Clinical Characteristics and Diagnosis of Nonaccelerating MDS/MPN-U Patient with Granulocyte Dysplasia.

BACKGROUND: The goal was to improve the clinical cognition of nonaccelerating myelodysplastic/myeloproliferative neoplasms-unclassifiable (MDS/MPN-U) and avoid misdiagnosis or delayed diagnosis. METHODS: The clinical manifestations, laboratory indicators, histopathology, and therapeutic effects of a patient with nonaccelerating MDS/MPN-U were analyzed and the relevant literature was reviewed. RESULTS: Blood routine: white blood cell 98.48 x 109/L, red blood cell 3.20 x 1012/L, basophils 0.42 x 109/L, eosinophils 1.31 x 109/L, hemoglobin 112 g/L, and platelet 113 x 109/L. Blood smears showed granulocytosis and cells at various stages, polylobular granulocytes also can be seen. Bone marrow images show granulocytosis and dysplastic neutrophils, such as binuclear granulocyte, cyclic nuclear granulocyte, nuclear punch, cytoplasm vacuoles, polylobular granulocytes and so on. Bone marrow biopsy: Bone marrow proliferation tumor, combined with cell morphology and molecular biochemistry is recommended. Gene test showed Jak-2 positive, BCR/ABL and MPL negative. Chromosome examination indicated the presence of 46, XY, add (2)(p25), del (12) (p11.2p13)[16]/46, XY. CONCLUSIONS: MDS/MPN-U with granulocytosis and dysplastic neutrophils is rare, mostly in the elderly, and the diagnosis should be made except for other myeloid tumors. Currently, there is no uniform treatment guideline or expert consensus. The treatment options are limited and need to be further confirmed by more studies. MDS/ MPN-U with granulocytosis and dysplastic neutrophils has adverse prognostic factors such as advanced age, increase of bone marrow original cells and related gene mutations. Whether the adverse prognosis is related to specific gene mutations and cytogenetic variation remains to be clarified by more research data.

Assessment of Total Antioxidant Capacity, 8-Hydroxy-2'-deoxy-guanosine, the Genetic Landscape, and Their Associations in BCR::ABL-1-Negative Chronic and Blast Phase Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs), namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal stem cell disorders defined by an excessive production of functionally mature and terminally differentiated myeloid cells. MPNs can transform into secondary acute myeloid leukemia (sAML/blast phase MPN) and are linked to alterations in the redox balance, i.e., elevated concentrations of reactive oxygen species and markers of oxidative stress (OS), and changes in antioxidant systems. We evaluated OS in 117 chronic phase MPNs and 21 sAML cases versus controls by measuring total antioxidant capacity (TAC) and 8-hydroxy-2'-deoxy-guanosine (8-OHdG) concentrations. TAC was higher in MPNs than controls (p = 0.03), particularly in ET (p = 0.04) and PMF (p = 0.01). MPL W515L-positive MPNs had higher TAC than controls (p = 0.002) and triple-negative MPNs (p = 0.01). PMF patients who had treatment expressed lower TAC than therapy-free subjects (p = 0.03). 8-OHdG concentrations were similar between controls and MPNs, controls and sAML, and MPNs and sAML. We noted associations between TAC and MPNs (OR = 1.82; p = 0.05), i.e., ET (OR = 2.36; p = 0.03) and PMF (OR = 2.11; p = 0.03), but not sAML. 8-OHdG concentrations were not associated with MPNs (OR = 1.73; p = 0.62) or sAML (OR = 1.89; p = 0.49). In conclusion, we detected redox imbalances in MPNs based on disease subtype, driver mutations, and treatment history.

Neutrophil-specific expression of JAK2-V617F or CALRmut induces distinct inflammatory profiles in myeloproliferative neoplasia.

BACKGROUND: Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. METHODS: Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1ß. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. RESULTS: Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. CONCLUSIONS: Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

Mathematical modelling of stem and progenitor cell dynamics during ruxolitinib treatment of patients with myeloproliferative neoplasms.

