Isolated Upward Rotation of the Fetal Cerebellar Vermis (Blake's Pouch Cyst) Is a Normal Variant: An Analysis of 111 Cases.

INTRODUCTION: The objective of the study was to provide more detailed data about fetal isolated upward rotation of the cerebellar vermis rotation (Blake's pouch cyst) in particular regarding pregnancy outcome. METHODS: This is a retrospective study of all cases of fetal isolated upward rotation of the cerebellar vermis (URCV) diagnosed in 3 referral centers in Italy from January 2009 to November 2019. Whenever possible, prenatal magnetic resonance imaging (MRI) was performed and a fetal karyotype was obtained. A detailed follow-up was obtained by consultation of medical records, interview with the parents, and the pediatricians. RESULTS: Our study population included 111 patients with a prenatal diagnosis of isolated URCV made at a median gestational age of 21 weeks +3 days (interquartile range (IQR) 21 + 0-22 + 2). The median brain stem-vermis (BV) angle was 27° (IQR 24-29°). In 37.9% of the cases, a regression of the finding with restoration of normal anatomy was noted at a follow-up scan or at postnatal checks. A BV angle of 25° or less predicted regression with a probability in excess of 90%. MRI was performed in utero or at birth in 101 patients and always confirmed sonographic diagnosis. Fetal CGH array and/or karyotype was available in 97 cases and was always normal, but in 1 case. A postnatal follow-up was available in 102 infants (mean 7 months, range 0-10 years of age) and documented a normal neurologic development in all the cases. CONCLUSIONS: Isolated URCV is most likely a normal variant of fetal anatomy without clinical consequences, at least at an early follow-up. A BV angle of 25° or less predicts intrauterine regression of the finding, but the outcome is good in all the cases. When a confident sonographic diagnosis is made, MRI is not mandatory. The risk of a chromosomal anomaly in these cases is probably low.

First prenatal case of Noonan syndrome with SOS2 mutation: Implications of early diagnosis for genetic counseling.

RASopathies are a group of syndromes with partially overlapping clinical features caused by germline mutations of the RAS/MAPK signaling pathway genes. The most common disorder is Noonan syndrome (NS; MIM 163950). We report the first prenatal case of NS with SOS2 (NM_006939.4) mutation in a euploid fetus with a severe increase in nuchal translucency (NT > 12 mm). Trio-based custom next-generation sequencing detected a de novo heterozygous missense mutation in the SOS2 gene: c.800 T > A (p.Met267Lys). Owing to the marked variable expressivity of NS and the scarcity of SOS2 mutation-related NS cases reported in the literature, it is difficult to provide appropriate genetic counseling. Several issues such as the best management technique and optimal NT cutoff have been discussed. In addition, in general, the fine balance between the advantages of an early prenatal diagnosis and the challenge of determining if the detected gene variant is pathogenic and, primarily, the stress of the counselees when providing a genetic counseling with limited information on the prenatal phenotype have been discussed. A prenatal path comprising examinations and multidisciplinary counseling is essential to support couples in a shared decision-making process.

Cleft Palate with or without Cleft Lip: The Role of Retronasal Triangle View and Maxillary Gap at 11-14 Weeks.

OBJECTIVE: To evaluate the presence of maxillary gap (MG) and abnormal retronasal triangle (RT) as markers of cleft palate (CP) with and without cleft lip in the first trimester and to assess their association with the type of orofacial cleft (OC). METHODS: The RT and the mid-sagittal view of the face were evaluated retrospectively by two operators in 26 fetuses with OC and in 80 normal controls to detect abnormal RT and/or MG. The agreement between operators was calculated. RESULTS: Amongst the 26 fetuses, there were 15 cases of bilateral, 6 cases of unilateral, and 4 cases of median cleft lip and palate, and 1 case of CP alone. The MG was observed in 18 cases by operator 1 and in 17 cases by operator 2; an abnormal RT was detected in 21 cases by operator 1 and in 22 cases by operator 2. Great agreement between operators was obtained. In controls, MG or abnormal RT was suspected in 6 and 2-4% of cases, respectively. CONCLUSIONS: RT seems to be more sensitive compared to MG; however, the latter showed an additional diagnostic ability when the secondary palate was involved. Both approaches in combination could be useful in detecting OC in the first trimester.

