Incidence and risk factors for myelofibrotic ... - Wiley Online Library

RESEARCH ARTICLE

A JH

Incidence and risk factors for myelofibrotic transformation among 272 chinese patients with JAK2-mutated polycythemia vera Jie Bai,1 Limei Ai,2 Lei Zhang,1 Feng-Chun Yang,3 Yuan Zhou,1* and Yanping Xue1* Post-polycythemia vera myelofibrosis (post-PV MF) is a critical hematologic evolution of polycythemia vera (PV). The main purpose of the present study was to identify the possible risk factors for the occurrence and prognosis of post-PV MF in Chinese patients with PV. A cohort of 272 Chinese PV patients with JAK2V617F or exon12 mutation was retrospectively analyzed. Of the 272 patients with PV, 63 developed post-PV MF. Platelet count >550 3 109/L and splenomegaly were identified as independent risk factors for post-PV MF. The median duration of survival for post-PV MF patients was 8 years. Anemia and age >65 years at diagnosis of post-PV MF were identified as significant predictors for the poor prognosis of post-PV MF. In conclusion, platelet counts and splenomegaly were significant predictors for the transformation to post-PV MF, while anemia (hemoglobin levels 65 years were significant predictors for poor prognosis of post-PV MF in Chinese PV patients with JAK2V617F or exon12 mutation. C 2015 Wiley Periodicals, Inc. Am. J. Hematol. 90:1116–1121, 2015. V

䊏 Introduction Polycythemia vera (PV) is a chronic progressive myeloproliferative neoplasm (MPN) characterized by pancytosis, especially the overproduction of red blood cells (RBCs), and therefore often associated with thrombocytosis and leukocytosis. It is estimated that PV will evolve into lifethreatening myelofibrosis (post-PV MF) in up to 20% of patients [1–3]. Passamonti et al. reported that leukocytosis at diagnosis of PV was a risk factor for the progression of PV to post-PV MF [2]. However, the risk factors remain unclear in Chinese patients with PV. The survival of PV patients declines considerably after evolution to post-PV MF, and the reported median survival of patients with post-PV MF varies widely [2,3]. Unfortunately, no generally accepted predictive model is currently available to stratify the risk factors in patients with post-PV MF, and treatment decisions are primarily based on the International Prognostic Scoring System (IPSS) or Dynamic International Prognostic Scoring System (DIPSS) for primary MF (PMF) [4–6]. Passamonti et al. developed a dynamic prognostic model to predict the survival of patients with PV who developed post-PV MF based on hemoglobin level, platelet count, and leukocyte count, which was useful for predicting the progression of PV to post-PV MF [2]. This model has been recently updated based on age >65 years, time to secondary MF >15 years, previous thrombosis, constitutional symptoms, hemoglobin 18 years; and (3) available for follow-up. The clinical data for all patients were confirmed by three or more physicians before the patients were included in the study. Cytogenetic examination. Chromosomes from aspirated bone marrow (BM) were prepared with conventional techniques. When evaluable metaphases could not be obtained in direct preparations, a 48-hr marrow culture was also studied. Chromosome identification was performed after photographic enlargement, and karyotypes were classified according to the International System for Human Cytogenetic [10]. A clone was defined as at least two cells with the same additional numerical and/or structural abnormality or three cells with a loss of the same chromosome. Cytogenetic examinations were performed in 158 PV patients. None of these patients received myelosuppressive therapy before the first examination. Analysis of JAK2 mutation V617F and exon 12. Routine genetic testing for JAK2 mutation V617F was performed. Peripheral blood granulocytes of patients were separated by Ficoll-Paque gradient centrifugation, followed by hypotonic lysis to remove contaminating red cells. Cell pellets were preserved for future use at 2208C. DNA was extracted, and JAK2 mutation V617F was detected at diagnosis with nested allele-specific polymerase chain reaction (PCR) as described previously [11]. Samples with low DNA content were pre-amplified and 1.0 lL of product was amplified by nested PCR. If the results were positive for JAK2 mutation V617F, DNA was re-extracted to rule out any possible sampling error. JAK2V617F allele burden (V617F %) was further determined by quantitative real-time PCR using primers and probes as previously described [12]. If the JAK2 mutation V617F were negative, PCR amplification of JAK2 exon12 is performed by amplifying the whole exon 12 of JAK2 using exon 12 primers (forward: 50 -CTCCTCTTTGGAGCAATTCA-30 ; reverse: 50 -GAGAACTTGGGAGTTGCGATA-30 ), followed by Sanger sequencing. Statistical analysis. Overall survival (OS) of PV or post-PV MF was defined as the time from diagnosis of PV or post-PV MF to death or was censored at the last follow-up. MF-free survival (MFS) was defined as the time from diagnosis of PV to the occurrence of post-PV MF or death, or was censored at the last follow-up. Leukemia-free survival in patients with post-PV MF was defined as the time from diagnosis of post-PV MF to the occurrence of leukemia or death, or was censored at the last follow-up. Standardized mortality ratios (SMRs) were calculated by dividing the number of observed deaths in the study cohort by the number of expected deaths in the age-matched, sex-matched, and calendar year-matched general Chinese population. Receiver-operating characteristics (ROC) curves were used to determine the cutoff levels of continuous variables of interest [3,13]. Hazard ratios (HRs) were used to measure the relative risk over time. Statistical analyses were performed using Fisher’s exact test for categorical variables and independent sample t-test or Mann–Whitney U test for continuous variables. Kaplan–Meier univariate and multivariate Cox analyses were performed to identify the prognostic factors for the OS of patients at the diagnosis of PV and postPV MF. Univariate analyses and multivariate Cox regression analyses were further applied to identify the risk factors for the occurrence of post-PV MF, based on the clinical characteristics at diagnosis of PV, including age, sex, splenomegaly, platelet count, WBC count, V617F%, and abnormal karyotypes in all 272 patients. Kaplan– Meier analyses and multivariate Cox regression analyses were also performed in 90 PV patients who were carried out JAK2V617F allele burden analysis to determine the effect of V617F% on the myelofibrotic transformation. All data were analyzed using SPSS 21.0 (IBM, Armonk, NY, USA). To determine the precision of the results, 95% confidence intervals (95% CIs) for the variables of interest were calculated. Two-sided P values 0.05 were considered statistically significant.

