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JAK2V617F is the most recurrent genetic mutation in Philadelphia-negative chronic Myeloproliferative Neoplasms (MPNs). Since the JAK2 locus is located on Chromosome 9, we hypothesized that Chromosome 9 copy number abnormalities may be a disease modifier in JAK2V617F-mutant MPN patients. In this study, we identified a subset of MPN patients with partial or complete Chromosome 9 trisomy (+9p patients), who differ from JAK2V617F-homozygous MPN patients as they carry three JAK2 alleles as well as three copies of all neighboring gene loci, including CD274, encoding immunosuppressive Programmed death-ligand 1 (PD-L1) protein. Investigation of the clonal hierarchy revealed that the JAK2V617F occurs first, followed by +9p. Functionally, CD34+ cells from +9p MPN patients demonstrated increased clonogenicity, generating a greater number of primitive colonies, due to high OCT4 and NANOG expression, with knock-down of these genes leading to a genotype-specific decrease in colony numbers. Moreover, our analysis revealed increased PD-L1 surface expression in malignant monocytes from +9p patients, while analysis of the T cell compartment unveiled elevated levels of exhausted cytotoxic T cells. Overall, here we identify a distinct novel subgroup of MPN patients, who feature a synergistic interplay between +9p and JAK2V617F that shapes immune escape characteristics and increased stemness in CD34+ cells., (© 2024. The Author(s).)

The variant allele frequency (VAF) of driver mutations (JAK2, CALR) in myeloproliferative neoplasms is associated with features of advanced disease and complications. Ruxolitinib and interferon were reported to variably reduce the mutant VAF, but the long-term impact of molecular responses (MR) remains debated. We prospectively measured changes in JAK2 and CALR VAF in 77 patients with polycythemia vera and essential thrombocythemia, treated with ruxolitinib for a median of 8 years, and assessed correlation with complete clinical and hematological response (CCHR) and outcomes. At last observation time, JAK2 VAF reduced overall from a median of 68% (range, 20%-99%) to 3.5% (0%-98%). A profound and durable MR (DMR; defined as a VAF stably ≤2%), including complete MR in 8%, was achieved in 20% of the patients, a partial MR (PMR; VAF reduction >50% of the baseline level) in 25%, and 56% had no molecular response (NMR). A CCHR was reached by 69% overall, independently of any degree of MR achieved; conversely, a DMR correlated with longer duration of CCHR and, most importantly, with reduced rate of progression to myelofibrosis and with longer myelofibrosis-free, event-free and progression-free survival. Achievement of PMR also had some favorable impact on outcomes, compared to NMR. A baseline JAK2 VAF <50%, and a VAF reduction of ≥35% after 2 years of treatment, predicted for the achievement of DMR and reduced progression to myelofibrosis. Overall, these findings support the clinical value of achieving profound, durable MR and its consideration as surrogate endpoint in future clinical trials., (© 2024 Wiley Periodicals LLC.)

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal disorders complicated mainly by vascular events and transformation to myelofibrosis (for PV and ET) or leukemia. Although secondary malignancies, in particular, lymphoproliferative disorders (LPNs), are rare, they occur at a higher frequency than found in the general population, and there has been recent scientific discussion regarding a hypothetical relationship between treatment with JAK inhibitors in MPN and the risk of development of LPN. This has prompted increased interest regarding the coexistence of MPN and LPN. This review focuses on the role of JAK2 and the JAK/STAT pathway in MPN and LPN, whether there is a role for the genetic background in the occurrence of both MPN and LPN and whether there is a role for cytoreductive drugs in the occurrence of both MPN and LPN. Furthermore, whether an increased risk of lymphoma development is limited to patients who receive the JAK inhibitor ruxolitinib, is a more general phenomenon that occurs following JAK1/2 inhibition or is associated with preferential JAK1 or JAK2 targeting is discussed., (© 2019 John Wiley & Sons Ltd.)

The 2016 revision of the World Health Organization (WHO) classification of myeloproliferative neoplasms defines 2 stages of primary myelofibrosis (PMF): prefibrotic/early (pre-PMF) and overt fibrotic (overt PMF) phase. In this work, we studied the clinical and molecular features of patients belonging to these categories of PMF. The diagnosis of 661 PMF patients with a bone marrow biopsy at presentation was revised according to modern criteria; clinical information and annotation of somatic mutations in both driver and selected nondriver myeloid genes were available for all patients. Compared with pre-PMF, overt PMF was enriched in patients with anemia, thrombocytopenia, leukopenia, higher blast count, symptoms, large splenomegaly, and unfavorable karyotype. The different types of driver mutations were similarly distributed between the 2 categories, whereas selected mutations comprising the high mutation risk (HMR) category (any mutations in ASXL1 , SRSF2 , IDH1/2 , EZH2 ) were more represented in overt PMF. More patients with overt PMF were in higher International Prognostic Scoring System risk categories at diagnosis, and the frequency increased during follow-up, suggesting greater propensity to disease progression compared with pre-PMF. Median survival was significantly shortened in overt PMF (7.2 vs 17.6 years), with triple negativity for driver mutations and presence of HMR mutations representing independent predictors of unfavorable outcome. The findings of this "real-life" study indicate that adherence to 2016 WHO criteria allows for identification of 2 distinct categories of patients with PMF where increased grades of fibrosis are associated with more pronounced disease manifestations, adverse mutation profile, and worse outcome, overall suggesting they might represent a phenotypic continuum., (© 2017 by The American Society of Hematology.)

Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV-MF) and essential thrombocythemia (PET-MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV-MF and PET-MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV-MF and/or PET-MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET-MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET-MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV-MF and PET-MF. Am. J. Hematol. 91:681-686, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Vascular events in essential thrombocythemia (ET) are associated with advanced age and thrombosis history. Recent information suggests additional effect from the presence of specific mutations., Objectives: To examine the influence of age and thrombosis history on the reported association between mutational status and thrombosis-free survival in ET., Patients and Methods: Analysis was performed using a Mayo Clinic cohort of 300 ET patients, and key findings were reanalyzed by including additional 102 Italian patients., Results: Among 300 Mayo patients with ET (median age 55 yr, 60% females), mutational frequencies were 53% JAK2, 32% CALR, 3% MPL, and 12% JAK2, CALR and MPL wild type. One hundred and six (35%) patients experienced arterial (n = 75) or venous (n = 43) events, before (n = 55) or after (n = 71) diagnosis. In univariate analysis, compared to JAK2-mutated cases, JAK2, CALR and MPL wild type (HR 0.31, 95% CI 0.11-0.86), and CALR-mutated (0.53, 95% CI 0.30-0.92) patients displayed better thrombosis-free survival. JAK2, CALR, and MPL wild type remained significant (P = 0.03; HR 0.32, 95% CI 0.11-0.9) during multivariable analysis that included age (P = 0.01) and thrombosis history (P = 0.0006); a favorable impact from CALR mutations was of borderline significance (P = 0.1; HR 0.62, 95% CI 0.35-1.1), but became significant (P = 0.02) when multivariable analysis including thrombosis history (P = 0.02) was performed on patients younger than 60 yr of age., Conclusions: The favorable impact of mutational status on thrombosis-free survival in ET might be most evident for JAK2, CALR, and MPL wild type patients, whereas the favorable effect from CALR mutations might be confined to young patients., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

CALR (calreticulin) trails JAK2 as the second most mutated gene in essential thrombocythemia (ET). Mutant CALR in ET is a result of frameshift mutations, caused by exon 9 deletions or insertions; type-1, 52-bp deletion (p.L367fs*46), and type-2, 5-bp TTGTC insertion (p.K385fs*47) variants constitute more than 80% of these mutations. The current study includes a total of 1027 patients divided into test (n = 402) and validation (n = 625) cohorts. Among the 402 ET patients in the test cohort, 227 (57%) harbored JAK2, 11 (3%) Myeloproliferative leukemia virus oncogene (MPL), and 114 (28%) CALR mutations; 12% were wild-type for all three mutations (i.e., triple-negative). Among the 114 patients with CALR mutations, 51 (45%) displayed type-1 and 44 (39%) type-2 variants; compared to mutant JAK2, both variants were associated with higher platelet and lower hemoglobin and leukocyte counts. However, male sex was associated with only type-1 (P = 0.005) and younger age with type-2 (P = 0.001) variants. Notably, platelet count was significantly higher in type-2 vs. type-1 CALR-mutated patients (P = 0.03) and the particular observation was validated in the validation cohort that included 111 CALR-mutated ET patients (P = 0.002). These findings, coupled with the recent demonstration of preferential expression of mutant and wild-type CALR in megakaryocytes, suggest differential effects of CALR variants on thrombopoiesis., (© 2014 Wiley Periodicals, Inc.)

An association has been reported between a specific haplotype of the JAK2 gene, the homozygous 46/1 haplotype, and a predisposition to the development of chromosome Philadelphia-negative myeloproliferative neoplasms. Concerning myelofibrosis (MF), controversy remains on the relationship between the above JAK2 haplotype and the patients' clinicohematological features and survival. Among 132 patients with MF (60 % primary MF, 20 % postpolycythemia vera MF, 20 % post-essential thrombocythemia MF; 59 % JAK2V617F positive) who were analyzed for the JAK2 46/1 haplotype, 29 were found to be homozygous and 53 heterozygous. The homozygous 46/1 haplotype was more often observed in JAK2V617F-positive patients (29.5 versus 11 %, p = 0.012). Moreover, among JAK2V617F-positive patients, those who were homozygous for the 46/1 haplotype had a higher allele burden than the remainder (92 versus 48 %, p = 0.0017). Overall, patients with homozygous 46/1 haplotype showed significantly higher hemoglobin values and higher leukocyte counts, but no association was seen with other clinicohematological features. Finally, no relationship was observed between the JAK2 46/1 haplotype and either the patients' prognostic score or survival.

We genotyped 370 subjects with primary myelofibrosis (PMF) and 148 with postpolycythemia vera/postessential thrombocythemia (PPV/PET) MF for mutations of EZH2. Mutational status at diagnosis was correlated with hematologic parameters, clinical manifestations, and outcome. A total of 25 different EZH2 mutations were detected in 5.9% of PMF, 1.2% of PPV-MF, and 9.4% of PET-MF patients; most were exonic heterozygous missense changes. EZH2 mutation coexisted with JAK2V617F or ASXL1 mutation in 12 of 29 (41.4%) and 6 of 27 (22.2%) evaluated patients; TET2 and CBL mutations were found in 2 and 1 patients, respectively. EZH2-mutated PMF patients had significantly higher leukocyte counts, blast-cell counts, and larger spleens at diagnosis, and most of them (52.6%) were in the high-risk International Prognostic Score System (IPSS) category. After a median follow-up of 39 months, 128 patients (25.9%) died, 81 (63.3%) because of leukemia. Leukemia-free survival (LFS) and overall survival (OS) were significantly reduced in EZH2-mutated PMF patients (P = .028 and P < .001, respectively); no such impact was seen for PPV/PET-MF patients, possibly due to the low number of mutated cases. In multivariate analysis, survival of PMF patients was predicted by IPSS high-risk category, a < 25% JAK2V617F allele burden, and EZH2 mutation status. We conclude that EZH2 mutations are independently associated with shorter survival in patients with PMF.