ORIGINAL ARTICLE. A Tefferi 1, TL Lasho 1, J Huang 1, C Finke 1, RA Mesa 1,CYLi 2,WWu 3, CA Hanson 2 and A Pardanani 1

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1 (28) 22, & 28 Nature Publishing Group All rights reserved /8 $3. ORIGINAL ARTICLE, compared to either a higher allele burden or unmutated status, is associated with inferior overall and leukemia-free survival A Tefferi, TL Lasho, J Huang, C Finke, RA Mesa,CYLi 2,WWu 3, CA Hanson 2 and A Pardanani Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA; 2 Division of Hematopathology, Department of Laboratory Medicine, Mayo Clinic, Rochester, MN, USA and 3 Division of Health Sciences Research, Department of Biostatistics, Mayo Clinic, Rochester, MN, USA The clinical relevance of JAK2V67F allele burden in primary myelofibrosis (PMF) has not been previously studied. Bone marrow-derived DNA from 99 patients with PMF was subjected to qualitative (n ¼ 99) and quantitative (n ¼ 29) analysis for V67F. Mutational frequency was 58% and median mutant allele burden ratio in V67F-positive patients was 29% (range, 74%). Multivariable analysis identified older age, platelet count X 9 l and peripheral blood blast percentage o3% as being associated with a positive mutational status. The mere presence of the mutation did not affect the incidence of thrombosis (P ¼.78), overall survival (P ¼.22) or leukemiafree survival (P ¼.5). The 29 patients with allele burden information were divided into four groups: V67F-negative (n ¼ 53) and V67F-positive with mutant allele burden in the lower quartile (n ¼ 9), middle quartiles (n ¼ 38) or upper quartile (n ¼ 9) range. Kaplan Meier plots revealed significantly shortened overall (P ¼.8) and leukemia-free (P ¼.) survival for the lower quartile, but not for upper quartile allele burden group; independent prognostic relevance was validated by multivariable analysis. We conclude that low V67F allele burden in PMF might indicate the presence of an overriding V67F-negative clone that confers a more aggressive disease phenotype. (28) 22, ; doi:.38/sj.leu.24597; published online 24 January 28 Keywords: JAK2; V67F; JAK2V67F; myelofibrosis; prognosis Introduction JAK2V67F is arguably the most significant recent discovery regarding the molecular basis of BCR-ABL-negative myeloproliferative neoplasms. The mutation is myeloid neoplasm specific and its frequency is estimated at above 9% in polycythemia vera (PV); 2 in the range of 3 7% in essential thrombocythemia (ET), 3 primary myelofibrosis (PMF), 4 or refractory anemia with ringed sideroblasts and marked thrombocytosis; 5 and less than 5% in myelodysplastic syndrome 6 or de novo acute myeloid leukemia. 7 Two other JAK-STAT-relevant mutations have recently been described in patients with JAK2V67F-negative PV (JAK2 exon 2 mutations) 8 or PMF/ET (MPLW55L/K). 9 As a result, it is currently touted that all patients with PV and the majority with either ET or PMF carry a JAK2 or related mutation; JAK2V67F screening has become an essential diagnostic test in PV 2 and mutational Correspondence: Professor A Tefferi, Division of Hematology, Department of Medicine, Mayo Clinic, 2 First Street, SW, Rochester, MN 5595, USA. tefferi.ayalew@mayo.edu Received 27 November 27; revised 6 December 27; accepted 8 December 27; published online 24 January 28 status is now formally incorporated into the revised World Health Organization diagnostic criteria for PV, ET and PMF. 3 Several studies have also looked into clinical correlates and/or prognostic relevance of JAK2V67F presence or its allele burden in ET, PV and PMF. In ET, the presence of JAK2V67F has been associated with advanced age, higher hemoglobin level, increased leukocyte count, decreased platelet count and venous thrombosis. 4 Furthermore, in mutation-positive patients with ET, a Mayo Clinic study has demonstrated a direct correlation between bone marrow mutant allele burden and both leukocyte and platelet count. 5 Similar results were obtained by a French group. 6 The aforementioned study from the Mayo Clinic also showed a higher prevalence of palpable splenomegaly and venous thrombosis in patients with a higher mutant allele burden. More importantly, however, overall survival (OS) or transformation rates to myelofibrosis or acute myeloid leukemia were not affected by either the presence of the mutation or its allele burden. 