䊏 Results Clinical characteristics at presentation and overall outcome Two hundred and seventy-two JAK2-mutated PV patients (147 males and 125 females) with a median age of 54 years (range: 27–83 years) were followed up for a median average of 6 years (range: 1–31 years). Their clinical characteristics at presentation are presented in Table I. The 10-year, 15-year, and 20-year OS was 86.3%, 75.3%, and 57.1%, respectively. Importantly, OS was significantly lower in patients with PV than in the general Chinese population matched by age, sex, and calendar-year, with an SMR of 7.896 (95% CI: 5.745– 10.851, P < 0.001, Supporting Information Fig. S1A). The incidence of thrombosis, post-PV MF, and acute myeloid leukemia (AML) was 82.8/1000 (95% CI: 69.2–98.9), 31.39/1000 (95% CI: 24.12–40.16), and 6.56/1000 (95% CI: 3.67–10.82) person/year, respectively. doi:10.1002/ajh.24191

Incidence and risk factors for post-polycythemia vera myelofibrosis TABLE I. Clinical characteristics at diagnosis of PV and events during the clinical course of 272 patients with JAK2-mutated PV

Characteristics and events Age at diagnosis (years) Male/female Median follow-up (years) Leukemic transformations Post-PV MF WBC count (3109/L) Hemoglobin level (g/L) Platelet count (3109/L) Abnormal karyotype (n 5 158) Causes of death (n 5 38) Acute leukemia Thrombotic complications Second malignancies Multiple organ failure Infection Bleeding Unknown Thrombosis Arterial thrombosis Venous thrombosis Major hemorrhage Aspirin therapy Cytoreductive drug IFNa alone Hydroxyurea alone Alkylating agent

Case number (%) or median (range) 54 147/125 6 15 63 13.60 200 420 19 14 10 1 7 1 3 1 120 104 16 16 218 253 92 150 11

(27–83) (54.0%/46.0%) (1–31) (5.5%) (23.2%) (4.09–43.80) (165–260) (78–1493) (12.0%) (36.8%) (26.3%) (2.6%) (21.2%) (2.6%) (7.9%) (2.6%) (44.1%) (38.2%) (5.9%) (5.9%) (80.0%) (93.0%) (33.8%) (55.1%) (4.0%)

Post-PV MF, post-polycythemia vera myelofibrosis; WBC, white blood cell; IFN-a, interferon-a.

