantly higher than in children. The known increase of clonality incidence with age (19), can not exclude that our children might develop a clonal pattern over time. At present, however, 4 out of the 10 girls with a polyclonal pattern have been followed for more than 8 years, 2 patients being now older than 20 (table 1). The somatic mutation of JAK2, considered a primary event in MPD (20), was reported in most patients with polycythemia vera (PV) and in a subgroup of ET patients. In our study, the V617FJAK2 mutation was significantly less frequent in children (4/20) than in adults (28/47) (p=0.009). Our results for adults are consistent with data reported elsewhere (6,21). It is relevant to note that, in addition to the sequencing method, we used the allele-specific-pcr, which is considered a highly sensitive technique to JAK2 mutations (21). Neither children nor adults showed homozygous JAK2 mutations. The association of JAK2 mutations with thrombohemorrhagic events is still a matter of debate (6,7, 20,22). Increased frequency of venous thrombosis in uncommon sites in adults presenting the V617FJAK2 mutation was reported for large cohorts (21) and confirmed by our cases. Among adults, 7 out of the 9 unusual site thromboses occurred in mutated patients, irrespectively of their clonality status. It is worthwhile noting that one pediatric case with both JAK2 mutation and clonal disease presented at 9 months with Budd-Chiari syndrome (Table 2). Whether this single case represents a random association or hints at a pathogenetic implication is not clear at present. In fact, another girl showed a monoclonal pattern and the V617FJAK2 mutation but no clinical symptoms. The simultaneous proof of clonal myelopoiesis on XCIP analysis and JAK2 somatic mutation could be suggestive of a primitive MPD which may have a more severe clinical course (4). JAK2 mutation in ET may imply the existence of a disease closely related to PV. Campbell et al. (21) recently suggested that mutation-positive and mutation-negative ET represent distinct disorders. Our study includes a number of children significant for a rare disease and thus our data indeed confirm the possible existence of different types of ET. Rare cases are characterized by monoclonal pattern and/or JAK2 mutation and possibly by a severe clinical behavior. The other cases may actually have developed a disease different from adult ET as previously hypothesized (8). Moreover, the difference between children and adult ET is underlined when, as suggested by Kaladjian (23), we combine the results of clonality assays and JAK2 evaluation: only 30% of children seem to have a clonal proliferation in comparison of 72% of adult cases.

5 Because criteria for the treatment of ET are not specific for children, therapeutic choices are highly heterogeneous (table 1). Thus new diagnostic and prognostic classifications might help shaping improved therapeutic recommendations, with wide implications (24). In conclusion, the heterogeneous molecular features found in our large set of pediatric ET cases may help in defining various subgroups, allowing to predict the clinical course mainly for thrombotic risks. This is necessary to assess which child with ET really deserves cytoreductive therapy, as already defined for adults. Larger samples are obviously needed to confirm our results thus further prompting the use of biological markers for diagnosis and treatment of ET words

6 REFERENCES 1) Shafer AI. Thrombocytosis. N Engl J Med 2004;350: ) Spivak JL. The chronic myeloproliferative disorders. Clonality and clinical heterogeneity. Semin Hematol 2004;4:1-5 3) Fialkow JP, Faguet GB, Jacobson RJ, Vaidya M, Murphy S. Evidence that essential thrombocythemia is a clonal disorder with origin in a multipotent stem cell. Blood 1981;158: ) 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;993: ) Vannucchi AM, Grossi A, Pancrazzi A, et al. PRV-1, erythroid colonies and platelet Mpl are unrelated to thrombosis in essential thrombocythemia. Br J Hematol 2004;127: ) Kralovics R, Passamonti F, Buser AS, et al: A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352: ) Baxter EJ, Scott LM, Campbell PJ, et al. Cancer Genome Project: Acquired mutation of the tyrosine kinase Jak2 in human myeloproliferative disorders. Lancet 2005;365: ) Randi ML, Putti MC, Fabris F, Sainati L, Zanesco L, Girolami A. Features of essential thrombocythemia in childhood: a study of five children. Br J Hematol 2000;108, ) Murphy S, Iland H, Rosenthal D, Laszlo J. Essential thrombocythemia: an interim report from the polycythemia vera study group. Semin Hematol 1986;23: ) Randi ML, Putti MC, Pacquola E, Luzzatto G, Zanesco L, Fabris F. Normal thrombopoietin and its receptor (c-mpl) genes in children with essential thrombocythemia. Pediatr Blood Cancer 2004;43:1-4 11) Teofili L, Martini M, Luongo M, et al. Overexpression of the Polycythemia Rubra Vera-1 gene in Essential Thrombocythemia. J Clin Oncol 2002;20: ) Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16: ) Kopp P, Jaggi R, Tobler A. et al. Clonal X-inactivation analysis of human tumours using the human androgen receptor gene (HUMARA) polymorphism: a non-radioactive and semiquantitative strategy applicable to fresh and archival tissue. Mol Cell Probes 1997;11:

