Calreticulin Mutations and Long-Term Survival in Essential Thrombocythemia

ORIGINAL ARTICLE Calreticulin mutations and long-term survival in essential thrombocythemia

A Tefferi1, EA Wassie1, TL Lasho1, C Finke1, AA Belachew1, RP Ketterling2, CA Hanson3, A Pardanani1, N Gangat1 and AP Wolanskyj1

The impact of calreticulin (CALR) mutations on long-term survival in essential thrombocythemia (ET) was examined in 299 patients whose diagnosis predated 2006. Mutational frequencies were 53% for Janus kinase 2 (JAK2), 32% for CALR and 3% for MPL; the remaining 12% were ‘triple-negative’. We confirmed the association of mutant CALR (vs JAK2V617F) with younger age (P ¼ 0.002), male sex (P ¼ 0.01), higher platelet count (0.0004), lower hemoglobin (Po0.0001), lower leukocyte count (0.02) and lower incidence of recurrent thrombosis (0.04). Triple-negative patients were also younger than their JAK2-mutated counterparts (P ¼ 0.003) and displayed lower hemoglobin (P ¼ 0.003), lower leukocyte count (o0.0001) and lower thrombotic events (P ¼ 0.02). Median follow- up time was 12.7 years and 47% of the patients were followed until death. Survival was the longest for triple-negative and shortest for MPL-mutated patients. Median survival was 19 years for JAK2 and 20 years for CALR-mutated cases (P ¼ 0.32); the corresponding figures for patients of age p65 years were 26 and 32 years (P ¼ 0.56). The two mutational categories were also similar for leukemic (P ¼ 0.28) and fibrotic (P ¼ 0.28) progression rates. The current study is uniquely characterized by its very long follow-up period and provides accurate estimates of long-term survival in ET and complements current information on mutation-specific phenotype and prognosis.

Leukemia (2014) 28, 2300–2303; doi:10.1038/leu.2014.148

INTRODUCTION MATERIALS AND METHODS Janus kinase 2 (JAK2), calreticulin (CALR) and myeloproliferative The current study was approved by the Mayo Clinic institutional review leukemia virus oncogene (MPL) mutations are recurrent and board. to obtain mature survival data, we queried an institutional database of patients with ET, whose diagnosis predated 2006 (diagnosis period was mutually exclusive in myeloproliferative neoplasms; their reported 10 frequencies were 95–100%, 0 and 0% for polycythemia vera (PV), between 1956 and 2005). From this database, we identified consecutive 59–64%, 15–24% and 4% for essential thrombocythemia (ET) and patients (the current study population) in who archived bone marrow or peripheral blood DNA was available. All bone marrow specimens as well as 53–58%, 25–35% and 6–8% for primary myelofibrosis (PMF), 1–5 cytogenetic studies were either performed or reviewed at our institution to respectively. In ET, mutant CALR (vs JAK2V617F) was associated ensure accuracy of diagnosis. Patients who received a diagnosis of ET before with younger age, male sex, higher platelet count, lower the formal publications of the World Health Organization (WHO) criteria 1–4 hemoglobin level and lower leukocyte count. In