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were observed in 11 of the 118 (9%) V617F positive and 5 of the 73 (7%) V617F negative patients (Table 2). .... 46,XX,inv(9)(p12;q13)[30]. ET7. Positive. 46,XX ...
CORRESPONDENCE JAK2 V617F mutation in classic chronic myeloproliferative diseases: a report on a series of 349 patients TO THE EDITOR Several groups have recently reported the presence of a unique gain-of-function JAK2 point mutation (V617F) in the majority of cases of classic cMPD as well as in a minority of atypical MPDs and in occasional cases of AML. To date, this mutation has not been reported in normal controls, lymphoid disorders or in patients with secondary erythrocytosis (1-10). In this study, we have analyzed bone marrow or peripheral blood samples from 349 patients (PV, n = 84; ET, n = 243; IMF, n = 22) referred to our diagnostic service for cytogenetic analysis and/or exclusion of the BCR-ABL fusion from several hospitals in the north of Spain between 1996 and 2005. Our study group thus includes the largest series of ET cases reported to date for JAK2 analysis. DNA was extracted and tested for V617F by a sensitive amplification refractory mutation system (ARMS) as described previously (6). Results were obtained from all samples and V617F was detected in 80%, 62% and 68% of cases of PV, ET and IMF, respectively (Table 1). These frequencies are comparable to other studies for PV and IMF, but somewhat higher than expected for ET, for which 23-57% of cases have been reported as V617F positive. Part of the reason for this is probably attributable to technical differences in the sensitivity of the V617F detection. Most other studies used sequencing, which can underestimate the proportion of positive patients (2,6), whereas allele-specific PCR assay is capable of detecting a mutation present in 1-2% of cells (6). Of the 17 V617F negative PV cases our series, only two were female (P=0.044, contingency χ2=4.058). This supports the previous observation of a significant male excess in mutation negative PV cases (6). In addition a relative risk ratio estimate (RR=1.26; confidence interval 95%=1.04-1.52) of our data indicated that V617F is 26% more frequent in PV females compared to PV males (Table 1). PV and IMF are both considered to be clonal diseases, but for ET only approximately half of cases are clonal, the remainder being polyclonal and therefore presumably reactive. It has been suggested (6) that the great majority of clonal ET cases must be V617F positive, but it remains to be determined if any V617F ET cases are also clonal. To address this question, we compared karyotypes between mutation negative and mutation positive cases. Of the 191 ET cases for whom cytogenetic analysis had been successfully performed, clonal cytogenetic abnormalities

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were observed in 11 of the 118 (9%) V617F positive and 5 of the 73 (7%) V617F negative patients (Table 2). Although the frequency of chromosome abnormalities is lower in V617F negative ET cases, these data show that at least some of these cases are indeed clonal. Karyotypes were also available for 79 of the 85 PV patients (V617F positive, n = 62; V617F negative, n = 17) and 19 of the 22 IMF cases (V617F positive, n = 12; V617F negative, n = 7) (Table 2). For PV, 11 V617F positive patients had cytogenetic abnormalities, of which three showed del(20q), four showed +9 and four had -Y or +8. A single V617F negative PV patient was karyotypically abnormal with del(20q). For IMF patients, all of V617F positive cases had a normal karyotype whereas two of the negative V617F patients showed aberrations (Table 2). Thus, on the basis of cytogenetics, it is clear that some V617F negative cMPD cases have a clonal disease, suggesting the existence of uncharacterised mutation/s that may or may not lie in the same signal transduction pathway. Currently, it is not clear if JAK2 V617F is the initiating, disease causing mutation or an important secondary event. However our finding of known recurrent cytogenetic abnormalities in both V617F positive and negative cases supports the notion that the development of cMPDs is a multistep pathogenetic process.

Acknowledgements We thank Virginia Cebrián, Iñigo Landa and Beatriz Sánchez for technical assistance. Funding Supported by grant 05/2003 from the Department of Health of the Government of Navarra, Spain; and by grant FIS 04/0037 from Instituto de Salud Carlos III. References 1. James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, Garcon L, Raslova H, Berger R, Bennaceur-Griscelli A, Villeval JL, Constantinescu SN, Casadevall N, Vainchenker W. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005; 434: 1144-1148. 2. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, Vassiliou GS, Bench AJ, Boyd EM, Curtin N, Scott MA, Erber WN, Green AR; Cancer Genome Project. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054-1061.

