Key Points CALR mutations occur in half of JAK2 and MPL wt patients with ET and associate with some distinctive phenotypic traits. Patients with ET harboring CALR mutations are at significantly lower risk of thrombosis compared with JAK2- and MPL-mutated patients.

Key Points• More than half of patients with PV or ET harbor DNA mutations/ variants other than JAK2/CALR/MPL.• The presence of some of these mutations adversely affects overall, leukemia-free, or myelofibrosis-free survival.Polycythemia vera (PV) is characterized by JAK2 and essential thrombocythemia (ET) by JAK2, calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) mutations; we describe the occurrence and prognostic relevance of DNA sequence variants/mutations other than JAK2/CALR/MPL. A myeloid neoplasm-relevant 27-gene panel was used for nextgeneration sequencing of bone marrow or whole blood DNA and conventional tools were used for analysis. "Adverse variants/mutations" were identified by age-adjusted multivariable analysis of impact on overall, leukemia-free, or myelofibrosis-free survival. Fifty-three percent of 133 Mayo Clinic patients with PV and 53% of 183 with ET harbored 1 or more sequence variants/mutations other than JAK2/CALR/MPL; the most frequent were TET2 and ASXL1. "Adverse variants/mutations" in PV included ASXL1, SRSF2, and IDH2 and in ET SH2B3, SF3B1, U2AF1, TP53, IDH2, and EZH2; combined prevalence was 15% and 15%, respectively. Adverse variants/mutations were associated with inferior survival in both PV (median, 7.7 vs 16.9 years) and ET (median, 9 vs 22 years) and the effect was independent of conventional prognostic models with respective hazard ratio (95% confidence interval) of 2.8 (1.5-5.1) and 2.6 (1.4-4.8); these observations were validated in 215 Italian patients with PV and 174 with ET. In both Mayo Clinic and Italian cohorts, leukemic or fibrotic progression was also predicted by adverse variants/mutations. Number of mutations did not provide additional prognostic information. We conclude that targeted deep sequencing in PV and ET allows for genetic risk stratification that is independent of clinically derived prognostic models.

We recently defined a high-molecular risk category (HMR) in primary myelofibrosis (PMF), based on the presence of at least one of the five 'prognostically detrimental' mutated genes (ASXL1, EZH2, SRSF2 and IDH1/2). Herein, we evaluate the additional prognostic value of the 'number' of mutated genes. A total of 797 patients were recruited from Europe (n=537) and the Mayo Clinic (n=260). In the European cohort, 167 (31%) patients were HMR: 127 (23.6%) had one and 40 (7.4%) had two or more mutated genes. The presence of two or more mutations predicted the worst survival: median 2.6 years (hazard ratio (HR) 3.8, 95% confidence interval (CI) 2.6-5.7) vs. 7.0 years (HR 1.9, 95% CI 1.4-2.6) for one mutation vs 12.3 years for no mutations. The results were validated in the Mayo cohort and prognostic significance in both cohorts was independent of International Prognostic Scoring System (IPSS; HR 2.4, 95% CI 1.6-3.6) and dynamic IPSS (DIPSS)-plus (HR 1.9, 95% CI 1.2-3.1), respectively. Two or more mutations were also associated with shortened leukemia-free survival (HR 6.2, 95% CI 3.5-10.7), also Mayo validated. Calreticulin mutations favorably affected survival, independently of both number of mutations and IPSS/DIPSS-plus. We conclude that the 'number' of prognostically detrimental mutations provides added value in the combined molecular and clinical prognostication of PMF.

Key Points Differential diagnosis of pre-PMF and overt PMF by 2016 WHO criteria underscores uniqueness in disease presentation and outcome. Patterns of driver and nondriver myeloid gene mutations contribute to prognosis in both pre-PMF and overt PMF.

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. (Blood. 2011;118(19):5227-5234) IntroductionThe identification of the JAK2V617F mutation 1-4 represented a seminal discovery in the field of Philadelphia-chromosomenegative chronic myeloproliferative neoplasms (MPNs), 5 providing clues to the pathogenesis, 6 prompting a revision of the diagnostic criteria, 7 and culminating in the development of clinical trials with JAK2 (and JAK1) inhibitors. 8,9 The JAK2V617F mutation occurs in almost all patients with polycythemia vera (PV) and in 50%-70% of those with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Soon after the identification of the JAK2V617F mutation, mutations in JAK2 exon 12 were described in rare patients with JAK2V617F-negative PV and mutations in MPL were reported in 5%-10% of ET or PMF subjects. The complexity of the molecular pathogenesis of MPNs is reinforced by discovery of additional mutations in TET2, 10 ASXL1, 11 CBL, 12 IDH1/IDH2, 13 and IKZF1. 14 These mutations are detected in a minority of patients at different phases of the disorder, including leukemic transformation, and are variably associated each other and with JAK2 or MPL mutations.We recently identified novel loss-of-function mutations in EZH2 in 1 of 30 (3%) PV and in 4 of 30 PMF patients (13%), as well as in 11%-25% of patients with myelodysplastic syndromes (MDS) and in 10% of patients with MDS/MPN. 15 Mutations were spread throughout the gene and included missense, nonsense, and premature stop codons; both monoallelic and biallelic mutations were described. Among patients with MDS/MPN, survival was significantly worse in those with EZH2 muta...

Key Points• Differential gene and miRNA expression analysis in PMF granulocytes identifies new biomarkers and putative therapeutic targets.• Activation of the miR-155/ JARID2 axis in PMF CD341 cells results in overproduction of MK precursors.Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte (MK) hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with somatic mutations in JAK2, MPL, or CALR. Previous studies have shown that abnormal MKs play a central role in the pathophysiology of PMF. In this work, we studied both gene and microRNA (miRNA) expression profiles in CD34 1 cells from PMF patients. We identified several biomarkers and putative molecular targets such as FGR, LCN2, and OLFM4. By means of miRNA-gene expression integrative analysis, we found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, we identified a network gathering several miRNAs with oncogenic potential (eg, miR-155-5p) and targeted genes whose abnormal function has been previously associated with myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Because the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, we studied the function of this axis in normal and PMF CD34 1 cells. We showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41 1 MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF. (Blood. 2014;124(13):e21-e32)

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Intrauterine growth restriction is a condition fetus does not reach its growth potential and associated with perinatal mobility and mortality. Intrauterine growth restriction is caused by placental insufficiency, which determines cardiovascular abnormalities in the fetus. This condition, moreover, should prompt intensive antenatal surveillance of the fetus as well as follow-up of infants that had intrauterine growth restriction as short and long-term sequele should be considered.