Introduction: The Philadelphia chromosome-negative myeloproliferative neoplasms are a group of slowly progressing haematological malignancies primarily characterised by an overproduction of myeloid blood cells. Patients are treated with various drugs, including the JAK1/2 inhibitor ruxolitinib. Mathematical modelling can help propose and test hypotheses of how the treatment works. Materials and methods: We present an extension of the Cancitis model, which describes the development of myeloproliferative neoplasms and their interactions with inflammation, that explicitly models progenitor cells and can account for treatment with ruxolitinib through effects on the malignant stem cell response to cytokine signalling and the death rate of malignant progenitor cells. The model has been fitted to individual patients' data for the JAK2 V617F variant allele frequency from the COMFORT-II and RESPONSE studies for patients who had substantial reductions (20 percentage points or 90% of the baseline value) in their JAK2 V617F variant allele frequency (n = 24 in total). Results: The model fits very well to the patient data with an average root mean square error of 0.0249 (2.49%) when allowing ruxolitinib treatment to affect both malignant stem and progenitor cells. This average root mean square error is much lower than if allowing ruxolitinib treatment to affect only malignant stem or only malignant progenitor cells (average root mean square errors of 0.138 (13.8%) and 0.0874 (8.74%), respectively). Discussion: Systematic simulation studies and fitting of the model to the patient data suggest that an initial reduction of the malignant cell burden followed by a monotonic increase can be recapitulated by the model assuming that ruxolitinib affects only the death rate of malignant progenitor cells. For patients exhibiting a long-term reduction of the malignant cells, the model predicts that ruxolitinib also affects stem cell parameters, such as the malignant stem cells' response to cytokine signalling.

[Recent findings and advances in treatment of myeloproliferative neoplasms].

Many novel agents have been developed for BCR::ABL1-negaive myeloproliferative neoplasms (MPN), namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Some of these agents not only achieve hematologic complete response, reduce spleen size, and alleviate constitutional symptoms, but also induce molecular response, which means that they reduce the allele burden of driver gene mutations. These agents also prevent and alleviate fibrosis in bone marrow, which reduces the incidence of thrombotic events and disease progression and might improve prognosis. This article discusses the latest findings and promising treatments, including ongoing clinical trials, in PV, ET, and PMF.

NGS panel enhance precise diagnosis of myeloid neoplasms under WHO-HAEM5 and International Consensus Classification: An observational study.

This study aimed to assess hematological diseases next-generation sequencing (NGS) panel enhances the diagnosis and classification of myeloid neoplasms (MN) using the 5th edition of the WHO Classification of Hematolymphoid Tumors (WHO-HAEM5) and the International Consensus Classification (ICC) of Myeloid Tumors. A cohort of 112 patients diagnosed with MN according to the revised fourth edition of the WHO classification (WHO-HAEM4R) underwent testing with a 141-gene NGS panel for hematological diseases. Ancillary studies were also conducted, including bone marrow cytomorphology and routine cytogenetics. The cases were then reclassified according to WHO-HAEM5 and ICC to assess the practical impact of these 2 classifications. The mutation detection rates were 93% for acute myeloid leukemia (AML), 89% for myelodysplastic syndrome (MDS), 94% for myeloproliferative neoplasm (MPN), and 100% for myelodysplasia/myeloproliferative neoplasm (MDS/MPN) (WHO-HAEM4R). NGS provided subclassified information for 26 and 29 patients with WHO-HAEM5 and ICC, respectively. In MPN, NGS confirmed diagnoses in 16 cases by detecting JAK2, MPL, or CALR mutations, whereas 13 "triple-negative" MPN cases revealed at least 1 mutation. NGS panel testing for hematological diseases improves the diagnosis and classification of MN. When diagnosed with ICC, NGS produces more classification subtype information than WHO-HAEM5.

CSF3R mutated myeloid neoplasms: Beyond chronic neutrophilic leukemia.