5 In another ET study from the Italian Group for Malignant Hematologic Disorders (GIMEMA), JAK2V67F homozygous patients, compared to those with a wild-type or heterozygous mutational status, displayed older age, higher leukocyte count, higher hemoglobin level, larger spleen size and higher incidences of thrombosis and fibrotic transformation. 7 In PV, the first study that compared JAK2V67F homozygous and heterozygous patients associated the former with higher hemoglobin level, presence of pruritus and higher rate of fibrotic transformation. 2 A subsequent study by GIMEMA confirmed these findings and also demonstrated a higher leukocyte count and lower platelet count in homozygous patients. 7 The same Italian group also studied the clinical relevance of JAK2V67F allele burden measured by quantitative PCR-based assay using granulocyte-derived DNA; a high mutant allele burden correlated with higher hemoglobin level and leukocyte count, larger spleen size, presence of pruritus and an increased propensity to require chemotherapy or experience cardiovascular events. 8 The direct correlation of allele burden with leukocyte count and hemoglobin level was also demonstrated by a French study. 6 Our own PV study using bone marrow-derived DNA for allele burden measurement also found a correlation with leukocyte count and pruritus, but not with hemoglobin level, spleen size, thrombotic complications or need for cytoreductive therapy. 9 Clinical differences between JAK2V67F-positive and -negative patients have also been examined in PMF. The first study in this regard showed an association between the presence of the mutation and an older age at diagnosis, history of thrombosis and pruritus. 4 This particular study did not identify JAK2V67F mutational status as an independent prognostic factor for survival. However, another multicenter study from Europe

2 Table Presenting features between JAK2V67F-positive and -negative cases among 99 patients with primary myelofibrosis 757 Clinical parameters All patients (n ¼ 99) JAK2V67F-positive cases (n ¼ 5) JAK2V67F-negative cases (n ¼ 84) Age (years; median, range) 6 (28 82) 64 (28 82) 56 (34 79).7 Female sex (%) 79 (4) 45 (39) 34 (4).85 Hemoglobin level (median and range; g per ml).2 ( ).4 ( ) ( ) 9 Leukocyte count (median and range; 9 l ) 8.4 ( ) 8.9 (.8 6) 7.2 ( ).6 Platelet count (median and range; 9 l ) 266 (2 96) 249 (2 96) 325 (3 955).2 Monocyte count (median and range; 9 l ).36 ( 5.9).34 ( 3.2) ( 5.9) 3 PB blast percentage (median and range) (.5) ( 5) (.5).34 No. of patients with hemoglobin o g per ml (%) 54 (27) 32 (28) 22 (26).8 No. of patients with leukocyte count o4 or 39 (2) 2 (8) 8 (2) l (%) No. of patients with platelet count o 9 l (%) 3 (6) 3 () 8 (2).5 No. of patients with AMC X 9 l (%) 25 (3) 5 (3) (2).82 No. of patients with PB blasts X% (%) 74 (37) 4 (36) 33 (39).6 No. of patients with PB blasts X3% (%) 3 (7) 2 (.7) (3).4 No. of patients with hypercatabolic symptoms 54 (27) 33 (29) 2 (25).56 No. of patients with Dupriez high/intermediate/low PSS (%) 2/63/5 (6/32/7) 69/39/7 (6/34/6) 52/24/8 (62/29/9).54 Abbreviations: AMC, absolute monocyte count; PB, peripheral blood; PSS, prognostic scoring system. Mutation analysis was performed by allele-specific PCR assay. Bold values indicate significance Po.5. reported inferior survival in JAK2V67F-positive patients with PMF; the presence of the mutation was also associated with higher leukocyte count. 2 A more recent GIMEMA study found that JAK2V67F homozygous patients with PMF displayed a higher leukocyte count, larger spleen size and were more likely to experience pruritus, leukemic transformation and require chemotherapy or splenectomy. 2 In addition, mutation-positive, as opposed to mutation-negative, patients displayed higher hemoglobin level, whereas a significantly higher platelet count was seen only in heterozygous patients. In the current study, we sought to clarify the discrepant observations among the aforementioned PMF studies as well as address, for the first time, the clinical and prognostic relevance of quantitatively measured JAK2V67F allele burden in PMF. Materials and methods Diagnosis of PMF was based on World Health Organization criteria 22 and study eligibility included the availability of bone marrow-derived DNA that was collected either at time of diagnosis or within one year of diagnosis but prior to any therapeutic intervention. Approval was obtained from the Mayo Clinic Institutional Review Board prior to reviewing medical records. Clinical and laboratory data, including bone marrow histology, were re-reviewed to confirm PMF diagnosis. Patients with post-pv/et myelofibrosis were excluded. Leukemic transformation was considered in the presence of blast phase PMF, which was defined according to the criteria by the International Working Group for Myelofibrosis Research and Treatment: the presence of either 2% blasts in the bone marrow or 3% in the peripheral blood. 