Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F-positive myeloproliferative neoplasms.

ABSTRACT: Pegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F-positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α-treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

Genetic basis and molecular profiling in myeloproliferative neoplasms.

BCR::ABL1-negative myeloproliferative neoplasms (MPNs) are clonal diseases originating from a single hematopoietic stem cell that cause excessive production of mature blood cells. The 3 subtypes, that is, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are diagnosed according to the World Health Organization (WHO) and international consensus classification (ICC) criteria. Acquired gain-of-function mutations in 1 of 3 disease driver genes (JAK2, CALR, and MPL) are the causative events that can alone initiate and promote MPN disease without requiring additional cooperating mutations. JAK2-p.V617F is present in >95% of PV patients, and also in about half of the patients with ET or PMF. ET and PMF are also caused by mutations in CALR or MPL. In ∼10% of MPN patients, those referred to as being "triple negative," none of the known driver gene mutations can be detected. The common theme between the 3 driver gene mutations and triple-negative MPN is that the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway is constitutively activated. We review the recent advances in our understanding of the early events after the acquisition of a driver gene mutation. The limiting factor that determines the frequency at which MPN disease develops with a long latency is not the acquisition of driver gene mutations, but rather the expansion of the clone. Factors that control the conversion from clonal hematopoiesis to MPN disease include inherited predisposition, presence of additional mutations, and inflammation. The full extent of knowledge of the mutational landscape in individual MPN patients is now increasingly being used to predict outcome and chose the optimal therapy.

CALR-mutated patients with low allele burden represent a specific subtype of essential thrombocythemia: A study on behalf of FIM and GBMHM.

A low allele burden (i.e., <20%) of the CALR driver mutation is found in 10.8% of CALR-mutated MPNs, mostly in essential thrombocythemia, and correlates with a milder phenotype and a more indolent evolution compared to patients with an allele burden ≥20%.

JAK2-V617F and interferon-α induce megakaryocyte-biased stem cells characterized by decreased long-term functionality.

We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.

Relationship between comorbidities, mutational profile, and outcome after intensive chemotherapy in patients older than 60 years with acute myeloid leukemia: Assessment of different risk scores.

The aim of this study was to evaluate how comorbidities and molecular landscape relate to outcome in patients with acute myeloid leukemia (AML) aged 60 years or older who received intensive induction therapy. In 91 patients, 323 mutations were identified in 77 genes by next-generation sequencing, with a median of four mutations per patient, with NPM1, FLT3, TET2, and DNMT3A being the most frequently mutated genes. A multistate model identified FLT3, IDH2, RUNX1, and TET2 mutations as associated with a higher likelihood of achieving complete remission while STAG2 mutations were associated with primary refractory disease, and DNMT3A, FLT3, IDH2, and TP53 mutations with mortality after relapse. Ferrara unfitness criteria and performance status were the best predictors of short-term outcome (area under the curve = 82 for 2-month survival for both parameters), whereas genomic classifications better predicted long-term outcome, with the Patel risk stratification performing the best over the 5-year follow-up period (C-index = 0.63 for event-free and overall survival). We show that most genomic prognostic classifications, mainly used in younger patients, are useful for classifying older patients, but to a lesser extent, because of different mutational profiles. Specific prognostic classifications, incorporating performance status, comorbidities, and cytogenetic/molecular data, should be specifically designed for patients over 60 years.

Diagnosis and prognosis are supported by integrated assessment of next-generation sequencing in chronic myeloid malignancies. A real-life study.

Next-generation sequencing (NGS) is used to investigate the presence of somatic mutations. The utility of incorporating routine sequencing to guide diagnosis and therapeutic decisions remains unclear. We report the findings of an observational, multicenter study that aimed to assess the impact of somatic mutation testing by NGS in a reallife setting of chronic myeloid malignancies. A total of 177 patients were enrolled, partitioned into two overlapping groups. In group A (n=94), the indication was to search for clonal hematopoiesis, in a context of suspected myelodysplastic syndrome or myeloproliferative neoplasia. In group B (n=95), the theranostic impact of somatic mutations was studied. A panel of 34 genes was used on DNA extracted from blood or bone marrow samples. Within group A, the detection of clonal hematopoiesis supported the diagnosis of chronic myeloid malignancies for 31 patients while the absence of clonal hematopoiesis ruled out the suspected diagnosis in 47 patients. Within group B, NGS identified prognostically relevant somatic mutations in 32 patients, which had a therapeutic impact in 18 cases. By determining the presence or absence of somatic mutations, the application of NGS in daily practice was found to be useful for an integrated final diagnosis in 83% of the patients. Moreover, the search for somatic mutations had a prognostic impact that led to treatment modification in 19% of the cases. This study outlines the fact that adequate implementation of new investigations may have a significant positive medico-economic impact by enabling appropriate management of patients.

