Proof of concept of triple COMBI therapy to prohibit MPN progression to AML.

Patients with accelerated or blast phase myeloproliferative neoplasms have a dismal prognosis. The report by de Castro et al. provides important information on the rationale and prospect for a novel therapeutic approach combining interferon-alpha2 with 5-azacytidine and a JAK1-2 inhibitor (ruxolitinib) to be explored in well-designed clinical trials. Commentary on: Castro et al. Ratio of stemness to interferon signalling as a biomarker and therapeutic target of myeloproliferative neoplasm progression to acute myeloid leukaemia. Br J Haematol 2024;204:206-220.

COVID-19 severity in patients with chronic lymphocytic leukemia treated with venetoclax: a single-center observational cohort study.

Patients with chronic lymphocytic leukemia (CLL) are at high risk of developing severe COVID-19. The present study was undertaken to elucidate COVID-19 related morbidity and mortality in CLL patients treated with venetoclax. We present a single-center study of 108 patients with small lymphocytic lymphoma or CLL treated with venetoclax. Primary outcome was 30-day COVID-19 mortality. Secondary outcomes included COVID-19 severity and hospitalization rate. Forty-eight (44%) patients had PCR-verified SARS-COV-2 between March 2020 and January 2023. Thirty-six patients (75%) presented with asymptomatic/mild COVID-19 and 12 (25%) with severe/critical disease. The hospitalization rate was 46% with a 30-day mortality rate of only 4% and severe comorbidities as the primary cause of death. COVID-19 severity and mortality were similar before and during the Omicron era. High CIRS-scores (P < 0.02) and thrombocytopenia (P < 0.01) were more frequent in patients with severe/critical disease. In real-world data, most venetoclax treated patients presented with mild COVID-19. Hospitalization and mortality rates were low compared to data of general CLL populations. Our data indicate that venetoclax was a safe treatment option for CLL patients during the pandemic.

JAK2V617F drives gut microbiota differences in patients with myeloproliferative neoplasms.

BACKGROUND: Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF) are myeloproliferative neoplasms (MPN). Inflammation is involved in the initiation, progression, and symptomology of the diseases. The gut microbiota impacts the immune system, infection control, and steady-state hematopoiesis. METHODS: We analyzed the gut microbiota of 227 MPN patients and healthy controls (HCs) using next-generation sequencing. We expanded our previous results in PV and ET patients with additional PV, pre-MF, and MF patients which allowed us to compare MPN patients collectively, MPN sub-diagnoses, and MPN mutations (separately and combined) vs. HCs (N = 42) and compare within MPN sub-diagnoses and MPN mutation. RESULTS: MPN patients had a higher observed richness (median, 245 [range, 49-659]) compared with HCs (191.5 [range, 111-300; p = .003]) and a lower relative abundance of taxa within the Firmicutes phylum; for example, Faecalibacterium (6% vs. 14%, p < .001). The microbiota of CALR-positive patients (N = 30) resembled that of HCs more than that of patients with JAK2V617F (N = 177). In JAK2V617F-positive patients, only minor differences in the gut microbiota were observed between MPN sub-diagnoses, illustrating the importance of this mutation. CONCLUSION: The gut microbiota in MPN patients differs from HCs and is driven by JAK2V617F, whereas the gut microbiota in CALR patients resembles HCs more.

Whole blood transcriptional profiling reveals highly deregulated atherosclerosis genes in Philadelphia-chromosome negative myeloproliferative neoplasms.