Prospective detection and differential diagnosis of cystic posterior fossa anomalies by assessing posterior brain at 11-14 weeks.

BACKGROUND: The role of the first-trimester scan has expanded from aneuploidy screening to the diagnosis of fetal malformations. Abnormal appearance of the posterior brain at 11-14 weeks gestation is a marker of cerebral anomalies; in fact an increased amount of fluid, particularly when the choroid plexus of the fourth ventricle is not visible and only 2 brain spaces instead of 3 are seen, may indicate the presence of cystic or cyst-like posterior fossa anomalies, such as Blake's pouch cyst or Dandy-Walker malformation. OBJECTIVE: The purpose of this study was to assess the role of ultrasound scanning in the identification of cystic posterior fossa anomalies at 11-14 weeks gestation. STUDY DESIGN: A prospective cohort study of fetuses with cystic appearance of the posterior fossa at 11-14 weeks gestation was performed. In all cases and in a control group of 40 normal fetuses, the brainstem-tentorium angle was also measured. The presence or absence of cystic posterior anomalies was determined at birth or at postmortem evaluation. RESULTS: In the period 2014-2018, 32 fetuses with an increased brainstem-occipital bone distance and/or failure to visualize the choroid plexus of fourth ventricle (2 brain spaces) were seen. Of these, 18 fetuses were terminated in the first trimester because of associated anomalies and were excluded from the study because of unavailable autoptic findings. The remaining 14 fetuses eventually were found to have a Dandy-Walker malformation in 4 cases, a Blake's pouch cyst in 8 cases, and normal brain anatomy in 2 cases. Two brain spaces were seen in all cases with Dandy-Walker malformation and in 2 of 8 cases with Blake's pouch cyst. Both brainstem-occipital bone measurement and brainstem-tentorium angle were significantly different in fetuses with Dandy-Walker malformation, Blake's pouch cyst, and control subjects (P<.0001). The brainstem-occipital bone z-scores of fetuses with Dandy-Walker malformation and Blake's pouch cyst were always +3 or more and +1.7 or more, respectively. The brainstem-tentorium angle z-scores were always -5 or less and -0.1 or less, respectively. CONCLUSION: Our study confirms that sonography of the posterior brain at 11-14 weeks gestation allows the identification of cystic posterior fossa anomalies. A large brainstem-occipital bone predicts Dandy-Walker malformation or Blake's pouch cyst. The presence of 2 brain spaces and a small brainstem-tentorium angle are correlated significantly with the presence of Dandy-Walker malformation.

Recurrent activating STAT5B N642H mutation in myeloid neoplasms with eosinophilia.

Determining the underlying cause of persistent eosinophilia is important for effective clinical management but remains a diagnostic challenge in many cases. We identified STAT5B N642H, an established oncogenic mutation, in 27/1715 (1.6%) cases referred for investigation of eosinophilia. Of the 27 mutated cases, a working diagnosis of hypereosinophilic syndrome (HES; n = 7) or a myeloid neoplasm with eosinophilia (n = 20) had been made prior to the detection of STAT5B N642H. Myeloid panel analysis identified a median of 2 additional mutated genes (range 0-4) with 4 cases having STAT5B N642H as a sole abnormality. STAT5B N642H was absent in cultured T cells of 4/4 positive cases. Individuals with SF3B1 mutations (9/27; 33%) or STAT5B N642H as a sole abnormality had a markedly better overall survival compared to cases with other additional mutations (median 65 months vs. 14 months; hazard ratio = 8.1; P < 0.001). The overall survival of STAT5B-mutated HES cases was only 30 months, suggesting that these cases should be reclassified as chronic eosinophilic leukemia, not otherwise specified (CEL-NOS). The finding of STAT5B N642H as a recurrent mutation in myeloid neoplasia with eosinophilia provides a new diagnostic and prognostic marker as well as a potential target for therapy.