23 Quantitative 24 and/or qualitative 25 allele-specific PCR was utilized to screen for V67F and measure mutant allele burden in DNA derived from unfractionated bone marrow aspirates. Assay sensitivity in both instances was between.5 and %. Quantitative PCR amplification and detection were performed on an ABI Prism 79 HT Analyzer (Applied Biosystems, Foster City, CA, USA). Mutant allele burden was reported as the percentage of total JAK2 represented by JAK2V67F (that is JAK2V67F/JAK2V67F þ JAK2 wild type). This JAK2V67F percentage was obtained from a standard curve for JAK2V67F/total JAK2 against DC t (C tjak2v67f C tjak2wt ), which was constructed using various proportions of genomic DNA from the cell lines K562 (homozygous for wild-type JAK2) and HEL (homozygous for JAK2V67F). Two microliters of each standard mixture (25 ng ml ) and each sample (25 ng ml ) were aliquoted into separate wells of the 96-well plate. Standards and samples were run in triplicate and the mean DC t for each standard mixture and sample was used to plot the JAK2V67F/ JAK2 total percentage. As illustrated in Table, all parameters used for statistical analysis were those obtained at the time of diagnosis and before any therapeutic intervention. Conspicuous of its absence from this list was cytogenetic information at diagnosis, which was available in very few patients and not always represented by an adequate number of metaphases. Follow-up information was obtained from medical records and telephone calls were made or letters sent to patients and/or their physicians in those patients whose last follow-up visit at the Mayo Clinic exceeded 6 months. Statistical procedures utilized were conventional and all data were analyzed by using StatView (SAS Institute, Cary, NC, USA). All s were two-tailed and statistical significance was set at the level of Po.5. Continuous variables were summarized as medians and ranges. Categorical variables were described as count and relative frequency (%). Comparison between categorical variables was performed by w 2 statistics. Linear regression analysis was used for testing associations between continuous variables. Comparison between categorical and continuous variables was performed by either the Mann Whitney U-test or Kruskal Wallis test. The association of variables selected from univariate analysis was explored using logistic regression models. OS and leukemia-free survival (LFS) curves were constructed by Kaplan Meier method taking the interval from the date of diagnosis to death or last contact for OS or to time of leukemic transformation or last contact/death for LFS. Log-rank test was used to test the homogeneity of survival curves over different groups. Cox proportional hazards model was utilized to determine the impact of various clinical and laboratory variables on OS or LFS.

3 758 Results A total of 99 patients (6% men; median age 6 years) met the above stipulated criteria for inclusion in the study and were suitable for analysis of comparisons between V67F-positive and -negative disease. JAK2V67F was detected in 5 cases for a mutational frequency of 58%. Several clinical and laboratory parameters in these 5 patients were compared with those of the remaining 84 (42%) patients who were JAK2V67Fnegative. The results are outlined in Tables and 2. analysis of variables at presentation identified older age (P ¼.7), platelet count X 9 l (P ¼.5) and PB blast percentage o3% (P ¼.4) as being associated with a positive mutational status; all three variables sustained their significance during multivariate analysis. In contrast, the borderline significance attached to leukocyte count was lost during multivariable analysis. As summarized in Table 2, the