BACKGROUND: The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are associated with a huge comorbidity burden, including an increased risk of cardiovascular diseases. Recently, chronic inflammation has been suggested to be the driving force for clonal evolution and disease progression in MPN but also potentially having an impact upon the development of accelerated (premature) atherosclerosis. OBJECTIVES: Since chronic inflammation, atherosclerosis, and atherothrombosis are prevalent in MPNs and we have previously shown oxidative stress genes to be markedly upregulated in MPNs, we hypothesized that genes linked to development of atherosclerosis might be highly deregulated as well. METHODS: Using whole blood gene expression profiling in patients with essential thrombocythemia (ET; n = 19), polycythemia vera (PV; n = 41), or primary myelofibrosis (PMF; n = 9), we herein for the first time report aberrant expression of several atherosclerosis genes. RESULTS: Of 84 atherosclerosis genes, 45, 56, and 46 genes were deregulated in patients with ET, PV, or PMF, respectively. Furthermore, BCL2L1, MMP1, PDGFA, PTGS1, and THBS4 were progressively significantly upregulated and BCL2 progressively significantly downregulated from ET over PV to PMF (all FDR <0.05). CONCLUSIONS: We have for the first time shown massive deregulation of atherosclerosis genes in MPNs, likely reflecting the inflammatory state in MPNs in association with in vivo activation of leukocytes, platelets, and endothelial cells being deeply involved in the atherosclerotic process.

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.

Neutrophil-to-lymphocyte ratio and all-cause mortality with and without myeloproliferative neoplasms-a Danish longitudinal study.

The neutrophil-to-lymphocyte ratio(NLR) is increased in chronic inflammation and myeloproliferative neoplasms (MPN). We hypothesize that NLR is associated with all-cause mortality and mortality by comorbidity burden in the general population and individuals with MPN. We included 835,430 individuals from The Danish General Suburban Population Study, general practitioners, and outpatient clinics. We investigated NLR on mortality stratified by prevalent and incident MPN, essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), comorbidity burden (CCI-score), and the Triple-A risk score using hazard ratio (HR) and 95% confidence interval (95%CI). NLR 1-1.9 was the reference level. During a median follow-up of 11.2 years, 197,802 deaths were recorded. All-cause mortality increased for a stepwise increasing NLR with a HR (95%CI) for NLR ≥ 6 of 2.06(2.03-2.09) for the whole population and 2.93(2.44-3.50) in prevalent MPN. ET, PV, and MF had a HR (95%CI) for NLR ≥ 2 of 2.14(1.71-2.69), 2.19(1.89-2.54), and 2.31(1.91-2.80). Results were similar for incident MPN. Mortality was higher for stepwise increasing NLR and CCI-score(pinteraction < 2×10-16), with a HR for NLR ≥ 6 of 2.23(2.17-2.29), 4.10(4.01-4.20), and 7.69(7.50-7.89), for CCI-score 0, 1-2, or ≥3. The Triple-A risk score demonstrated alignment with NLR. Increasing NLR and comorbidity burden were associated with lower survival in individuals without MPN but were even worse in prevalent and incident MPN, ET, PV, and MF.

JAK2V617F mutation is highly prevalent in patients with ischemic stroke: a case-control study.

Ischemic stroke has a high recurrence rate despite treatment. This underlines the significance of investigating new possible cerebrovascular risk factors, such as the acquired gene mutation JAK2V617F found in 3.1% of the general population. We aimed to investigate the prevalence of the JAK2V617F mutation in a population with ischemic stroke compared with that in matched controls. We enrolled 538 consecutive Danish patients with ischemic stroke (mean age, 69.5 ± 10.9 years; 39.2% female) within 7 days of symptom onset. Using multiple-adjusted conditional logistic regression analysis, we compared the prevalence of JAK2V617F with that in age- and sex-matched controls free of ischemic cerebrovascular disease (ICVD) from the Danish General Suburban Population Study. DNA was analyzed for JAK2V617F mutation using sensitive droplet digital polymerase chain reaction in patients and controls. Of the 538 patients with ischemic stroke, 61 (11.3%) had JAK2V617F mutation. There were no differences in patient demographics or cerebrovascular comorbidities between the patients with and without mutations. Patients with ischemic stroke were more likely to have the JAK2V617F mutation than matched controls, in whom the JAK2V617F prevalence was 4.4% (odds ratio, 2.37; 95% confidence interval, 1.57-3.58; P < .001). A subanalysis stratified by smoking history revealed that the association was strongest in current smokers (odds ratio, 4.78; 95% confidence interval, 2.22-10.28; P < .001). Patients with ischemic stroke were 2.4 times more likely to have the JAK2V617F mutation than matched controls without ICVD when adjusting for other cerebrovascular risk factors. This finding supports JAK2V617F mutation as a novel cerebrovascular risk factor.

