Exacerbation of thrombo-inflammation by JAK2V617F mutation worsens the prognosis of cerebral venous sinus thrombosis.

Cerebral venous sinus thrombosis (CVST) is an uncommon venous thromboembolic event accounting for <1% of strokes resulting in brain parenchymal injuries. JAK2V617F mutation, the most frequent driving mutation of myeloproliferative neoplasms has been reported to be associated with worse clinical outcomes in patients with CVST. We investigated whether hematopoietic JAK2V617F expression predisposes to specific pathophysiological processes and/or worse prognosis after CVST. Using an in vivo mouse model of CVST, we analyzed clinical, biological and imaging outcomes in mice with hematopoietic-restricted Jak2V617F expression, compared to Jak2WT mice. In parallel, we studied a human cohort of JAK2V617F-positive or negative CVST. Early after CVST, mice with hematopoietic Jak2V617F expression had increased adhesion of platelets and neutrophils in cerebral veins located in the vicinity of CVST. On day 1, Jak2V617F mice had a worse outcome characterized by significantly more frequent and severe intracranial hemorrhages (ICH) and higher mortality rates. Peripheral neutrophil activation was enhanced, as indicated by higher circulating platelet-neutrophil aggregates, upregulated CD11b expression, and higher myeloperoxydase (MPO) plasma level. Concurrently, immunohistological and brain homogenates analysis showed higher neutrophil infiltration and increased blood-brain-barrier disruption. Similarly, JAK2V617F-positive CVST patients tended to present higher thrombotic burden and had significantly higher SII, a systemic thrombo-inflammatory marker, compared to JAK2V617F-negative patients. In mice with CVST, our study corroborates that Jak2V617F mutation leads to a specific pattern including increased thrombotic burden, ICH and mortality. The exacerbated thrombo-inflammatory response, observed both in mice and JAK2V617F-positive patients, could contribute to hemorrhagic complications.

Comparative clinical and molecular landscape of primary and secondary myelofibrosis: Superior performance of MIPSS70+ v2.0 over MYSEC-PM.

Comparative clinical characteristics, molecular landscape and prognosis scoring for primary (PMF) and secondary myelofibrosis (SMF).

Genomic and functional impact of Trp53 inactivation in JAK2V617F myeloproliferative neoplasms.

Classical myeloproliferative neoplasms (MPNs) are characterized by the proliferation of myeloid cells and the risk of transformation into myelofibrosis or acute myeloid leukemia (AML) and TP53 mutations in MPN patients are linked to AML. However, JAK2V617F has been reported to impact the TP53 response to DNA damage, suggesting potential overlapping role of TP53 inactivation in MPN. We established a mouse model showing that JAK2V617F/Vav-Cre/Trp53-/- mice displayed a similar phenotype to JAK2V617F/Vav-Cre mice, but their proliferation was outcompeted in competitive grafts. RNA-Seq revealed that half of the genes affected by JAK2V617F were affected by p53-inactivation, including the interferon pathway. To validate this finding, mice were repopulated with a mixture of wild-type and JAK2V617F (or JAK2V617F/Vav-Cre/Trp53-/-) cells and treated with pegylated interferonα. JAK2V617F-reconstituted mice entered complete hematological remission, while JAK2V617F/Vav-Cre /Trp53-/--reconstituted mice did not, confirming that p53 loss induced interferon-α resistance. KEGG and Gene Ontology analyses of common deregulated genes showed that these genes were mainly implicated in cytokine response, proliferation, and leukemia evolution, illustrating that in this mouse model, the development of MPN is not affected by TP53 inactivation. Taken together, our results show that many genetic modifications induced by JAK2V617F are influenced by TP53, the MPN phenotype may not be. Trp53 loss alone is insufficient to induce rapid leukemic transformation in steady-state hematopoiesis in JAK2V617F MPN, and Trp53 loss may contribute to interferon resistance in MPN.

Distinct clinico-molecular arterial and venous thrombosis scores for myeloproliferative neoplasms risk stratification.