Univariate and multivariate analyses of the prognosis factors for the OS of patients with PV were performed, including platelet count, age, sex, splenomegaly, thrombosis, abnormal karyotype, and V617F%. The significant parameters are presented in Supporting Information Table S1. Multivariate analysis showed that WBC count >25 3 109/L (HR 5 5.496, 95% CI: 1.509–20.015, P 5 0.010), thrombosis (HR 5 4.109, 95% CI: 1.039–16.252, P 5 0.044) and patient age >65 years at diagnosis of PV (HR 5 5.734, 95% CI: 1.210–27.178, P 5 0.028) were independent risk factors for poor survival in patients with JAK2-mutated PV. During the course of PV, 120 (44.1%) patients experienced 164 thrombotic events. Among them, 27(22.5%) patients suffered from twice or more embolism, 15 (12.5%) suffered two or more sites of thrombosis. Ischemic strokes and myocardial infarction accounted for 52.8% and 16.9%, respectively. Eighty-three (69.2%) patients experienced thrombosis before or at the diagnosis of PV. Major hemorrhage was observed in 16 (5.9%) patients, and gastrointestinal bleeding and cerebral hemorrhage accounted for 31.3% and 25% among them, respectively (Supporting Information Table S2). Karyotype analyses were carried out in 158 patients with JAK2-mutated PV. The cytogenetic results and the clinical events including myelofibrotic and leukemic transformation in 19 patients with abnormal karyotype are shown in Supporting Information Table S3.

Post-PV MF transformation In the present study, 63 of the 272 patients with PV (23.16%) developed post-PV MF, with 10-year, 15-year, and 20-year incidence of post-PV MF transformation of 27.4%, 39.9%, and 61.6%, respectively. The clinical characteristics of patients with post-PV MF are summarized in Table II. OS was also significantly lower in patients with post-PV MF than in the general Chinese population matched by age, sex, and calendar year, with an SMR of 18.78 (range: 12.879– 27.386, P < 0.001) (Supporting Information Fig. S1B).

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Supporting Information Table S4 presented the univariate and multivariate analyses of the risk factors for post-PV MF transformation. Univariate analyses showed that splenomegaly (P < 0.001), WBC count >13 3 109/L (P < 0.001), platelet count >550 3 109/L (P < 0.001), age >61 years (P 5 0.044), V617F% 50% (P 5 0.005), and abnormal karyotype (P 5 0.016) were associated with the transformation to post-PV MF. Multivariate analysis revealed that splenomegaly (HR 5 6.087, 95% CI: 1.603–28.911, P 5 0.009) and platelet count >550 3 109/L (HR 5 2.335, 95% CI: 1.385–4.936, P 5 0.001) were independent risk factors for post-PV MF transformation. Patients with WBC count >13 3 109/L had a tendency to be associated with the transformation to post-PV MF (HR 5 2.045, 95% CI: 0.896–4.668, P 5 0.089).

Correlation of disease duration and JAK2V617F allele burden V617F% was examined in 90 patients with adequate DNA samples. Correlation analysis revealed that high V617F% (V617% 50%) was strongly associated with elevated WBC count (P 5 0.034), thrombocytosis (P 5 0.010), incidence of thrombosis (P 5 0.032), and postPV MF (P 5 0.018), but was not related to age (P 5 0.273) and sex (P 5 0.676). The median V617F% was 96.99% and 43.80% for patients with and without post-PV MF, respectively (P < 0.01, Fig. 1A). Kaplan–Meier analysis showed that MFS was significantly lower in PV patients with V617F% 50% than in patients with V617F% 65 years old (P 5 0.007) were associated with poor prognosis of post-PV MF, while circulating blast cells 1% only showed a tendency to be associated (P 5 0.082). Multivariate analysis showed that anemia (hemoglobin level 65 years at diagnosis of post-PV MF (HR 5 2.778, 95% CI: 1.153– 6.689, P 5 0.023) were risk factors for poor survival in patients with post-PV MF. Patients with these two risk factors had the lowest survival rates, with a 5-year survival rate of only 17.3% and a median survival of only 3 years (range: 1.587–4.413 years). In contrast, none patients without these two risk factors died during the follow-up. The cumulative survival rate of patients with one of these two risk factors was intermediate, and the prognosis was worse in patients with anemia than in patients aged >65 years (Fig. 2A). The 63 post-PV MF patients were also grouped into four groups according to the IPSS or DIPSS criteria for PMF: low risk, intermediate-1 risk, intermediate-2 risk, and high risk, respectively. In IPSS criteria-based grouping, there was no significant difference in survival between intermediate-2 risk and high-risk patients, but low-