A novel integrated biomarker index for the assessment of hematological responses in MPNs during treatment with hydroxyurea and interferon-alpha2.

BACKGROUND: Conventional cytoreductive therapy for patients with chronic Philadelphia-negative myeloproliferative neoplasms (MPNs) includes hydroxyurea (HU), interferon-alpha2 (IFN), and anagrelide. HU is worldwide the most used cytoreductive agent, which lowers elevated blood cell counts within days in the large majority of patients. However, some patients may experience rebound cytosis when HU is reduced due to cytopenia, thereby potentially giving rise to fluctuating cell counts during therapy. Such rapid oscillations may be harmful and potentially elicit thrombosis. Treatment with IFN gradually lowers elevated cell counts within weeks and when the dosage is reduced, the cell counts do not rapidly increase but are sustained within the normal range in the large majority of patients. Conventional hematological response criteria are among others based upon single absolute cell count values and do not take into account the relative decreases toward normal for each cell count. MATERIALS, METHODS & RESULTS: Using serial data from the Danish DALIAH trial, we herein describe a novel integrated biomarker index for the assessment of hematological and molecular (JAK2V617F) responses in patients with MPNs during treatment with IFN or HU. DISCUSSION: This novel tool convincingly displays the superiority of IFN versus HU in normalizing elevated cell counts. Our results need to be validated in larger studies but already now call for studies of the safety and efficacy of combination therapy during the initial treatment of patients with MPNs.

Revisiting Circulating Extracellular Matrix Fragments as Disease Markers in Myelofibrosis and Related Neoplasms.

Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) arise due to acquired somatic driver mutations in stem cells and develop over 10-30 years from the earliest cancer stages (essential thrombocythemia, polycythemia vera) towards the advanced myelofibrosis stage with bone marrow failure. The JAK2V617F mutation is the most prevalent driver mutation. Chronic inflammation is considered to be a major pathogenetic player, both as a trigger of MPN development and as a driver of disease progression. Chronic inflammation in MPNs is characterized by persistent connective tissue remodeling, which leads to organ dysfunction and ultimately, organ failure, due to excessive accumulation of extracellular matrix (ECM). Considering that MPNs are acquired clonal stem cell diseases developing in an inflammatory microenvironment in which the hematopoietic cell populations are progressively replaced by stromal proliferation-"a wound that never heals"-we herein aim to provide a comprehensive review of previous promising research in the field of circulating ECM fragments in the diagnosis, treatment and monitoring of MPNs. We address the rationales and highlight new perspectives for the use of circulating ECM protein fragments as biologically plausible, noninvasive disease markers in the management of MPNs.

The gut microbiota in patients with polycythemia vera is distinct from that of healthy controls and varies by treatment.

Chronic inflammation is believed to play an important role in the development and disease progression of polycythemia vera (PV). Because an association between gut microbiota, hematopoiesis, and inflammation is well established, we hypothesized that patients with PV have a gut microbiota distinct from healthy control participants (HCs). Recombinant interferon alfa 2 (IFN-α2)-treatment of patients with PV is reportedly disease modifying in terms of normalization of elevated blood cell counts in concert with a reduction in the JAK2V617F allelic burden. Therefore, we hypothesized that patients treated with IFN-α2 might have a composition of the gut microbiota toward normalization. Herein, via amplicon-based next-generation sequencing of the V3 to V4 regions of the 16S ribosomal RNA gene, we report on an abnormal gut microbiota in 102 patients with PV compared with 42 HCs. Patients with PV had a lower alpha diversity and a lower relative abundance of several taxa belonging to Firmicutes (45%) compared with HCs (59%, P <.001). Furthermore, we report the composition of the gut microbiota to differ between the treatment groups (IFN-α2, hydroxyurea, no treatment, and combination therapy with IFN-α2 and ruxolitinib) and the HCs. These observations are highly interesting considering the potential pathogenetic importance of an altered gut microbiota for development of other diseases, including chronic inflammatory diseases. Our observations call for further gut microbiota studies to decipher potential causal associations between treatment and the gut microbiota in PV and related neoplasms.