Current recommended risk scores to predict thrombotic events associated with myeloproliferative neoplasms (MPN) do not discriminate between arterial and venous thrombosis despite their different physiopathology. To define novel stratification systems, we delineated a comprehensive landscape of MPN associated thrombosis across a large long-term follow-up MPN cohort. Prior arterial thrombosis, age >60 years, cardiovascular risk factors and presence of TET2 or DNMT3A mutations were independently associated with arterial thrombosis in multivariable analysis. ARTS, an ARterial Thrombosis Score, based on these four factors, defined low- (0.37% patients-year) and high-risk (1.19% patients-year) patients. ARTS performance was superior to the two-tiered conventional risk stratification in our training cohort, across all MPN subtypes, as well as in two external validation cohorts. Prior venous thrombosis and presence of a JAK2V617F mutation with a variant allelic frequency ≥50% were independently associated with venous thrombosis. The discrimination potential of VETS, a VEnous Thrombosis Score based on these two factors, was poor, similar to the two-tiered conventional risk stratification. Our study pinpoints arterial and venous thrombosis clinico-molecular differences and proposes an arterial risk score for more accurate patients' stratification. Further improvement of venous risk scores, accounting for additional factors and considering venous thrombosis as a heterogeneous entity is warranted.

Myeloproliferative neoplasms and splanchnic vein thrombosis: Contemporary diagnostic and therapeutic strategies.

Myeloproliferative neoplasms (MPNs) are the most common etiologies of primary splanchnic vein thrombosis, present in almost forty percent of patients with Budd-Chiari syndrome or portal vein thrombosis. Diagnosis of MPNs can be difficult in these patients because key characteristics, such as elevated blood cell counts and splenomegaly, are confounded by portal hypertension or bleeding complications. In recent years, diagnostic tools have improved to provide more accurate diagnosis and classification of MPNs. Although bone marrow biopsy findings remain a major diagnostic criterion, molecular markers are playing an increasing role not only in diagnosis but also in better estimating prognosis. Therefore, though screening for JAK2V617F mutation should be the starting point of the diagnostic workup performed in all patients with splanchnic vein thrombosis, a multidisciplinary approach is needed to accurately diagnose the subtype of myeloproliferative neoplasm, recommend the useful additional tests (bone marrow biopsy, search for an additional mutation using targeted next-generation sequencing), and suggest the best treatment strategy. Indeed, providing a specific expert care pathway for patients with splanchnic vein thrombosis and underlying myeloproliferative neoplasm is crucial to determine the optimal management to reduce the risk of both hematological and hepatic complications.

Clonal architecture evolution in Myeloproliferative Neoplasms: from a driver mutation to a complex heterogeneous mutational and phenotypic landscape.

Myeloproliferative neoplasms are characterized by the acquisition at the hematopoietic stem cell level of driver mutations targeting the JAK/STAT pathway. In addition, they also often exhibit additional mutations targeting various pathways such as intracellular signalling, epigenetics, mRNA splicing or transcription. The natural history of myeloproliferative neoplasms is usually marked by a chronic phase of variable duration depending on the disease subtype, which can be followed by an accelerated phase or transformation towards more aggressive diseases such as myelofibrosis or acute leukemia. Besides, recent studies revealed important new information about the rates and mechanisms of sequential acquisition and selection of mutations in hematopoietic cells of myeloproliferative neoplasms. Better understanding of these events has been made possible in large part with the help of novel techniques that are now available to precisely decipher at the single cell level both the clonal architecture and the mutation-induced cell modifications. In this review, we will summarize the most recent knowledge about the mechanisms leading to clonal selection, how clonal architecture complexity can explain disease heterogeneity, and the impact of clonal evolution on clinical evolution.

Impact of TP53 on outcome of patients with myelofibrosis undergoing hematopoietic stem cell transplantation.