rates of disease complications, including thrombohemorrhagic events, the need for chemotherapy or splenectomy and leukemic transformation were not significantly affected by the presence of JAK2V67F. Accordingly, both OS (Figure ) and LFS (Figure 2) were similar between mutation-positive and -negative cases. The amount of stored DNA was adequate enough to perform quantitative measurement of V67F allele burden in 29 of the total 99 patients; the Dupriez prognostic scoring system stratification at time of diagnosis among the 29 patients in whom allele burden data were available compared to the remaining 7 patients without such information was not statistically different (P ¼.38). The 29 patients with allele burden information were subsequently divided into four groups: V67F negative (n ¼ 53) and V67F positive with mutant allele burden in the lower quartile (n ¼ 9), middle quartile (n ¼ 38) or upper quartile (n ¼ 9) ranges (median and range of V67F allele burden ratio are listed in Table 3). As is evident in Tables 3 and 4, comparison across all four groups revealed significant associations between lower quartile allele burden and older age (P ¼.4), upper quartile allele burden and higher leukocyte count (P ¼.7) and higher occurrence rate for circulating blastsx3% in patients with wild-type mutational status (P ¼.). When the same series of analyses was performed only in mutation-positive patients, only the association between upper quartile allele burden and higher leukocyte count was sustained (P ¼.3), although age maintained a borderline significance (P ¼.). The only other significant association that became evident in the latter cascade of analysis restricted to mutation-positive patients was that of upper quartile allele burden and fewer reports of constitutional symptoms (P ¼.5). In multivariable analysis, the significant association of upper quartile allele burden with younger age and higher leukocyte count was sustained. Kaplan Meier plots revealed significantly shortened OS (P ¼.8; Figure 3) and LFS (P ¼.; Figure 4) associated with lower quartile allele burden. Cox analysis disclosed that Survival V67F-negative; n = 84 V67F-positive; n = 5 P = Figure Overall survival comparison between 5 and 84 JAK2V67F-negative cases among 99 patients with primary myelofibrosis. Survival V67F-positive; n = 5 AML events = 7 V67F-negative; n = 84 AML events = P = Figure 2 -free survival comparison between 5 JAK2V67F-positive and 84 JAK2V67F-negative cases among 99 patients with primary myelofibrosis. AML, acute myeloid leukemia. Table 2 Clinical course and outcome of JAK2V67F-positive and JAK2V67F-negative cases among 99 patients with primary myelofibrosis Clinical course or outcome All patients (n ¼ 99) JAK2V67F-positive cases (n ¼ 5) JAK2V67F-negative cases (n ¼ 84) Median follow-up (months; range) 23 ( 266) 23 ( 265) 25 (. 266) 4 No. of patients with any treatment (%) 44 (72) 88 (77) 56 (67).3 No. of patients with cytoreductive therapy (%) 82 (4) 49 (43) 33 (39).64 No. of patients with splenectomy (%) 3 (5) 6 (4) 4 (7).6 No. of patients with thrombosis (5) 8 (7) 2 (2.4).5 No. of patients with bleeding 9 (4.5) 4 (3.5) 5 (6) No. of patients with AML (%) 7 (8.5) 7 (6) (2).5 No. of patients who have died (%) 57 (29) 34 (3) 23 (27).74 Median survival in months (Kaplan Meier estimates) Abbreviation: AML, acute myeloid leukaemia. Mutation analysis was performed by allele-specific PCR assay.

4 Table 3 Presenting features among JAK2V67F-negative and JAK2V67F-positive cases with lower, middle and upper quartile mutant allele burden ranges among 29 patients with primary myelofibrosis 759 Clinical parameters JAK2V67F negative (n ¼ 53) lower quartile (n ¼ 9) middle quartiles (n ¼ 38) upper quartile (n ¼ 9) V67F allele burden (%; median, range) NA 2% ( 2) 29% (2 55) 68% (56 74) N/A Age (years, median, range) 57 (38 79) 7 (44 77) 66 (38 82) 56 (39 75).4 Female sex (%) 22 (42) 8 (42) 2 (32) 7 (37).78 Hemoglobin level (median and range; g per ml).8 ( ).2 ( ).4 ( ).5 ( ).5 Leukocyte count (median and range; 9 l ) 6.6 (2. 57) 6.3 ( ) 8.6 (.8 3.2) 4.6 ( ).7 Platelet count (median and range; 9 l ) 27 (3 785) 24 (4 66) 242 (2 96) 33 (64 637).5 Monocyte count (median and range; 9 l ) (.2 5.9).3 (.) (. 