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TABLE II. Demographic and hematologic characteristics of 63 patients with post-PV MF

Characteristics and events

Case number (%) or median (range)

Age at diagnosis, years 60 (32–85) Male/Female 39/24 (61.9%/38.1%) 9 WBC count (310 /L) 17.95 (2.55–66.91) Hemoglobin level (g/L) 120 (30–148) Platelet count (3109/L) 350 (14–1185) IWG-MRT required criteria 1. Previous diagnosis of polycythemia 63 (100%) vera (WHO criteria) 2. Bone marrow fibrosis grade 2–3 (on 0–3 scale) 63 (100%) IWG-MRT additional criteria (2 are required) a 1. Anemia 39 (61.9%) or sustained loss of requirement of 24 (38.1%) phlebotomy or cytoreduction 2. Leukoerythroblastic PB picture 47 (74.6%) 3. Increasing splenomegalyb 63 (100%) 4. Development of more than one of the 26 (41.9%) constitutional symptoms IPSS Low 8 (12.7%) Intermediate-1 12 (19.0%) Intermediate-2 16 (25.4%) High 27(42.9%) DIPSS Low 8 (12.7%) Intermediate-1 9 (14.3%) Intermediate-2 22 (34.9%) High 24 (38.1%) post-PV MF, post-polycythemia vera myelofibrosis; IWG-MRT, international working group for myelofibrosis research and treatment; IPSS, International Prognostic Scoring System for PMF; DIPSS, Dynamic International Prognostic Scoring System for PMF. a Defined as hemoglobin value 15 3 109/L) at diagnosis has been reported to be a risk factor for the evolution of post-PV MF [2]. In the present study, leukocytosis at diagnosis of PV was identified as a


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significant risk factor for post-PV MF in the univariate analysis, but the result was insignificant in the multivariate analysis. In the present study, platelet count >550 3 109/L at diagnosis of PV was also identified as a significant risk factor for post-PV MF. Platelet-derived growth factor, basic fibroblast growth factor-2, and vascular endothelial growth factor secreted by megakaryocytes are involved in the pathogenesis of MF by altering the hematopoietic microenvironment, perpetuating BM fibrosis, and increasing BM microvessel density [21]. Barbui et al. also reported that patients with masked PV (a subclinical manifestation of PV) had higher platelet counts, increased incidence of post-PV MF, and poorer prognosis [22]. Therefore, the increase in megakaryocytes and thrombocytosis might contribute to the progression of post-PV MF, and it is not surprising that patients with platelet counts >550 3 109/L are at a higher risk for post-PV MF transformation. A previous study showed that the median survival was 5.7 years with an SMR of 6.5 in Italian patients with PV who developed postPV MF [2]. However, in the present study, the median survival of Chinese patients with post-PV MF was 8 years (range: 4.13–11.87 years) with an SMR of 18.78. This could be attributed to the fact that transformation into post-PV MF occurs at an earlier age and a higher incidence in Chinese patients than in Western patients. Cervantes et al. developed a prognostic scoring system for PMF based on the study of International Working Group for Myelofibrosis Research and Treatment [4], in which age >65 years, presence of constitutional symptoms, hemoglobin levels 25 3 109/L, and circulating blast cells 1.0% were identified as predictors of shortened survival. Passamonti et al. developed a dynamic prognostic model to predict the survival of European patients with post-PV MF, which confers a higher prognostic power to anemia (hemoglobin levels 65 years, time to secondary MF >15 years, previous thrombosis, constitutional symptoms, hemoglobin 65 years and hemoglobin level