TP53 mutations (TP53MTs) have been associated with poor outcomes in various hematologic malignancies, but no data exist regarding its role in patients with myelofibrosis undergoing hematopoietic stem cell transplantation (HSCT). Here, we took advantage of a large international multicenter cohort to evaluate the role of TP53MT in this setting. Among 349 included patients, 49 (13%) had detectable TP53MT, of whom 30 showed a multihit configuration. Median variant allele frequency was 20.3%. Cytogenetic risk was favorable (71%), unfavorable (23%), and very high (6%), with complex karyotype present in 36 patients (10%). Median survival of patients with TP53MT was 1.5 vs 13.5 years for those with wild-type TP53 (TP53WT; P < .001). Outcome was driven by multihit TP53MT constellation (P < .001), showing 6-year survival of 56% for individuals with single-hit vs 25% for those with multihit TP53MT vs 64% for those with TP53WT. Outcome was independent of current transplantation-specific risk factors and conditioning intensity. Similarly, cumulative incidence of relapse was 17% for single-hit vs 52% for multihit vs 21% for TP53WT. Ten patients with TP53MT (20%) presented as leukemic transformation vs only 7 (2%) in the TP53WT group (P < .001). Out of the 10 patients with TP53MT, 8 showed multihit constellation. Median time to leukemic transformation was shorter for multihit and single-hit TP53MT (0.7 and 0.5 years, respectively) vs 2.5 years for TP53WT. In summary, multihit TP53MT represents a very high-risk group in patients with myelofibrosis who are undergoing HSCT, whereas single-hit TP53MT alone showed similar outcome to patients with nonmutated TP53, informing prognostication for survival and relapse together with current transplantation-specific tools.

Comprehensive in silico and functional studies for classification of EPAS1/HIF2A genetic variants identified in patients with erythrocytosis.

Gain-of-function mutations in the EPAS1/HIF2A gene have been identified in patients with hereditary erythrocytosis that can be associated with the development of paraganglioma, pheochromocytoma and somatostatinoma. In the present study, we describe a unique European collection of 41 patients and 28 relatives diagnosed with an erythrocytosis associated with a germline genetic variant in EPAS1. In addition we identified two infants with severe erythrocytosis associated with a mosaic mutation present in less than 2% of the blood, one of whom later developed a paraganglioma. The aim of this study was to determine the causal role of these genetic variants, to establish pathogenicity, and to identify potential candidates eligible for the new hypoxia-inducible factor-2 α (HIF-2α) inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2b variants has been studied by using canonical and real-time reporter luciferase assays. These functional assays consisted of a novel edited vector containing an expanded region of the erythropoietin promoter combined with distal regulatory elements which substantially enhanced the HIF-2α-dependent induction. Altogether, our studies allowed the classification of 11 mutations as pathogenic in 17 patients and 23 relatives. We described four new mutations (D525G, L526F, G527K, A530S) close to the key proline P531, which broadens the spectrum of mutations involved in erythrocytosis. Notably, we identified patients with only erythrocytosis associated with germline mutations A530S and Y532C previously identified at somatic state in tumors, thereby raising the complexity of the genotype/phenotype correlations. Altogether, this study allows accurate clinical follow-up of patients and opens the possibility of benefiting from HIF-2α inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.

Reduced murine double minute 2 and 4 protein, but not messenger RNA, expression is associated with more severe disease in myelodysplastic syndromes and acute myeloblastic leukaemia.

The human homologues of murine double minute 2 (MDM2) and 4 (MDM4) negatively regulate p53 tumour suppressor activity and are reported to be frequently overexpressed in human malignancies, prompting clinical trials with drugs that prevent interactions between MDM2/MDM4 and p53. Bone marrow samples from 111 patients with acute myeloblastic leukaemia, myelodysplastic syndrome or chronic myelomonocytic leukaemia were examined for protein (fluorescence-activated cell sorting) and messenger RNA (mRNA) expression (quantitative polymerase chain reaction) of MDM2, MDM4 and tumour protein p53 (TP53). Low protein expression of MDM2 and MDM4 was observed in immature cells from patients with excess of marrow blasts (>5%) compared with CD34+ /CD45low cells from healthy donors and patients without excess of marrow blasts (<5%). The mRNA levels were indistinguishable in all samples examined regardless of disease status or blast levels. Low MDM2 and MDM4 protein expression were correlated with poor survival. These data show a poor correlation between mRNA and protein expression levels, suggesting that quantitative flow cytometry analysis of protein expression levels should be used to predict and validate the efficacy of MDM2 and MDM4 inhibitors. These findings show that advanced disease is associated with reduced MDM2 and MDM4 protein expression and indicate that the utility of MDM2 and MDM4 inhibitors may have to be reconsidered in the treatment of advanced myeloid malignancies.

High Molecular and Cytogenetic Risk in Myelofibrosis Does Not Benefit From Higher Intensity Conditioning Before Hematopoietic Cell Transplantation: An International Collaborative Analysis.