Mutation landscape in patients with myelofibrosis receiving ruxolitinib or hydroxyurea.

Refractoriness to ruxolitinib in patients with myelofibrosis (MF) was associated with clonal evolution; however, whether genetic instability is promoted by ruxolitinib remains unsettled. We evaluated the mutation landscape in 71 MF patients receiving ruxolitinib (n = 46) and hydroxyurea (n = 25) and correlated with response. A spleen volume response (SVR) was obtained in 57% and 12%, respectively. Highly heterogenous patterns of mutation acquisition/loss and/or changes of variant allele frequency (VAF) were observed in the 2 patient groups without remarkable differences. In patients receiving ruxolitinib, driver mutation type and high-molecular risk profile (HMR) at baseline did not impact on response rate, while HMR and sole ASXL1 mutations predicted for SVR loss at 3 years. In patients with SVR, a decrease of ≥ 20% of JAK2V617F VAF predicted for SVR duration. VAF increase of non-driver mutations and clonal progression at follow-up correlated with SVR loss and treatment discontinuation, and clonal progression also predicted for shorter survival. These data indicate that (i) ruxolitinib does not appreciably promote clonal evolution compared with hydroxyurea, (ii) VAF increase of pre-existing and/or (ii) acquisition of new mutations while on treatment correlated with higher rate of discontinuation and/or death, and (iv) reduction of JAK2V617F VAF associated with SVR duration.

Driver mutations and prognosis in primary myelofibrosis: Mayo-Careggi MPN alliance study of 1,095 patients.

The 2013 discovery of calreticulin (CALR) mutations in myeloproliferative neoplasms was attended by their association with longer survival in primary myelofibrosis (PMF). Subsequent studies have suggested prognostic distinction between type 1/like and type 2/like CALR mutations and detrimental effect from triple-negative mutational status. Among 709 Mayo Clinic patients with PMF, 467 (66%) harbored JAK2, 112 (16%) CALR type 1/like, 24 (3.4%) CALR type 2/like, 38 (5.4%) MPL mutations and 68 (10%) were triple-negative. Survival was longer with type 1/like CALR, compared to JAK2 (HR 2.6, 95% CI 1.9-3.5), type 2/like CALR (HR 2.5, 95% CI 1.4-4.5), MPL (HR 1.8, 95% CI 1.1-2.9) and triple-negative mutational status (HR 2.4, 95% CI 1.6-3.6), but otherwise similar between the non-type 1/like CALR mutational states (P = .41). In multivariable analysis, the absence of type 1/like CALR (P < .001; HR 2, 95% CI 1.4-2.7), presence of ASXL1/SRSF2 mutations (P < .001; HR 1.9, 95% CI 1.5-2.4) and DIPSS-plus (P < .001) were each predictive of inferior survival. Furthermore, among 210 patients with ASXL1/SRSF2 mutations, survival was significantly longer in the presence vs. absence of type 1/like CALR mutations (median 5.8 vs. 2.9 years; P < .001). Triple-negative status did not disclose additional prognostic information for overall or leukemia-free survival. The observations regarding the prognostic distinction between CALR mutation variants were validated in an external cohort of 386 patients from the University of Florence Careggi hospital. We conclude that type 1/like CALR mutations in PMF not only predict superior survival, but also partially amend the detrimental effect of high molecular risk mutations.

CALR mutational status identifies different disease subtypes of essential thrombocythemia showing distinct expression profiles.

Polycythemia vera (PV) and essential thrombocythemia (ET) are Philadelphia-negative myeloproliferative neoplasms (MPNs) characterized by erythrocytosis and thrombocytosis, respectively. Approximately 95% of PV and 50-70% of ET patients harbor the V617F mutation in the exon 14 of JAK2 gene, while about 20-30% of ET patients carry CALRins5 or CALRdel52 mutations. These ET CALR-mutated subjects show higher platelet count and lower thrombotic risk compared to JAK2-mutated patients. Here, we showed that CALR-mutated and JAK2V617F-positive CD34+ cells display different gene and miRNA expression profiles. Indeed, we highlighted several pathways differentially activated between JAK2V617F- and CALR-mutated progenitors, i.e., mTOR, MAPK/PI3K, and MYC pathways. Furthermore, we unveiled that the expression of several genes involved in DNA repair, chromatin remodeling, splicing, and chromatid cohesion are decreased in CALR-mutated cells. According to the low risk of thrombosis in CALR-mutated patients, we also found the downregulation of several genes involved in thrombin signaling and platelet activation. As a whole, these data support the model that CALR-mutated ET could be considered as a distinct disease entity from JAK2V617F-positive MPNs and may provide the molecular basis supporting the different clinical features of these patients.