PMF, CALR- were re-evaluated retrospectively and included in the current study only if mutated patients were also younger and presented with higher they also fulfilled the 2001 or 2008 WHO criteria for ET.11,12 Special care was platelet count, lower risk scores as per the dynamic international undertaken to avoid inadvertent inclusion of patients with either ‘prefibrotic’ prognostic scoring system (DIPSS)-plus6 and lower frequencies of PMF or PV.13 The occurrence of leukemic or fibrotic disease progression was anemia, leukocytosis and spliceosome mutations.5 annotated by the treating physician and reviewed by the authors, 11,14 CALR mutations have been associated with superior thrombosis- wherever possible, for compliance to formal criteria. Treatment free survival in ET1–4 and overall survival in PMF;5 the latter was approaches reflected the individual physician’s best clinical judgment and independent of both ASXL1 mutational status7 and DIPSS-plus.6,8 included anti-platelet and cytoreductive agents, such as hydroxyurea, anagrelide, interferon alpha, busulfan, chlorambucil and radiophosphorus Mutational status according to both CALR and ASXL1 and the (P-32). Follow-up information was updated in February 2014. number of prognostically-detrimental mutations have recently 8,9 Previously published methods were used for CALR, JAK2 and MPL been shown to enable robust molecular prognostication in PMF. mutation analysis.5 All statistical analyses considered clinical and laboratory In contrast, the observations regarding CALR mutation impact on parameters obtained at the time of diagnosis. Differences in the distribution overall survival in ET were inconclusive owing to inadequate of continuous variables between categories were analyzed by either follow-up time (median 5.2–6 years)3,4 and low number of events Mann–Whitney (for comparison of two groups) or Kruskal–Wallis (12.1%).3 The particular scenario also creates overly optimistic (for comparison of three or more groups) test. Patient groups with nominal 2 survival estimates because of excess early censoring. In the current variables were compared by w -test. Overall survival analysis was considered study, we sought to address these issues by querying an from the date of diagnosis to the date of death (uncensored) or last contact institutional database of patients with ET, whose diagnosis (censored). Leukemia-free survival calculation considered leukemic transformation as the uncensored variable. Survival curves were prepared by the predated 2006, thus ensuring a minimum follow-up time of 8 Kaplan–Meier method and compared by the log-rank test. Cox proportional years; the main objectives were to provide accurate estimates of hazard regression model was used for multivariable analysis. P-values less long-term survival in ET and clarify the survival impact of CALR than 0.05 were considered significant. The Stat View (SAS Institute, Cary, NC, mutations in such a setting. USA) statistical package was used for all calculations.