Case Report: First longitudinal study of a patient with CALR positive clonal hematopoiesis of indeterminate potential developing into pre-fibrotic myelofibrosis.

Initial diagnosis of overt myeloproliferative neoplasms (MPNs) represents the juncture during clonal evolution when symptoms or complications prompt an afflicted individual to seek medical attention. In 30-40% of the MPN subgroups essential thrombocythemia (ET) and myelofibrosis (MF), somatic mutations in the calreticulin gene (CALR) are drivers of the disease resulting in constitutive activation of the thrombopoietin receptor (MPL). In the current study, we describe a healthy CALR mutated individual during a 12 year follow-up from initial identification of CALR clonal hematopoiesis of indeterminate potential (CHIP) to the diagnosis of pre-MF. The pre-diagnostic exponential development dynamics of the malignant clone demonstrated close correlation with the platelet counts, neutrophil-to-lymphocyte (NLR) ratio, and inversely correlated to hemoglobin and erythrocyte counts. Backward extrapolation of the growth rate indicated the potential for discovery of the malignant clone many years prior to presentation of overt disease, opening a window of opportunity for early treatment intervention. We did not find any additional mutations associated with MPNs and the current case report provides novel information regarding the development of a driver mutation and the association with blood cell counts prior to clinical manifestation of symptoms suggesting that pre-diagnostic dynamics may supplement future diagnostic criteria for early diagnosis and intervention in MPN patients.

Pronounced gut microbiota signatures in patients with JAK2V617F-positive essential thrombocythemia.

Essential thrombocythemia (ET) is part of the Philadelphia chromosome-negative myeloproliferative neoplasms. It is characterized by an increased risk of thromboembolic events and also to a certain degree hypermetabolic symptoms. The gut microbiota is an important initiator of hematopoiesis and regulation of the immune system, but in patients with ET, where inflammation is a hallmark of the disease, it is vastly unexplored. In this study, we compared the gut microbiota via amplicon-based 16S rRNA gene sequencing of the V3-V4 region in 54 patients with ET according to mutation status Janus-kinase 2 (JAK2V617F)-positive vs JAK2V617F-negative patients with ET, and in 42 healthy controls (HCs). Gut microbiota richness was higher in patients with ET (median-observed richness, 283.5; range, 75-535) compared with HCs (median-observed richness, 191.5; range, 111-300; P < 0.001). Patients with ET had a different overall bacterial composition (beta diversity) than HCs (analysis of similarities [ANOSIM]; R = 0.063, P = 0.004). Patients with ET had a significantly lower relative abundance of taxa within the Firmicutes phylum compared with HCs (51% vs 59%, P = 0.03), and within that phylum, patients with ET also had a lower relative abundance of the genus Faecalibacterium (8% vs 15%, P < 0.001), an important immunoregulative bacterium. The microbiota signatures were more pronounced in patients harboring the JAK2V617F mutation, and highly similar to patients with polycythemia vera as previously described. These findings suggest that patients with ET may have an altered immune regulation; however, whether this dysregulation is induced in part by, or is itself inducing, an altered gut microbiota remains to be investigated. IMPORTANCE Essential thrombocythemia (ET) is a cancer characterized by thrombocyte overproduction. Inflammation has been shown to be vital in both the initiation and progression of other myeloproliferative neoplasms, and it is well known that the gut microbiota is important in the regulation of our immune system. However, the gut microbiota of patients with ET remains uninvestigated. In this study, we characterized the gut microbiota of patients with ET compared with healthy controls and thereby provide new insights into the field. We show that the gut microbiota of patients with ET differs significantly from that of healthy controls and the patients with ET have a lower relative abundance of important immunoregulative bacteria. Furthermore, we demonstrate that patients with JAK2V617F-positive ET have pronounced gut microbiota signatures compared with JAK2V617F-negative patients. Thereby confirming the importance of the underlying mutation, the immune response as well as the composition of the microbiota.

Recombinant Interferon-ß in the Treatment of Polycythemia Vera and Related Neoplasms: Rationales and Perspectives.