7 14) Gilliland DG, Blanchard KL. Levy J, Perrin S, Bunn HF. Clonality in myeloproliferative disorders: analysis by means of the polymerase chain reaction. Proc Natl Acad Sci U S A. (HUMARA) on chromosome X. Nucl Acid Res 1994,22: ) Liu Y, Phelan J, Go R C P, Prchal J F, Prchal J. Rapid determination of clonality by detection of two closely-linked X chromosome exonic polymorphisms using allele- specific PCR. J Clin Invest 1997; 99: ) Bellanné-Chantelot C, Chaumarel I, et al. Genetic and clinical implications of the Val617Phe Jak2 mutation in 72 families with myeloproliferative disorders. Blood e-pub march 14, ) Dror Y, Zipursly A, Blanchette VS. Essential thrombocythemia in children. J Ped Hematol Oncol 1999;21: ) 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: ) Gale RE, Fielding AK, Harrison C, Linch DC. Acquired skewing of X-chromosome inactivation patterns in myeloid cells of the elderly suggests stochastic clonal loss with age. Br J Hematol 1997;98: ) Wolanskyj AP, Lasho TL, Schwager SM, et al. JAK2 V617F mutation in essential thrombocythemia: clinical associations and long-term prognostic relevance. Br J Hematol 2005;131: ) Campbell PJ, Scott LM, Buck G, et al. Definition of subtypes of essential thromboythemia and relation to polycythemia vera based on JAK2 V617F mutation status: a prospective study. Lancet 2005;366: ) Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cells 2005;7: ) Kiladjian JJ, Elkassar N, Cassinat B, et al. Essential thrombocythemia without V617F JAK2 mutation are clonal hematopoietic stem cell disorders. Leukemia e-pub April 6, ) Randi ML, Putti MC. Essential thrombocythemia in children: is a treatment needed? Expert Opin Pharmacother 2004;5:

8 Table 1: Main clinical findings of children with essential thrombocythemia. They were followed in 7 different pediatric units. The median clinical follow up was 7.5 years (range 18 months 24 years). The age of patients at diagnosis and at the moment of the analyses are reported. Treatments were chosen by the referring physicians, upon local criteria. Patient n 1 underwent orthotopic liver transplantation (OLT) at 7 years of age for Budd Chiari syndrome presented at diagnosis. Patient n 12 was initially treated with interferon alpha (IFN) with partially transitory response and then shifted to low dose aspirin (ASA). Patients n 16 and 20 were treated with Anagrelide (ANAGR) for only 6 months each. No relation between age and molecular findings are recognizable. patient n /sex Clonal/JAK2 Age at diagnosis (years) Age of analysis (years) Signs and symptoms 1/F Yes/V617F 0,8 9 Budd Chiari syndrome & inferior vena cava thrombosis Treatment OLT + warfarin 2/F No/WT 2 2 none none 3/F No/WT 3 14 none ASA 4/F No/WT 4 6 none none 5/M WT 4 15 none ANAGR 6/F No/WT 6 7 Epistaxis, headache ASA 7/M WT 6 7 none ANAGR 8/M V617F 6 7 headache ASA 9/F Yes/WT 7 8 none ASA + ANAGR 10/F No/WT 7 11 headache ANAGR 11/F No/WT 8 9 none ANAGR 12/F No/WT 8 20 headache IFN / ASA 13/F No/WT 8 31 none ASA 14/M V617F 9 15 none ANAGR 15/F No/WT headache ANAGR 16/F No/WT none ASA 17/M WT headache ASA 18/F Yes/WT none none 19/F No/WT headache ASA 20/F Yes/V617F none ANAGR

9 Table 2: Results of molecular studies in 20 children with ET, compared to 47 consecutive adult patients. Correlation of clonality pattern, JAK2 mutation and thrombotic complications is explored. Clonality JAK2 Major thrombotic complications CHILDREN 4 monoclonal 2 V617F 1 Budd Chiari (29% of informative cases) 2 WT 15 females 10 polyclonal (71% of informative 10 WT cases) 1/15 skewing 1 WT 5 males 2 V617F 3 WT Total: 20 children 4 V617F (20%) & 16 WT 1/20 ADULTS 9 monoclonal 3 V617F 1 cerebral vein, 1 Budd-Chiari (45% of informative cases) 6 WT 11 polyclonal 7 V617F 1 cerebral vein, 1 Budd-Chiari 36 females (55% of informative cases) 4 WT 9 skewing 7 V617F 1 peripheral artery, 1 DVT (25% of total ) 2 WT 7 homozygous 3 V617F 1 Budd-Chiari (19% of total) 4 WT 1 portal vein 11 males 8 V617F 2 Budd-Chiari, 1 MI 3 WT 1 cerebral vein Total: 47 adults 28 (60%) V617F & 19 WT 12/47 V617F= JAK2 mutation, WT = wild type MI= myocardial infarction, DVT= deep vein thrombosis

10 Prepublished online July 18, 2006; doi: /blood Pediatric patients with essential thrombocythemia are mostly polyclonal and V617FJAK2 negative Maria L Randi, Maria C Putti, Margherita Scapin, Enrica Pacquola, Fabio Tucci, Concetta Micalizzi, Luigi Zanesco and Fabrizio Fabris Information about reproducing this article in parts or in its entirety may be found online at: Information about ordering reprints may be found online at: Information about subscriptions and ASH membership may be found online at: Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in the paper journal (edited, typeset versions may be posted when available prior to final publication). Advance online articles are citable and establish publication priority; they are indexed by PubMed from initial publication. Citations to Advance online articles must include digital object identifier (DOIs) and date of initial publication. Blood (print ISSN , online ISSN ), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Copyright 2011 by The American Society of Hematology; all rights reserved.