1Division of Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA; 2Division of Cytogenetics, Laboratory Medicine, Mayo Clinic, Rochester, MN, USA and 3Division of Hematopathology, Laboratory Medicine, Mayo Clinic, Rochester, MN, USA. Correspondence: Professor A Tefferi, Division of Hematology, Department of Internal Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. E-mail: [email protected] Received 10 March 2014; revised 9 April 2014; accepted 11 April 2014; accepted article preview online 5 May 2014; advance online publication, 30 May 2014 CALR mutations and survival in ET A Tefferi et al 2301 RESULTS thrombosis was documented in 70 patients and was less frequent A total of 299 patients fulfilled the above-stipulated criteria for in CALR-mutated (P ¼ 0.04) and triple-negative (P ¼ 0.02) patients, inclusion in the current study. Clinical and laboratory features at compared to JAK2-mutated cases. Overall, survival was the longest presentation are outlined in Table 1. Median age at diagnosis in triple-negative patients (median not reached) and shortest in was 56 years (range 15–91) and women constituted 60% of the MPL-mutated cases (median 9 years) (P ¼ 0.007; Figure 2). patients. Median hemoglobin, leukocyte and platelet counts However, the differences were no longer significant when adjusted were 13.9 g/dl, 9.6 10(9)/l and 1000 10(9)/l, respectively. for age (P ¼ 0.42). Median survival was 19 years for JAK2 and 20 Palpable splenomegaly, mostly described as ‘tip palpable’ was years for CALR-mutated cases (P ¼ 0.32); the corresponding figures recorded in 68 (23%) patients. Thrombosis history before or at for patients of age p65 years were 26 and 32 years (P ¼ 0.56; the time of diagnosis was obtained in 54 (18%) patients. Twenty Figure 3). The two mutational categories were also similar for (6.7%) patients presented with major bleeding. Microcirculatory leukemic (P ¼ 0.28) and fibrotic (P ¼ 0.28) progression rates. symptoms were mentioned by 53 (18%) patients. Risk stratifica- Multivariable analysis that included age confirmed the lack of tion per the international prognostic scoring system for ET mutation effect on survival and instead confirmed the prognostic 15 (IPSET)15 were low in 109 (36%) patients, intermediate in 119 relevance of IPSET (Figure 4). (40%) and high in 71 (24%). Cytogenetic information was available in 218 patients and showed abnormal results in 19 (8.7%). DISCUSSION Mutational frequencies were 53% for JAK2, 32% for CALR and Considering the fact that JAK2, CALR and MPL mutations can be 3% for MPL. Approximately 13% of the patients were triple- present or absent in otherwise morphologically-defined ET, it is negative. Figure 1 lists all the CALR mutation variants seen; type 1 important to clarify their impact on disease characteristics and and type 2 mutations constituted 48 and 35% of all CALR-mutated patient outcome. The current study confirms previous observa- cases. As outlined in Table 1, we confirmed mutation-specific tions regarding the phenotypic differences between JAK2- differences in several clinical and laboratory characteristics at mutated, CALR-mutated and triple-negative patients with ET.3,4 presentation. Compared to JAK2V617F, CALR mutations were We show that CALR-mutated and triple-negative ET patients were associated with younger age (P ¼ 0.002), male sex (P ¼ 0.01), significantly younger than their JAK2-mutated counterparts, which higher platelet count (0.0004), lower hemoglobin level underscores the need to adjust for age when comparing overall (Po0.0001), lower leukocyte count (0.02) and lower incidence of and thrombosis-free survival. These two mutational categories microcirculatory symptoms (0.005). Triple-negative patients were were also similar in terms of displaying lower hemoglobin levels also younger than their JAK2-mutated counterparts (P ¼ 0.003) and leukocyte counts, compared with JAK2-mutated cases, but and displayed lower hemoglobin (P ¼ 0.003), lower leukocyte differed in their platelet counts and sex distribution. These count (o0.0001) and lower thrombotic events (P ¼ 0.02). observations suggest differences in underlying biological effects Compared to JAK2-mutated cases, CALR-mutated and triple-negative from mutant JAK2, which promotes erythroid and granulocyte patients also presented with significantly lower IPSET scores proliferation, and mutant CALR, which is currently believed to (P ¼ 0.04). There was no difference between JAK2-mutated and promote megakaryocytic proliferation or platelet production. triple-negative cases in terms of sex distribution (P ¼ 0.34), platelet In one of the first two seminal papers on CALR mutations in count (P ¼ 0.13) and incidence of microcirculatory symptoms myeloproliferative neoplasms,1 a survival advantage for CALR vs (P ¼ 0.13). The small number of informative cases did not justify JAK2-mutated patients was suggested for both ET and PMF.1 This MPL mutation comparisons. was subsequently confirmed in PMF5 but not in ET.3,4 However, Median follow-up time was 12.7 years. During this period, 141 the observations in ET were inconclusive because of inadequate (47%) deaths were documented and transformation rates for AML, follow-up time.3,4 The current study addresses the latter issue and MF and PV were 5.4, 8 and 5%, respectively (Table 1). Recurrent confirms the similarity of long-term survival between JAK2- and