Sequential mutational evaluation of CALR -mutated myeloproliferative neoplasms with thrombocytosis reveals an association between CALR allele burden evolution and disease progression.

In myeloproliferative neoplasms (MPN), JAK2V617F allele burden measurement has an impact on prognosis that helps in patient monitoring. Less is known about its usefulness in CALR-mutated cases. Additional mutations found by next-generation sequencing have also shown an impact on prognosis that may drive therapeutic choices, especially in myelofibrosis, but few studies focused on CALR-mutated patients. We performed a molecular evaluation combining next-generation sequencing with a myeloid panel and CALR allele burden measurement at diagnosis and during follow-up in a cohort of 45 patients with CALR-mutated essential thrombocythaemia. The bone marrow histology was also blindly reviewed in order to apply the WHO2016 classification. The most frequently mutated gene was TET2 (11/21 mutations). CALR type 1-like patients appear to have a more complex molecular landscape. We found an association between disease progression and CALR allele burden increase during follow-up, independently of additional mutations and WHO2016-reviewed diagnosis. Patients with disease progression at the time of follow-up showed a significant increase in CALR allele burden (+16·7%, P = 0·005) whereas patients without disease progression had a stable allele burden (+3·7%, P = 0·194). This result argues for clinical interest in CALR allele burden monitoring.

WT1 gene is overexpressed in myeloproliferative neoplasms, especially in myelofibrosis.

Classical Philadelphia-negative myeloproliferative neoplasms include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). They are characterized by the presence of driver mutations of JAK2, CALR or MPL genes. Overexpression of WT1 is used as a marker of minimal residual disease in acute myeloid leukemia, especially after allogeneic stem cell transplantation (SCT). We investigated WT1 expression at diagnosis in 152 MPN patients and showed that the WT1 transcript was overexpressed in PMFs and PVs compared to controls. In particular, WT1 transcript levels were higher in PMF than in ET and PV. WT1 transcript levels were significantly increased during myelofibrotic transformation of ET or PV. Using multivariate linear regression, high WT1 transcript levels in PMF were associated with age over 65, splenomegaly and thrombocytopenia. The ROC curve analysis showed that a level of WT1 transcript >10 WT1 copies/104ABL1 enabled the diagnosis of PMF with a specificity of 95.8% (PMF vs ET; ROC AUC = 0.91). In myelofibrosis, studying follow-ups of WT1 transcript showed that this marker is of interest after allogeneic SCT. These results demonstrate that WT1 overexpression is a simple marker of myelofibrosis in MPN and could be used during patient follow-up.

MiR-10a and HOXB4 are overexpressed in atypical myeloproliferative neoplasms.

BACKGROUND: Atypical Myeloproliferative Neoplasms (aMPN) share characteristics of MPN and Myelodysplastic Syndromes. Although abnormalities in cytokine signaling are common in MPN, the pathophysiology of atypical MPN still remains elusive. Since deregulation of microRNAs is involved in the biology of various cancers, we studied the miRNome of aMPN patients. METHODS: MiRNome and mutations in epigenetic regulator genes ASXL1, TET2, DNMT3A, EZH2 and IDH1/2 were explored in aMPN patients. Epigenetic regulation of miR-10a and HOXB4 expression was investigated by treating hematopoietic cell lines with 5-aza-2'deoxycytidine, valproic acid and retinoic acid. Functional effects of miR-10a overexpression on cell proliferation, differentiation and self-renewal were studied by transducing CD34+ cells with lentiviral vectors encoding the pri-miR-10a precursor. RESULTS: MiR-10a was identified as the most significantly up-regulated microRNA in aMPN. MiR-10a expression correlated with that of HOXB4, sitting in the same genomic locus. The transcription of these two genes was increased by DNA demethylation and histone acetylation, both necessary for optimal expression induction by retinoic acid. Moreover, miR-10a and HOXB4 overexpression seemed associated with DNMT3A mutation in hematological malignancies. However, overexpression of miR-10a had no effect on proliferation, differentiation or self-renewal of normal hematopoietic progenitors. CONCLUSIONS: MiR-10a and HOXB4 are overexpressed in aMPN. This overexpression seems to be the result of abnormalities in epigenetic regulation mechanisms. Our data suggest that miR-10a could represent a simple marker of transcription at this genomic locus including HOXB4, widely recognized as involved in stem cell expansion.

Sequential analysis of 18 genes in polycythemia vera and essential thrombocythemia reveals an association between mutational status and clinical outcome.