There is no direct evidence to recommend specific conditioning intensities in myelofibrosis undergoing allogeneic hematopoietic cell transplantation, especially in the molecular era. We aimed to compare outcomes of reduced intensity (RIC) or myeloablative conditioning (MAC) transplantation in myelofibrosis with molecular information. The study included 645 genetically annotated patients (with at least driver mutation status available), of whom 414 received RIC and 231 patients received MAC. The median follow-up time from transplantation was 6.0 years for RIC and 9.4 years for MAC. The 6-year overall survival rates for RIC and MAC were 63% (95% confidence interval [CI], 58%-68%) and 59% (95% CI, 52%-66%; P = 0.34) and progression-free survival was 52% (95% CI, 47%-57%) and 52% (95% CI, 45%-59%; P = 0.64). The 2-year cumulative incidence of nonrelapse mortality was 26% (95% CI, 21%-31%) for RIC and 29% (95% CI, 23%-34%) for MAC (P = 0.51). In terms of progression/relapse, the 2-year cumulative incidence was 10% (95% CI, 5%-19%) for RIC and 9% (95% CI, 4%-14%) for MAC (P = 0.46). Higher intensity conditioning did not seem to improve outcomes for higher-risk disease, according to mutational, cytogenetic, and clinical profile. In contrast, patients with reduced performance status, matched unrelated donors, and ASXL1 mutations appeared to benefit from RIC in terms of overall survival.

Reduced intensity hematopoietic stem cell transplantation for accelerated-phase myelofibrosis.

Accelerated-phase myelofibrosis, currently defined by circulating blasts 10% to 19%, usually confers very high risk for progression and poor outcome. The outcome of hematopoietic stem cell transplantation for accelerated-phase myelofibrosis has not been evaluated yet. We analyzed the outcome of 349 clinically and genetically annotated patients with primary or secondary myelofibrosis undergoing reduced intensity transplantation, of whom 35 had accelerated-phase myelofibrosis. In comparison with chronic-phase (<10% blasts) myelofibrosis, median leukocyte counts were higher, more patients had constitutional symptoms, and RAS mutations were detected more frequently in the accelerated-phase group. After a median follow-up of 5.9 years, estimated 5-year overall survival was 65% (95% confidence interval [CI], 49% to 81%) vs 64% (95% CI, 59% to 69%) for the chronic-phase group (P = .91), and median overall survival was not reached. In terms of relapse-free survival, estimated 5-year outcome for the accelerated-phase group was 49% (95% CI, 32% to 67%) vs 55% (95% CI, 50% to 61%) for the chronic-phase group (P = .65). Estimated 5-year nonrelapse mortality was 20% (95% CI, 8% to 33%) for the accelerated-phase group vs 30% (95% CI, 24% to 35%; P = .25) for the chronic-phase group. In terms of relapse, 5-year incidence was 30% (95% CI, 14% to 46%) for the accelerated-phase group vs 15% (95% CI, 11% to 19%) for the chronic-phase group (P = .02). Results were confirmed in multivariable analysis and propensity score matching. In conclusion, reduced intensity transplantation showed excellent survival but higher relapse for accelerated-phase myelofibrosis.

Single-cell analysis reveals selection of TP53-mutated clones after MDM2 inhibition.

The mechanisms of transformation of chronic myeloproliferative neoplasms (MPN) to leukemia are largely unknown, but TP53 mutations acquisition is considered a key event in this process. p53 is a main tumor suppressor, but mutations in this protein per se do not confer a proliferative advantage to the cells, and a selection process is needed for the expansion of mutant clones. MDM2 inhibitors may rescue normal p53 from degradation and have been evaluated in a variety of cancers with promising results. However, the impact of these drugs on TP53-mutated cells is underexplored. We report herein evidence of a direct effect of MDM2 inhibition on the selection of MPN patients' cells harboring TP53 mutations. To decipher whether these mutations can arise in a specific molecular context, we used a DNA single-cell approach to determine the clonal architecture of TP53-mutated cells. We observed that TP53 mutations are late events in MPN, mainly occurring in the driver clone, whereas clonal evolution frequently consists of sequential branching instead of linear consecutive acquisition of mutations in the same clone. At the single-cell level, the presence of additional mutations does not influence the selection of TP53 mutant cells by MDM2 inhibitor treatment. Also, we describe an in vitro test allowing to predict the emergence of TP53 mutated clones. Altogether, this is the first demonstration that a drug treatment can directly favor the emergence of TP53-mutated subclones in MPN.