CSF3R activating mutation is a genetic hallmark of chronic neutrophilic leukemia (CNL), and is also present in a subset of atypical chronic myeloid leukemia (aCML), but infrequent in other myeloid neoplasms. However, the occurrence of CSF3R mutations in various myeloid neoplasms is not well studied. Here we evaluate the spectrum of CSF3R mutations and the clinicopathologic features of CSF3R mutated myeloid neoplasms. We retrospectively identified CSF3R mutations in a variety of myeloid neoplasms: two CNL, three atypical chronic myeloid leukemia (aCML), nine acute myeloid leukemia (AML), one chronic myelomonocytic leukemia, and one myeloproliferative neoplasm. The prototypic T618I mutation was found in 50% of cases: CNL (2/2), aCML (2/3) and AML (4/9). We observed a new recurrent CSF3R mutation Q776* in 25% of cases, and a potential-germline mutation in a 20-year-old patient. Co-occurring mutations were often in epigenetic modifier and spliceosome. IDH/RUNX1 and tumor suppressor mutations were frequent in AML but absent in CNL/aCML. All CNL/aCML patients succumbed within 2-years of diagnosis. We demonstrate that CSF3R mutations are not restricted to CNL. CNL and aCML show similar clinicopathologic and molecular features, suggesting that CNL may be best classified as myelodysplastic/myeloproliferative neoplasm rather than myeloproliferative neoplasm.

[Relationship between DTA Mutations and Thromboembolism in Patients with Myeloproliferative Neoplasms].

OBJECTIVE: To analyze the DTA (DNMT3A, TET2, ASXL1) mutations in patients with myeloproliferative neoplasms (MPN), and preliminarily explore their correlation with thromboembolism. METHODS: Clinical characteristics of 62 patients diagnosed de novo MPN at Central Hospital Affiliated to Shandong First Medical University from September 2016 to September 2022 were retrospectively analyzed. Next-generation sequencing was used to detect 35 MPN-related genes, and the DTA mutations in MPN patients and their relationship with thromboembolic events were analyzed. RESULTS: 75.8% (47/62) of the patients presented pathogenic non-driver mutations, and the mean number of pathogenic non-driver mutations per patient was 1.08. Among them, the most frequently mutated non-driver genes were TET2 (38.7%, 24/62), DNMT3A (9.7%, 6/62) and ASXL1 (6.5%, 4/62). The presence of DTA gene mutations was 50% (31/62) in the total MPN patients, and mainly accompanied by driver mutations. The mutation rate of DTA in patients aged ≥60 years was significantly higher than that in patients <60 years old (P =0.039). The incidence of thromboembolism in patients with DTA mutation was 58.1% (18/31), which was significantly higher than that in patients without DTA mutation (19.4%, 6/31) (P =0.002). The TET2 gene mutation rate in MPN patients with thromboembolism was 66.7% (16/24), which was significantly higher than that in patients without thromboembolism (21.1%, 8/38) (P =0.00). CONCLUSION: Patients with MPN have a higher incidence of DTA mutations, which are mainly accompanied by driver gene mutations. The incidence of thromboembolism in MPN patients with DTA mutations is higher than that in patients without DTA mutations. Especially, the elderly (≥60 years) essential thrombocythemia(ET) and polycythemia vera(PV) patients with TET2 mutation should be vigilant for thromboembolic events.

Treatment approach and outcomes of patients with accelerated/blast-phase myeloproliferative neoplasms in the current era.

ABSTRACT: Progression of myeloproliferative neoplasms (MPNs) to accelerated or blast phase is associated with poor survival outcomes. Since 2017 there have been several therapies approved for use in acute myeloid leukemia (AML); these therapies have been incorporated into the management of accelerated/blast-phase MPNs (MPN-AP/BP). We performed a multicenter analysis to investigate outcomes of patients diagnosed with MPN-AP/BP in 2017 or later. In total, 202 patients were identified; median overall survival (OS) was 0.86 years. We also analyzed patients based on first-line treatment; the 3 most common approaches were intensive chemotherapy (n = 65), DNA methyltransferase inhibitor (DNMTi)-based regimens (n = 65), and DNMTi + venetoclax-based regimens (n = 54). Median OS was not significantly different by treatment type. In addition, we evaluated response by 2017 European LeukemiaNet AML criteria and 2012 MPN-BP criteria in an effort to understand the association of response with survival outcomes. We also analyzed outcomes in 65 patients that received allogeneic hematopoietic stem cell transplant (allo-HSCT); median OS was 2.30 years from time of allo-HSCT. Our study demonstrates that survival among patients with MPN-AP/BP is limited in the absence of allo-HSCT even in the current era of therapeutics and underscores the urgent need for new agents and approaches.