The three-vessel and trachea view (3VTV) in the first trimester of pregnancy: an additional tool in screening for congenital heart defects (CHD) in an unselected population.

OBJECTIVE: The aim of the study was to evaluate the feasibility of obtaining the three-vessel and trachea view (3VTV) in an unselected population undergoing first trimester screening for aneuploidy, and to investigate its role in the early detection of congenital heart defects (CHD). METHODS: Cardiac examination was performed by expert sonographers. Abnormal findings of 3VTV were classified in three different subgroups: number, size and spatial relationship of the vessels. RESULTS: We enrolled 6350 consecutive singleton pregnancies and included 5343 cases. Examination of 3VTV was feasible in 94% of cases. Fifty-seven (1%) CHD were present in the study period; 24 cases were excluded because parents opted for termination of pregnancy. Of the remaining 33 cases, 25 were suspected at the first trimester and eight were detected only at the mid-trimester. An abnormal 3VTV was suspected in 22 cases, and it was confirmed in 21. Five cases that were erroneously classified in the subgroup of abnormal vessel number were actually characterized by a diminutive size of one of the great arteries. The detection rate for CHD, including 4-CV and 3VTV, was 75.8%. CONCLUSIONS: Our study demonstrates that 3VTV is an easy plane to obtain by expert sonographers in an unselected population during first trimester. Typical suspicions include detection of abnormal number, size or spatial relationship of the vessels. © 2017 John Wiley & Sons, Ltd.

Presentation and outcome of patients with 2016 WHO diagnosis of prefibrotic and overt primary myelofibrosis.

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.

Safety and efficacy of ruxolitinib in splanchnic vein thrombosis associated with myeloproliferative neoplasms.

Splanchnic vein thrombosis (SVT) is one of the vascular complications of myeloproliferative neoplasms (MPN). We designed a phase 2 clinical trial to evaluate safety and efficacy of ruxolitinib in reducing splenomegaly and improving disease-related symptoms in patients with MPN-associated SVT. Patients diagnosed with myelofibrosis (12 cases), polycythemia vera (5 cases) and essential thrombocythemia (4 cases) received ruxolitinib for 24 weeks in the core study period. Spleen volume was assessed by magnetic resonance imaging (MRI) and splanchnic vein circulation by echo-Doppler analysis. Nineteen patients carried JAK2V617F, one had MPLW515L, and one CALRL367fs*46 mutation. Eighteen patients had spleno-portal-mesenteric thrombosis, two had Budd-Chiari syndrome, and one had both sites involved; 16 patients had esophageal varices. Ruxolitinib was well tolerated with hematological toxicities consistent with those of patients without SVT and no hemorrhagic adverse events were recorded. After 24 weeks of treatment, spleen volume reduction ≥35% by MRI was achieved by 6/21 (29%) patients, and a ≥50% spleen length reduction by palpation at any time up to week 24 was obtained by 13/21 (62%) patients. At week 72, 8 of the 13 (62%) patients maintained the spleen response by palpation. No significant effect of treatment on esophageal varices or in splanchnic circulation was observed. MPN-related symptoms, evaluated by MPN-symptom assessment form (SAF) TSS questionnaire, improved significantly during the first 4 weeks and remained stable up to week 24. In conclusion, this trial shows that ruxolitinib is safe in patients with MPN-associated SVT, and effective in reducing spleen size and disease-related symptoms.