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3. Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, Boggon TJ, Wlodarska I, Clark JJ, Moore S, Adelsperger J, Koo S, Lee JC, Gabriel S, Mercher T, D'Andrea A, Frohling S, Dohner K, Marynen P, Vandenberghe P, Mesa RA, Tefferi A, Griffin JD, Eck MJ, Sellers WR, Meyerson M, Golub TR, Lee SJ, Gilliland DG. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387-397. 4. Zhao R, Xing S, Li Z, Fu X, Li Q, Krantz SB, Li Z, Zhao ZJ. Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 2005; 280: 22788-22792. 5. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli A, Cazzola M, Skoda RC. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779-1790. 6. Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L, Score J, Seear R, Chase AJ, Grand FH, White H, Zoi C, Loukopoulos D, Terpos E, Vervessou EC, Schultheis B, Emig M, Ernst T, Lengfelder E, Hehlmann R, Hochhaus A, Oscier D, Silver RT, Reiter A, Cross NC. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 2005; 106: 2162-2168. 7. Steensma DP, Dewald GW, Lasho TL, Powell HL, McClure RF, Levine RL, Gilliland DG, Tefferi A. The JAK2 V617F activating tyrosine kinase mutation is an infrequent event in both "atypical" myeloproliferative disorders and myelodysplastic syndromes. Blood 2005; 106: 1207-1209. 8. Jelinek J, Oki Y, Gharibyan V, Bueso-Ramos C, Prchal JT, Verstovsek S, Beran M, Estey E, Kantarjian HM, Issa JP. JAK2 mutation 1849G>T is rare in acute leukemias but can be found in CMML, Philadelphia-chromosome negative CML and megakaryocytic leukemia. Blood 2005; 106: 3370-3373. 9. Goerttler PS, Steimle C, Marz E, Johansson PL, Andreasson B, Griesshammer M, Gisslinger H, Heimpel H, Pahl HL. The Jak2V617F mutation, PRV-1 overexpression and EEC formation define a similar cohort of MPD patients. Blood 2005; 106: 28622864. 10. Wolanskyj AP, Lasho TL, Schwager SM, McClure RF, Wadleigh M, Lee SJ, Gilliland DG, Tefferi A. JAK2 mutation in essential thrombocythaemia: clinical associations and long-term prognostic relevance. Br J Haematol 2005; 131: 208-213.

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José L. Vizmanos1, Cristina Ormazábal1, María J. Larráyoz1, Nicholas C. P. Cross2, María J. Calasanz1 1

Department of Genetics, School of Science, University of Navarra, Pamplona, Spain

2

Wessex Regional Genetics Laboratory, Salisbury and Human Genetics Division, University of Southampton, Southampton, United Kingdom.

Correspondence: Prof. José L. Vizmanos, PhD. Department of Genetics, School of Science. University of Navarra. C/ Irunlarrea 1, E-31008 Pamplona, Navarra, Spain. Phone: international +34.948.425600. Fax: international +34.948.425649. E-mail: [email protected]

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Table 1. JAK2 V617F mutation frequencies (separated by gender) Sex

V617F JAK2 positive

V617F JAK2 negative

Male (n = 57)

42 (74%)

15 (26%)

Female (n = 27)

25 (93%)

2 (7 %)

Male (n = 130)

85 (65%)

45 (35%)

Female (n = 113)

65 (58%)

48 (42%)

Male (n = 15)

11 (73%)

4 (27%)

Female (n = 7)

4 (57%)

3 (43%)

PV patients (n = 84)

ET patients (n = 243)

IMF patients (n = 22)

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Table 2. Karyotypic abnormalities observed. The number of metaphases are between brackets. V617F JAK2 status

Karyotype

PV patients (n = 12/79) PV1

Negative

46,XY/46,XY,del(20)(q13)

PV2

Positive

46,XY[28]/47,XY,+9[2]

PV3

Positive

46,XY[4]/45,X,-Y[26]

PV4

Positive

46,XY[9]/46,XY,del(20)(q13)[21]

PV5

Positive

46,XY[3]/47,XY,+9[27]

PV6

Positive

46,XY[19]/45,X,-Y[11]

PV7

Positive

46,XY[28]/47,XY,+9[2]

PV8

Positive

46,XY[2]/47,XY,+9[36]

PV9

Positive

46,XY[13]/46,XY,del(20)(q12)[17]

PV10

Positive

47,XY,+8[30]

PV11

Positive

45,X,-Y[30]

PV12

Positive

46,XY,del(20)(q12)[30]

ET patients (n = 16/191) ET1

Positive

46,XX[14]/45,XX,-5,del(9)(q22?),1dmin[16]

ET 2

Positive

46,XX,del(5)(q14q22)[31]

ET3

Negative

46,XY[4]/45,XY,-7[31]

ET4

Positive

46,XY[28]/47,XY,+8[2]

ET5

Positive

46,XX[17]/46,XX,+der(15)t(1;15)(q12;p13),-15[13]

ET6

Positive

46,XX,inv(9)(p12;q13)[30]

ET7

Positive

46,XX,del(20)(q3)[32]

ET8

Negative

46,X,-Y[30]

ET9

Positive

46,XX[21]/45,XX,-21[9]

ET10

Positive

46,XY[12]/46,XY,del(9)(q12q22)[23]

ET11

Positive

46,XY[24]/47,XY,+9[6]

ET12

Negative

46,X[29]/46,XY,del(5)(q23q33)[3]

ET13

Negative

46,XY[20]/47,XY,+8[15]

ET14

Positive

46,XY[24]/47,XY,+9[6]

ET15

Negative

46,XX [29]/47,XX,+12 [1]

ET16

Positive

47,XX,+9[30]

IMF patients (n = 2/22)

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IMF1

Negative

46,XY[7]/46,XY,del(1)(p33)[23]

IMF2

Negative

46,XX,t(12;12)(p11;q13)[30]

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