Revisiting Diagnostic performances of serum erythropoïetin level and JAK2 mutation for polycythemias: analysis of a cohort of 1090 patients with red cell mass measurement.

We assessed the diagnostic performances of erythropoietin and JAK2 mutations in 1,090 patients with suspected polycythemia who were referred for red cell mass (RCM) measurement. In patients with a high haematocrit and/or haemoglobin level, a low erythropoietin level (<=3·3 mUI/ml) and JAK2 mutation showed comparable positive predictive value (PPV) for true polycythemia (RCM>=125%), 92·1% and 90% respectively. A very-low erythropoietin level (<=1·99 mUI/ml) had a PPV of 100% for polycythemia vera (PV) diagnosis. We confirmed the correlations between RCM, erythropoietin and JAK2 variant allelic frequency in PV patients. This study prompts the need to revisit the role of EPO in PV diagnostic criteria.

Improvement of Standardization of Molecular Analyses in Hematology: The 10-year GBMHM French Experience.

Molecular tests have become an indispensable tool for the diagnosis and prognosis of hematological malignancies and are subject to accreditation according to the International Standard ISO 15189. National standardization of these techniques is essential to ensure that patients throughout France benefit from the same care. We report here on the experience of the GBMHM (Groupe des Biologistes Moléculaires des Hémopathies Malignes). By organizing External Evaluation of Quality (EEQ) programs and training meetings, the GBMHM has contributed to improvement and standardization of molecular tests in 64 French laboratories. A retrospective analysis of the quality-control results of 11 national campaigns spanning 10 years was performed for the 3 most frequently prescribed tests: BCR-ABL1, JAK2 V617F, and lymphoid clonality. For each test, particular attention was placed on comparing methodologies and their evolution throughout the period. The establishment of the BCR-ABL1, JAK2 V617F, and lymphoid clonality EEQ programs and the associated training meetings have initiated a process of collective standardization concerning the methods of implementation (JAK2 V617F) and the interpretation and formulation of results (lymphoid clonality). In addition, it resulted in objective improvement in technical performance (BCR-ABL1). Our evaluation of the impact of these EEQ programs demonstrates that it is possible to obtain reproducible values across different laboratories in France by applying national recommendations. To our knowledge, this is the first publication that evaluates the impact of a national quality assurance program on improving molecular results in hematology.

Inferring the dynamics of mutated hematopoietic stem and progenitor cells induced by IFNα in myeloproliferative neoplasms.

Classical BCR-ABL-negative myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells (HSCs) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon α (IFNα) has demonstrated some efficacy in inducing molecular remission in MPNs. To determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in patients with MPN by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured the clonal architecture of early and late hematopoietic progenitors (84 845 measurements) and the global variant allele frequency in mature cells (409 measurements) several times per year. Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSCs. Our data support the hypothesis that IFNα targets JAK2V617F HSCs by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSCs and increases with high IFNα dose in heterozygous JAK2V617F HSCs. We also found that the molecular responses of CALRm HSCs to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and a high dose of IFNα correlates with worse outcomes. Our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dose.

Dual role of EZH2 in megakaryocyte differentiation.

EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss-of-function mutations have been found in myeloproliferative neoplasms, particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and short hairpin RNA induces megakaryocyte (MK) commitment by accelerating lineage marker acquisition without change in proliferation. Later in differentiation, EZH2 inhibition blocks proliferation and polyploidization and decreases proplatelet formation. EZH2 inhibitors similarly reduce MK polyploidization and proplatelet formation in vitro and platelet levels in vivo in a JAK2V617F background. In transcriptome profiling, the defect in proplatelet formation was associated with an aberrant actin cytoskeleton regulation pathway, whereas polyploidization was associated with an inhibition of expression of genes involved in DNA replication and repair and an upregulation of cyclin-dependent kinase inhibitors, particularly CDKN1A and CDKN2D. The knockdown of CDKN1A and to a lesser extent CDKN2D could partially rescue the percentage of polyploid MKs. Moreover, H3K27me3 and EZH2 chromatin immunoprecipitation assays revealed that CDKN1A is a direct EZH2 target and CDKN2D expression is not directly regulated by EZH2, suggesting that EZH2 controls MK polyploidization directly through CDKN1A and indirectly through CDKN2D.