Table 1. Clinical and laboratory features of 299 patients with essential thrombocythemia stratiﬁed by the presence or absence of CALR, JAK2 and MPL mutations

Variables All patients JAK2-mutated CALR mutated Mpl mutated Triple-negative P-value (n ¼ 299) (n ¼ 159; 53%) (n ¼ 95; 32%) (n ¼ 8; 3%) (n ¼ 37; 12%)

Age in years; median (range) 56 (15–91) 59 (16–88) 47 (15–91) 66 (57–85) 42 (16–81) 0.0003 Age465 years; n (%) 102 (34.1%) 64 (42.3%) 26 (27.4%) 4 (50%) 8 (21.6%) 0.04 Females (%) 179 (59.9%) 103 (64.8%) 46 (48.4%) 3 (37.5%) 27 (73%) 0.01 Hemoglobin, g/dl; median (range) 13.9 (6.9–17.9) 14.3 (9.8–17.9) 13.3 (6.9–16.4) 12.9 (9.0–15.8) 13.0 (8.4–15.9) o0.0001 Leukocytes, 109/l; median (range) 9.6 (2.8–53.4) 10.0 (3.9–53.4) 8.6 (3.3–32.6) 7.0 (4.0–17.7) 7.2 (2.8–12.6) o0.0001 Platelets, 109/l; median (range) 1000 (454–3460) 960 (500–3000) 1082 (454–3460) 969 (685–2249) 1000 (557–3300) 0.005 Leukocytes X11 109/l; n (%) 100 (33.4) 62 (39%) 30 (31.6%) 2 (25%) 6 (16.2%) 0.04 Platelets 41000 109/l; n (%) 157 (52.5%) 71 (44.6%) 62 (65.2%) 4 (50%) 20 (54.1%) 0.02 Abnormal karyotype 19/218 (9%) 13/126 (10%) 5/56 (9%) 1/7 (14%) 0/29 (0%) 0.33 Palpable splenomegaly 68 (23%) 35 (22%) 24 (25%) 2 (25%) 7 (19%) 0.87 Major hemorrhage 20 (7%) 9 (6%) 7 (7.4%) 2 (25%) 2 (5.4%) 0.19 Microcirculatory symptoms 53 (18%) 33 (21%) 7 (7.4%) 1 (12.5%) 12 (32%) 0.004 Pre-diagnosis thrombosis 54 (18%) 31 (20%) 12 (13%) 2 (25%) 9 (24%) 0.34 Post-diagnosis thrombosis 71 (24%) 42 (26%) 17 (18%) 3 (38%) 4 (11%) 0.03 Median follow-up in years (range) 12.7 (0.1–43.6) 12.7 (0.6–43.6) 14.2 (1.6–34.1) 8.9 (6.7–21) 11.1 (0.1–36) 0.47 Deaths 141 (47%) 82 (52%) 42 (44%) 7 (88%) 10 (27%) 0.005 Leukemic conversions 18 (6%) 8 (5%) 8 (8.4%) 2 (25%) 0 (0%) 0.03 Fibrotic progression 28 (9.4%) 12 (7.5%) 11 (12%) 3 (38%) 2 (5.4%) 0.03 Polycythemic progression 8 (3%) 8 (5%) 0 (0%) 0 (0%) 0 (0%) 0.06 Bolded values indicate signiﬁcant differences.

& 2014 Macmillan Publishers Limited Leukemia (2014) 2300 – 2303 CALR mutations and survival in ET A Tefferi et al 2302

Figure 1. Calreticulin (CALR) mutation variants among 95 calreticulin-mutated patients with ET.

1 CALR mutated N=95 Median ~20 years 0.8 Triple-negative N=37 Not reached 0.6

JAK2 mutated N=159 Survival 0.4 Median ~19 years

MPL mutated 0.2 N=8 Median ~ 9 years P=0.007

0 0 5 10 15 20 25 30 35 40 45 Years Figure 2. Kaplan–Meier estimates of overall survival in 299 patients with ET stratiﬁed by mutation type.

CALR mutated and age 65 years 1 N=70 Median ~32 years

0.8

0.6 JAK2 mutated and age 65 yearsy N=98 Median ~26 years

Survival 0.4

JAK2 mutated and age >65 years N=61 0.2 Median ~12 years P=0.95 CALR mutated and age >65 years N=25 0 Median ~9 years 0 5 10 15 20 25 30 35 40 45 Years Figure 3. Kaplan–Meier estimates of overall survival in 254 JAK2- (n ¼ 159) or CALR-mutated patients (n ¼ 95) with ET stratiﬁed by age. Leukemia (2014) 2300 – 2303 & 2014 Macmillan Publishers Limited CALR mutations and survival in ET A Tefferi et al 2303 IPSET low 1 N=109 Median ~32 years

0.8

IPSET 0.6 intermediate N=119 Median ~18 years

Survival 0.4

IPSET high N=71 0.2 Median ~10 years P<0.0001

0 0 5 10 15 20 25 30 35 40 45 Years Figure 4. Kaplan–Meier estimates of overall survival in 299 patients with ET stratiﬁed by the IPSET: 2 points for patients of age X60 years and 1 point each for history of thrombosis or leukocyte count X11 10(9)/l: Low-risk, zero points; intermediate-risk, one or two points; high-risk, 3 or more points.