Trisomy 8 Defines a Distinct Subtype of Myeloproliferative Neoplasms Driven by the MYC-Alarmin Axis.

Despite advances in understanding the genetic abnormalities in myeloproliferative neoplasms (MPN) and the development of JAK2 inhibitors, there is an urgent need to devise new treatment strategies, particularly for patients with triple-negative (TN) myelofibrosis (MF) who lack mutations in the JAK2 kinase pathway and have very poor clinical outcomes. Here we report that MYC copy number gain and increased MYC expression frequently occur in TN-MF and that MYC-directed activation of S100A9, an alarmin protein that plays pivotal roles in inflammation and innate immunity, is necessary and sufficient to drive development and progression of MF. Notably, the MYC-S100A9 circuit provokes a complex network of inflammatory signaling that involves numerous hematopoietic cell types in the bone marrow microenvironment. Accordingly, genetic ablation of S100A9 or treatment with small molecules targeting the MYC-S100A9 pathway effectively ameliorates MF phenotypes, highlighting the MYC-alarmin axis as a novel therapeutic vulnerability for this subgroup of MPNs. Significance: This study establishes that MYC expression is increased in TN-MPNs via trisomy 8, that a MYC-S100A9 circuit manifest in these cases is sufficient to provoke myelofibrosis and inflammation in diverse hematopoietic cell types in the BM niche, and that the MYC-S100A9 circuit is targetable in TN-MPNs.

Increased CXCL10 (IP-10) is associated with advanced myeloproliferative neoplasms and its loss dampens erythrocytosis in mouse models.

Key studies in pre-leukemic disorders have linked increases in pro-inflammatory cytokines with accelerated phases of the disease, but the precise role of the cellular microenvironment in disease initiation and evolution remains poorly understood. In myeloproliferative neoplasms (MPNs), higher levels of specific cytokines have been previously correlated with increased disease severity (tumor necrosis factor-alpha [TNF-α], interferon gamma-induced protein-10 [IP-10 or CXCL10]) and decreased survival (interleukin 8 [IL-8]). Whereas TNF-α and IL-8 have been studied by numerous groups, there is a relative paucity of studies on IP-10 (CXCL10). Here we explore the relationship of IP-10 levels with detailed genomic and clinical data and undertake a complementary cytokine screen alongside functional assays in a wide range of MPN mouse models. Similar to patients, levels of IP-10 were increased in mice with more severe disease phenotypes (e.g., JAK2V617F/V617F TET2-/- double-mutant mice) compared with those with less severe phenotypes (e.g., CALRdel52 or JAK2+/V617F mice) and wild-type (WT) littermate controls. Although exposure to IP-10 did not directly alter proliferation or survival in single hematopoietic stem cells (HSCs) in vitro, IP-10-/- mice transplanted with disease-initiating HSCs developed an MPN phenotype more slowly, suggesting that the effect of IP-10 loss was noncell-autonomous. To explore the broader effects of IP-10 loss, we crossed IP-10-/- mice into a series of MPN mouse models and showed that its loss reduces the erythrocytosis observed in mice with the most severe phenotype. Together, these data point to a potential role for blocking IP-10 activity in the management of MPNs.

Molecular Pathology of Myeloid Neoplasms: Molecular Pattern Recognition.