Philadelphia-negative classical myeloproliferative neoplasms (MPN) are clonal diseases characterized by driver mutations of JAK2, MPL, or CALR. Additional mutations may occur in epigenetic regulators, signaling, or splicing genes that may be useful in the prognostic assessment of MPN patients. In primary myelofibrosis, molecular-based prognostic scoring systems have been recently proposed, but few data are available to date for polycythemia vera (PV) and essential thrombocythemia (ET). In this study, we used a next generation sequencing-based 18-gene panel in 50 JAK2V617F positive PV and JAK2V617F positive ET patients from an institutional cohort investigated at diagnosis and at 3-year follow-up (3y). Disease progression at 3y was defined by a composite criterion. Patients (28 PV and 22 ET) were included according to their clinical status, with or without disease progression. At diagnosis, we found 28 additional mutations in 21 of the 50 patients. Patients with disease progression were more likely to have at least one additional mutation. There was no difference between PV and ET. All patients with two or more additional mutations exhibited disease progression at 3y. No novel mutations appeared at 3y. The allele burden increase by at least one mutation at 3y was more frequent in patients with disease progression. Our data suggest that screening for additional mutations in PV and ET could identify patients at a higher risk of disease progression. © 2017 Wiley Periodicals, Inc.

Efficacy and safety of antibiotics targeting Gram-negative bacteria in nosocomial pneumonia: a systematic review and Bayesian network meta-analysis.

BACKGROUND: Multiple randomized controlled studies have compared numerous antibiotic regimens, including new, recently commercialized antibiotics in the treatment of nosocomial pneumonia (NP). The objective of this Bayesian network meta-analysis (NMA) was to compare the efficacy and the safety of different antibiotic treatments for NP. METHODS: We conducted a systematic search of PubMed, Medline, Web of Science, EMBASE and the Cochrane Library databases from 2000 through 2021. The study selection included studies comparing antibiotics targeting Gram-negative bacilli in the setting of NP. The primary endpoint was 28 day mortality. Secondary outcomes were clinical cure, microbiological cure and adverse events. RESULTS: Sixteen studies encompassing 4993 patients were included in this analysis comparing 13 antibiotic regimens. The level of evidence for mortality comparisons ranged from very low to moderate. No significant difference in 28 day mortality was found among all beta-lactam regimens. Only the combination of meropenem plus aerosolized colistin was associated with a significant decrease of mortality compared to using intravenous colistin alone (OR = 0.43; 95% credible interval [0.17-0.94]), based on the results of the smallest trial included. The clinical failure rate of ceftazidime was higher than meropenem with (OR = 1.97; 95% CrI [1.19-3.45]) or without aerosolized colistin (OR = 1.40; 95% CrI [1.00-2.01]), imipemen/cilastatin/relebactam (OR = 1.74; 95% CrI [1.03-2.90]) and ceftazidime/avibactam (OR = 1.48; 95% CrI [1.02-2.20]). For microbiological cure, no substantial difference between regimens was found, but ceftolozane/tazobactam had the highest probability of being superior to comparators. In safety analyses, there was no significant difference between treatments for the occurrence of adverse events, but acute kidney failure was more common in patients receiving intravenous colistin. CONCLUSIONS: This network meta-analysis suggests that most antibiotic regimens, including new combinations and cefiderocol, have similar efficacy and safety in treating susceptible Gram-negative bacilli in NP. Further studies are necessary for NP caused by multidrug-resistant bacteria. Registration PROSPERO CRD42021226603.

IL-1ß promotes MPN disease initiation by favoring early clonal expansion of JAK2-mutant hematopoietic stem cells.

ABSTRACT: JAK 2-V617F is the most frequent somatic mutation causing myeloproliferative neoplasm (MPN). JAK2-V617F can be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the prevalence of MPNs. The factors controlling the conversion of JAK2-V617F CHIP to MPN are largely unknown. We hypothesized that interleukin-1ß (IL-1ß)-mediated inflammation can favor this progression. We established an experimental system using bone marrow (BM) transplantations from JAK2-V617F and GFP transgenic (VF;GFP) mice that were further crossed with IL-1ß-/- or IL-1R1-/- mice. To study the role of IL-1ß and its receptor on monoclonal evolution of MPN, we performed competitive BM transplantations at high dilutions with only 1 to 3 hematopoietic stem cells (HSCs) per recipient. Loss of IL-1ß in JAK2-mutant HSCs reduced engraftment, restricted clonal expansion, lowered the total numbers of functional HSCs, and decreased the rate of conversion to MPN. Loss of IL-1R1 in the recipients also lowered the conversion to MPN but did not reduce the frequency of engraftment of JAK2-mutant HSCs. Wild-type (WT) recipients transplanted with VF;GFP BM that developed MPNs had elevated IL-1ß levels and reduced frequencies of mesenchymal stromal cells (MSCs). Interestingly, frequencies of MSCs were also reduced in recipients that did not develop MPNs, had only marginally elevated IL-1ß levels, and displayed low GFP-chimerism resembling CHIP. Anti-IL-1ß antibody preserved high frequencies of MSCs in VF;GFP recipients and reduced the rate of engraftment and the conversion to MPN. Our results identify IL-1ß as a potential therapeutic target for preventing the transition from JAK2-V617F CHIP to MPNs.

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.