CALR-mutated ETs, in both young and older patients. The study 3 Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L et al. Impact also illustrates the similar natural history between JAK2- and CALR- of calreticulin mutations on clinical and hematological phenotype and outcome mutated ETs in terms of transformation rates into AML or MF. As in essential thrombocythemia. Blood 2013; 123: 1552–1555. expected and not surprisingly, evolution into PV occurred only in 4 Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, Milosevic JD et al. JAK2 JAK2-mutated cases but the rate of transformation was only 5% or CALR mutation status defines subtypes of essential thrombocythemia with and not anywhere near the 29% projected at 15 years, in a recent substantially different clinical course and outcomes. Blood 2013; 123: 1544–1551. study.4 This suggests inadvertent inclusion of early cases of PV in 5 Tefferi A, Lasho TL, Finke CM, Knudson RA, Ketterling R, Hanson CH et al. CALR vs the particular study and does not support the notion that JAK2- JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia 2014; 28: 1472–1477. mutated ET and PV are different phenotypes of a single myeloid 6 Gangat N, Caramazza D, Vaidya R, George G, Begna K, Schwager S et al. DIPSS neoplasm. plus: a refined Dynamic International Prognostic Scoring System for primary The uniquely long follow-up time in the current study allowed myelofibrosis that incorporates prognostic information from karyotype, platelet accurate estimation of median survival in ET, which wasB26 years count, and transfusion status. J Clin Oncol 2011; 29: 392–397. in patients younger than age 66 years. Moreover, the significantly 7 Vannucchi AM, Lasho TL, Guglielmelli P, Biamonte F, Pardanani A, Pereira A et al. shorter survival in older patients (B10 years) was probably more Mutations and prognosis in primary myelofibrosis. Leukemia 2013; 27: of a reflection of their advanced age rather than their disease. Also 1861–1869. in the current study, despite the long follow-up time, we were 8 Tefferi A, Guglielmelli P, Lasho TL, Rotunno G, Finke C, Mannarelli C et al. CALR unable to show differences in fibrotic or leukemic transformation and ASXL1 mutations-based molecular prognostication in primary myelofibrosis: an international study of 570 patients. Leukemia 2014; 28: 1494–1500. rates. Incidentally, we did notice the apparently higher rates of 9 Guglielmelli P, Lasho TL, Rotunno G, Score J, Mannarelli C, Pancrazzi A et al. mortality and disease transformation in our MPL-mutated patients The number of prognostically detrimental mutations and prognosis in (Table 1), but the low number of cases did not allow additional primary myelofibrosis: an international study of 797 patients. Leukemia 2014; 28: comments in this regard, although the particular observation 1804–1810. warrants further investigation. 10 Gangat N, Wolanskyj AP, McClure RF, Li CY, Schwager S, Wu W et al. Risk stratification for survival and leukemic transformation in essential thrombocythemia: a single institutional study of 605 patients. Leukemia 2007; 21: 270–276. CONFLICT OF INTEREST 11 Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood 2002; 100: 2292–2302. The authors declare no conflict of interest. 12 Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid ACKNOWLEDGEMENTS neoplasms and acute leukemia: rationale and important changes. Blood 2009; 114: 937–951. This study was supported by the Mayo Clinic Harvey-Yulman Charitable Foundation 13 Kvasnicka HM, Thiele J. Prodromal myeloproliferative neoplasms: the 2008 WHO for Myelofibrosis Tissue Bank and the Clinical Database of Molecular and Biological classification. Am J Hematol 2010; 85: 62–69. Abnormalities. 14 Barosi G, Mesa RA, Thiele J, Cervantes F, Campbell PJ, Verstovsek S et al. Proposed criteria for the diagnosis of post-polycythemia vera and post-essential thrombocythemia myelofibrosis: a consensus statement from the International REFERENCES Working Group for Myelofibrosis Research and Treatment. Leukemia 2008; 22: 1 Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD et al. 437–438. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 15 Passamonti F, Thiele J, Girodon F, Rumi E, Carobbio A, Gisslinger H et al. 2013; 369: 2379–2390. A prognostic model to predict survival in 867 World Health Organization- 2 Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al. Somatic defined essential thrombocythemia at diagnosis: a study by the International CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. NEnglJ Working Group on Myelofibrosis Research and Treatment. Blood 2012; 120: Med 2013; 369: 2391–2405. 1197–1201.

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