3.2) ( 2.6).52 PB blast percentage (median and range) (.5) ( 4.) ( 2.) (.).8 No. of patients with hemoglobin o g per ml (%) 5 (28) 5 (26) (26) 7 (37).86 No. of patients with leukocyte count o4 or 4 (26) 5 (26) 5 (3) 4 (2) l (%) No. of patients with platelet count o 9 l (%) 6 (3) 4 (2) 5 (3) 2 ().5 No. of patients with AMC X 9 l (%) (9) (5) 7 (8) 3 (6).56 No. of patients with PB blasts X% (%) 22 (42) 6 (32) 7 (45) 7 (37).79 No. of patients with PB blasts X3% (%) 9 (7) (5) () (). No. of patients with hypercatabolic symptoms (%) 7 (32) 9 (47) 2 (32) 2 (). No. of patients with Dupriez high/intermediate/low PSS (%) 29/9/5 (55/36/9) /6/2 (57/32/) 24/3/ (63/34/3) /7/2 (52/37/).9 Abbreviations: AMC, arthrogryposis multiplex congenita; PB, peripheral blood; PSS, prognostic scoring system. Mutation analysis was performed by allele-specific, real-time quantitative PCR assay. Bold values indicate significance Po.5. Table 4 Clinical course and outcome among JAK2V67F-negative and JAK2V67F-positive cases with lower, middle and upper quartile mutant allele burden ranges among 29 patients with primary myelofibrosis Clinical course or outcome JAK2V67F negative (n ¼ 53) lower quartile (n ¼ 9) middle quartiles (n ¼ 38) upper quartile (n ¼ 9) Median follow-up (months; range) 2 (. 62) 4 (.5 6) 24 (.3 35) 39 (.3 32).23 No. of patients with any treatment (%) 39 (74) 8 (95) 3 (79) 3 (68).2 No. of patients with cytoreductive therapy (%) 24 (45) 7 (37) 6 (42) (53).8 No. of patients with splenectomy (%) (2) 4 (2) 6 (6) 5 (26).82 No. of patients with thrombosis (%) 2 (4) 2 () 3 (8) 2 ().65 No. of patients with bleeding (%) 5 (9.4) (5) 2 (5) ().5 No. of patients with AML (%) 6 () 3 (6) (3) (5).29 No. of patients who have died (%) 6 (3) 9 (47) 2 (32) 4 (2).36 Median survival in months (Kaplan Meier P ¼.8 estimates) Abbreviation: AML, acute myeloid leukaemia. Mutation analysis was performed by allele-specific (AS), real-time quantitative PCR assay..8 P =.8 Survival.6 Upper quartile allele burden n = 9.2 Lower quartile allele burden n = 9 V67F-negative; n = 53 Middle quartiles allele burden n = Figure 3 quartiles. Overall survival comparison among 29 patients with primary myelofibrosis stratified into wild-type and JAK2V67F allele burden

5 76 P =. -free Survival Lower quartile allele burden n = 9 V67F-negative n = 53 Middle quartiles allele burden n = 38 Upper quartile allele burden n = Figure 4 -free survival comparison among 29 patients with primary myelofibrosis stratified into wild-type and JAK2V67F allele burden quartiles. the association between lower quartile allele burden and inferior OS was sustained during multivariable analysis that included either age or each one of the three frequently used prognostic scoring systems for PMF (that is, Dupriez, Cervantes and Mayo prognostic scoring systems) were included as covariates. The independent prognostic value of lower quartile allele burden was further validated by entering in the multivariate model each one of the individual risk factors considered in the aforementioned prognostic scoring systems: hemoglobin o g ml, platelet count o 9 l, leukocyte count 43 or o4 9 l, absolute monocyte count of X 9 l, circulating blast percentage X and the presence of hypercatabolic (that is, constitutional) symptoms. Similarly, the significant association related to LFS was independent of recently described other risk factors for leukemic transformation in PMF, including platelet count o 9 l and circulating blast percentage X3. 26 The general observations stated above regarding the clinical correlates and prognostic relevance of JAK2V67F allele burden remained mostly unaltered when analysis was restricted to mutation-positive cases only. Discussion The most remarkable finding in the current study was the significantly shortened OS and LFS associated with a low JAK2V67F allele burden that was not accounted for by either other PMF-relevant risk factors or the older age range attached to the particular patient group. In contrast, the outcome of patients with higher mutant allele burden was not significantly different from that of JAK2V67F-negative cases. These observations are notably different from those recently communicated by GIMEMA, where JAK2V67F homozygosity, inferred from BsaXI restriction analysis, was associated with a more aggressive clinical phenotype. 