Despite the apparent complexity of the molecular genetic underpinnings of myeloid neoplasms, most myeloid mutational profiles can be understood within a simple framework. Somatic mutations accumulate in hematopoietic stem cells with aging and toxic insults, termed clonal hematopoiesis. These "old stem cells" mutations, predominantly in the epigenetic and RNA spliceosome pathways, act as "founding" driver mutations leading to a clonal myeloid neoplasm when sufficient in number and clone size. Subsequent mutations can create the genetic flavor of the myeloid neoplasm ("backseat" drivers) due to their enrichment in certain entities or act as progression events ("aggressive" drivers) during clonal evolution.

Phase 1 study of azacitidine in combination with quizartinib in patients with FLT3 or CBL mutated MDS and MDS/MPN.

We conducted a phase 1 study evaluating 3 dose levels of quizartinib (30 mg, 40 mg or 60 mg) in combination with azacitidine for HMA-naïve or relapsed/refractory MDS or MDS/MPN with FLT3 or CBL mutations. Overall, 12 patients (HMA naïve: n=9, HMA failure: n=3) were enrolled; 7 (58 %) patients had FLT3 mutations and 5 (42 %) had CBL mutations. The maximum tolerated dose was not reached. Most common grade 3-4 treatment-emergent adverse events were thrombocytopenia (n=5, 42 %), anemia (n=4, 33 %), lung infection (n=2, 17 %), skin infection (n=2, 17 %), hyponatremia (n=2, 17 %) and sepsis (n=2, 17 %). The overall response rate was 83 % with median relapse-free and overall survivals of 15.1 months (95 % CI 0.0-38.4 months) and 17.5 months (95 % CI NC-NC), respectively. FLT3 mutation clearance was observed in 57 % (n=4) patients. These data suggest quizartinib is safe and shows encouraging activity in FLT3-mutated MDS and MDS/MPN. This study is registered at Clinicaltrials.gov as NCT04493138.

Risk factors for ischemic stroke in patients with Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms.

BACKGROUND: Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-negative MPNs) are linked with various complications, notably ischemic stroke. The study aims to identify risk factors for ischemic stroke in Ph-negative MPNs patients. METHODS: Patients were categorized into two groups based on whether they had experienced ischemic stroke. Subsequently, an analysis of demographics, biochemical makers, and genetic mutations (JAK2V617F and CALR mutations), was conducted to identify potential associations with an elevated risk of ischemic stroke in individuals with Ph-negative MPNs. RESULTS: A total of 185 patients diagnosed with Ph-negative MPNs participated in the study, including 82 with essential thrombocythemia (ET), 78 with polycythemia vera (PV), and 25 with primary myelofibrosis (PMF). Among these, 57 patients (30.8 %) had a history of ischemic stroke. Independent risk factors associated with ischemic stroke in Ph-negative MPNs patients included hypertension (OR = 5.076) and smoking (OR = 5.426). Among ET patients, smoking (OR = 4.114) and an elevated percentage of neutrophils (OR = 1.080) were both positively correlated with ischemic stroke incidence. For PV patients, hypertension (OR = 4.647), smoking (OR = 6.065), and an increased percentage of lymphocytes (OR = 1.039) were independently associated with ischemic stroke. Regardless of the presence of the JAK2V617F mutation, hypertension was the sole positively and independently associated risk factor for ischemic stroke. The odds ratios for patients with the JAK2V617F mutation was 3.103, while for those without the mutation, it was 11.25. CONCLUSIONS: Hypertension was a more substantial factor associated with an increased incidence of ischemic stroke in Ph-negative MPNs patients.

Enhanced Molecular Response in Myeloproliferative Neoplasms with Complete JAK2V617F Inhibition.

SUMMARY: Dunbar, Bowman, and colleagues present here a novel genetic mouse model with inducible and reversible expression of the JAK2V617F mutation in the endogenous locus. Results from this study clearly demonstrate an absolute requirement for myeloproliferative neoplasm-initiating cells for this mutation in their survival and imply that more efficacious inhibitors could be curative for these patients even in the setting of additional cooperating mutations. See related article by Dunbar et al., p. 737 (8).