Prognostic impact of bone marrow fibrosis in primary myelofibrosis. A study of the AGIMM group on 490 patients.

The prognostic significance of bone marrow (BM) fibrosis grade in patients with primary myelofibrosis (PMF) is still debated. A fibrosis grade greater than 1 was shown to associate with higher risk of death, and addition of fibrosis grade to IPSS score resulted in a more accurate prediction of survival. The aim of this study was to analyze the prognostic impact of BM fibrosis in 490 patients with PMF, evaluated at diagnosis, molecularly annotated and with extensive follow-up information. We found that fibrosis grade 2 and greater on a 0-3 scale was associated with clinical characteristics indicative of a more advanced disease, such as anemia, leukopenia, thrombocytopenia, constitutional symptoms, larger splenomegaly and a higher IPSS risk category. Patients with higher grade of fibrosis were also more likely to have additional somatic mutations in ASXL1 and EZH2, that are prognostically adverse. Median survival was significantly reduced in patients with grade 2 and 3 fibrosis as compared with grade 1; this effect was maintained when analysis was restricted to younger patients. In multivariate analysis, fibrosis grade independently predicted for survival regardless of IPSS variables and mutational status; the adverse impact of fibrosis was noticeable especially in lower IPSS risk categories. Overall, results indicate that higher grades of fibrosis correlate with unique clinical and molecular aspects and represent an independent adverse variable in patients with PMF; these observations deserve confirmation in prospectively designed series of patients. Am. J. Hematol. 91:918-922, 2016. © 2016 Wiley Periodicals, Inc.

Clinical presentation and management practice of systemic mastocytosis. A survey on 460 Italian patients.

Systemic mastocytosis is a rare heterogeneous myeloproliferative neoplasm characterized by abnormal proliferation and activation of mast cells. We describe a large multicentre series of 460 adult patients with systemic mastocytosis, with a diagnosis based on WHO 2008 criteria, in a "real-life" setting of ten Italian centers with dedicated multidisciplinary programs. We included indolent forms with (n = 255) and without (n = 165) skin lesions, smouldering (n = 20), aggressive (n = 28), associated with other hematological diseases mastocytosis (n = 21) and mast cell leukemia (n = 1). This series was uniquely characterized by a substantial proportion of patients with low burden of neoplastic mast cells; notably, 38% of cases were diagnosed using only minor diagnostic criteria according to WHO 2008 classification, underlying the feasibility of early diagnosis where all diagnostic approaches are made available. This has particular clinical relevance for prevention of anaphylaxis manifestations, that were typically associated with indolent forms. In multivariate analysis, the most important features associated with shortened overall survival were disease subtype and age at diagnosis >60 years. Disease progression was correlated with mastocytosis subtype and thrombocytopenia. As many as 32% of patients with aggressive mastocytosis suffered from early evolution into acute leukemia. Overall, this study provides novel information about diagnostic approaches and current presentation of patients with SM and underlines the importance of networks and specialized centers to facilitate early diagnosis and prevent disease-associated manifestations. Am. J. Hematol. 91:692-699, 2016. © 2016 Wiley Periodicals, Inc.

Epidemiology and clinical relevance of mutations in postpolycythemia vera and postessential thrombocythemia myelofibrosis: A study on 359 patients of the AGIMM group.

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.

Integrative analysis of copy number and gene expression data suggests novel pathogenetic mechanisms in primary myelofibrosis.

Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, gene expression and copy number signals were integrated and several genomic abnormalities leading to a concordant alteration in gene expression levels were identified. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus was accompanied by a coordinated transcriptional up-regulation in PMF patients. PAOX inhibition resulted in rapid cell death of PMF progenitor cells, while sparing normal cells, suggesting that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival. Moreover, copy number loss in the chromatin modifier HMGXB4 gene correlates with a concomitant transcriptional down-regulation in PMF patients. Interestingly, silencing of HMGXB4 induces megakaryocyte differentiation, while inhibiting erythroid development, in human hematopoietic stem/progenitor cells. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterizes PMF patients.

Targeted deep sequencing in polycythemia vera and essential thrombocythemia.

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 next-generation 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.