About 30 years ago, the first clinical trials of the safety and efficacy of recombinant interferon-α2 (rIFN-α2) were performed. Since then, several single-arm studies have shown rIFN-α2 to be a highly potent anticancer agent against several cancer types. Unfortunately, however, a high toxicity profile in early studies with rIFN-α2 -among other reasons likely due to the high dosages being used-disqualified rIFN-α2, which was accordingly replaced with competitive drugs that might at first glance look more attractive to clinicians. Later, pegylated IFN-α2a (Pegasys) and pegylated IFN-α2b (PegIntron) were introduced, which have since been reported to be better tolerated due to reduced toxicity. Today, treatment with rIFN-α2 is virtually outdated in non-hematological cancers, where other immunotherapies-e.g., immune-checkpoint inhibitors-are routinely used in several cancer types and are being intensively investigated in others, either as monotherapy or in combination with immunomodulatory agents, although only rarely in combination with rIFN-α2. Within the hematological malignancies, rIFN-α2 has been used off-label for decades in patients with Philadelphia-negative chronic myeloproliferative neoplasms (MPNs)-i.e., essential thrombocythemia, polycythemia vera, and myelofibrosis-and in recent years rIFN-α2 has been revived with the marketing of ropeginterferon-α2b (Besremi) for the treatment of polycythemia vera patients. Additionally, rIFN-α2 has been revived for the treatment of chronic myelogenous leukemia in combination with tyrosine kinase inhibitors. Another rIFN formulation-recombinant interferon-ß (rIFN-ß)-has been used for decades in the treatment of multiple sclerosis but has never been studied as a potential agent to be used in patients with MPNs, although several studies and reviews have repeatedly described rIFN-ß as an effective anticancer agent as well. In this paper, we describe the rationales and perspectives for launching studies on the safety and efficacy of rIFN-ß in patients with MPNs.

Interferon-alpha2 treatment of patients with polycythemia vera and related neoplasms favorably impacts deregulation of oxidative stress genes and antioxidative defense mechanisms.

Chronic inflammation is considered a major driving force for clonal expansion and evolution in the Philadelphia-negative myeloproliferative neoplasms, which include essential thrombocythemia, polycythemia vera and primary myelofibrosis (MPNs). One of the key mutation drivers is the JAK2V617F mutation, which has been shown to induce the generation of reactive oxygen species (ROS). Using whole blood gene expression profiling, deregulation of several oxidative stress and anti-oxidative defense genes has been identified in MPNs, including significant downregulation of TP53, the NFE2L2 or NRF2 genes. These genes have a major role for maintaining genomic stability, regulation of the oxidative stress response and in modulating migration or retention of hematopoietic stem cells. Therefore, their deregulation might give rise to increasing genomic instability, increased chronic inflammation and disease progression with egress of hematopoietic stem cells from the bone marrow to seed in the spleen, liver and elsewhere. Interferon-alpha2 (rIFNα) is increasingly being recognized as the drug of choice for the treatment of patients with MPNs. Herein, we report the first gene expression profiling study on the impact of rIFNα upon oxidative stress and antioxidative defense genes in patients with MPNs (n = 33), showing that rIFNα downregulates several upregulated oxidative stress genes and upregulates downregulated antioxidative defense genes. Treatment with rIFNα induced upregulation of 19 genes in ET and 29 genes in PV including CXCR4 and TP53. In conclusion, this rIFNα- mediated dampening of genotoxic damage to hematopoietic cells may ultimately diminish the risk of additional mutations and accordingly clonal evolution and disease progression towards myelofibrotic and leukemic transformation.

[Elevated blood cell counts and vascular disease with the myeloproliferative neoplasms as model diseases].

Recent studies have shown the Philadelphia-negative myeloproliferative neoplasms (MPN) to be massively underdiagnosed and often preceded by a long pre-diagnostic phase of several years, in which many patients suffer serious vascular events. In this review, we focus on the urgent need for earlier diagnosis and treatment of MPN. Such efforts are foreseen to decrease morbidity and mortality for the individual patients and potentially reduce costs for health and social care systems.