2 In other words, unlike the GIMEMA study, we did not find higher rates of leukemic transformation, splenectomy or need for chemotherapy in our patients with high mutant allele burden. Although direct comparison of the two studies is not possible because of the different tissue sources and assay methods used for mutation screening, such methodological differences are less likely to account for the similarly discrepant results obtained from qualitative mutational stratification; the mere presence of the mutation, as opposed to its absence, did not carry any prognostic relevance in the current study. We submit that our key observation that links low JAK2V67F allele burden with aggressive disease phenotype requires validation by other studies. However, we are intrigued by the possibility that a low JAK2V67F allele burden is a surrogate for the coexistence of a more dominant JAK2V67F-negative clone with a higher propensity to undergo clonal evolution. Such a contention is supported by increasing evidence that point to JAK2V67F as a secondary event in the clonal hierarchy of myeloproliferative neoplasms 27 and is consistent with the development of mutation-negative leukemic transformation in mutation-positive patients. 28 Also in support of the scenario for competing clones, we have previously shown the concurrent occurrence of JAK2V67F and MPLW55L/K in some patients with PMF 29 and clonal dominance by the latter in such an instance. 3 Obviously, more studies are needed to clarify the issue further and it is prudent, in the meantime, to abstain from making definitive conclusions regarding the clinical implications of JAK2V67F mutational status or allele burden in PMF. Also, additional insight into the subject matter is expected from currently ongoing JAK2 inhibitor treatment trials. 3 References Levine RL, Pardanani A, Tefferi A, Gilliland DG. Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders. Nat Rev Cancer 27; 7: Tefferi A, Lasho TL, Schwager SM, Strand JS, Elliott M, Mesa R et al. The clinical phenotype of wild-type, heterozygous, and homozygous JAK2V67F in polycythemia vera. Cancer 26; 6: Antonioli E, Guglielmelli P, Pancrazzi A, Bogani C, Verrucci M, Ponziani V et al. Clinical implications of the JAK2 V67F mutation in essential thrombocythemia. 25; 9: Tefferi A, Lasho TL, Schwager SM, Steensma DP, Mesa RA, Li CY et al. The JAK2 tyrosine kinase mutation in myelofibrosis with myeloid metaplasia: lineage specificity and clinical correlates. Br J Haematol 25; 3: Wang SA, Hasserjian RP, Loew JM, Sechman EV, Jones D, Hao S et al. Refractory anemia with ringed sideroblasts associated with marked thrombocytosis harbors JAK2 mutation and shows over-

6 lapping myeloproliferative and myelodysplastic features. 26; 2: Steensma DP, Dewald GW, Lasho TL, Powell HL, McClure RF, Levine RL et al. The JAK2 V67F activating tyrosine kinase mutation is an infrequent event in both atypical myeloproliferative disorders and myelodysplastic syndromes. Blood 25; 6: Steensma DP, McClure RF, Karp JE, Tefferi A, Lasho TL, Powell HL et al. JAK2 V67F is a rare finding in de novo acute myeloid leukemia, but STAT3 activation is common and remains unexplained. 26; 2: Scott LM, Tong W, Levine RL, Scott MA, Beer PA, Stratton MR et al. JAK2 exon 2 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med 27; 356: Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M et al. MPLW55L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med 26; 3: e27. Pardanani A, Lasho TL, Finke C, Hanson CA, Tefferi A. 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Stem Cells 27; 25: Campbell PJ, Baxter EJ, Beer PA, Scott LM, Bench AJ, Huntly BJP et al. Mutation of JAK2 in the myeloproliferative disorders: timing, clonality studies, cytogenetic associations, and role in leukemic transformation. Blood 26; 8: Pardanani AD, Levine RL, Lasho T, Pikman Y, Mesa RA, Wadleigh M et al. MPL55 mutations in myeloproliferative and other myeloid disorders: a study of 82 patients. Blood 26; 8: Lasho TL, Pardanani A, McClure RF, Mesa RA, Levine RL, Gary Gilliland D et al. Concurrent MPL55 and JAK2V67F mutations in myelofibrosis: chronology of clonal emergence and changes in mutant allele burden over time. Br J Haematol 26; 35: Pardanani A, Hood J, Lasho T, Levine RL, Martin MB, Noronha G et al. TG29, a small molecule JAK2-selective kinase inhibitor potently inhibits myeloproliferative disorder-associated JAK2V67F and MPLW55L/K mutations. 27; 2: