Philadelphia Chromosome-negative Myeloproliferative Neoplasms Research Articles

Secondary Solid Cancers Among Patients with Philadelphia Chromosome-Negative Myeloproliferative Neoplasms: A Multicenter Study.

We investigated the occurrence and characteristics of secondary solid cancers (SSC) in Philadelphia chromosome-negative myeloproliferative neoplasm (Ph- MPN) patients from Türkiye. We identified the potential risk factors for SSC development including the impact of cytoreductive therapies and assess the influence of SSC on patient survival. 1013 Ph- MPN patients diagnosed between 1995 and 2022 was retrospectively analyzed. Data related to demographics, clinical and laboratory parameters, SSC development, cytoreductive therapy exposure and survival outcomes were collected. Statistical analyses were performed using SPSS 26.0 software. Of the Ph- MPN patients, 6.6% developed SSC, with carcinoma being the most common type. Older age at Ph- MPN diagnosis and male gender were associated with SSC occurrence. Ph- MPN patients diagnosed with SSC and patients with no diagnosis of SSC showed no significant difference for complete blood count, spleen size, Ph- MPN diagnostic groups and driver mutation frequencies. However, SSC patients showed a higher frequency of arterial thrombosis and tendency towards increased rate for total thrombosis (p=0.030, p=0.069; respectively). In multivariate analysis, arterial thrombosis was the sole independent risk factor and interferon (IFN)-based therapy the sole protective factor for SSC development. Median overall survival (OS) did not differ between patients with and without SSC except for polycythemia vera (PV) patients with SSC, who had shorter OS (175±15 and 321±26 months, respectively; p = 0.005). Our study highlights the prevalence and characteristics of SSC in Turkish patients diagnosed with Ph- MPN. Arterial thrombosis was associated with increased SSC risk while IFN-based therapy offered potential protection from SSC. Screening for SSC in Ph- MPN patients with arterial thrombosis may be relevant. These findings emphasize the importance of malignancy screening in Ph- MPN patients, especially in high-risk subgroups and call for further research to elucidate the underlying mechanisms and optimize treatment strategies.

Superior detection of low-allele burden Janus kinase 2 V617F mutation and monitoring clonal evolution in myeloproliferative neoplasms using chip-based digital PCR.

The JAK2 V617F is a prevalent driver mutation in Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPNs), significantly affecting disease progression, immunophenotype, and patient outcomes. The World Health Organization (WHO) guidelines highlight the JAK2 V617F mutation as one of the key diagnostic criterions for Ph-MPNs. In this study, we analyzed 283 MPN samples with the JAK2 V617F mutation to assess the effectiveness of three detection technologies: chip-based digital PCR (cdPCR), real-time quantitative PCR (qPCR), and next-generation sequencing (NGS). Additionally, we investigated the relationship between JAK2 V617F mutant allele burden (% JAK2 V617F) and various laboratory characteristics to elucidate potential implications in MPN diagnosis. Our findings demonstrated high conformance of cdPCR with qPCR/NGS for detecting % JAK2 V617F, but the mutant allele burdens detected by qPCR/NGS were lower than those detected by cdPCR. Moreover, the cdPCR exhibited high sensitivity with a limit of detection (LoD) of 0.08% and a limit of quantification (LoQ) of 0.2% for detecting % JAK2 V617F in MPNs. Clinical implications were explored by correlating % JAK2 V617F with various laboratory characteristics in MPN patients, revealing significant associations with white blood cell counts, lactate dehydrogenase levels, and particularly β2-microglobulin (β2-MG) levels. Finally, a case report illustrated the application of cdPCR in detecting low-allele burdens in a de novo chronic myeloid leukemia (CML) patient with a hidden JAK2 V617F subclone, which expanded during tyrosine kinase inhibitor (TKI) treatment. Our findings underscore the superior sensitivity and accuracy of cdPCR, making it a valuable tool for early diagnosis and monitoring clonal evolution.

Multi-omics differences in the bone marrow between essential thrombocythemia and prefibrotic primary myelofibrosis

Essential thrombocythemia (ET) and prefibrotic primary myelofibrosis (pre-PMF) are Philadelphia chromosome-negative myeloproliferative neoplasms. These conditions share overlapping clinical presentations; however, their prognoses differ significantly. Current morphological diagnostic methods lack reliability in subtype differentiation, underlining the need for improved diagnostics. The aim of this study was to investigate the multi-omics alterations in bone marrow biopsies of patients with ET and pre-PMF to improve our understanding of the nuanced diagnostic characteristics of both diseases. We performed proteomic analysis with 4D direct data-independent acquisition and microbiome analysis with 2bRAD-M sequencing technology to identify differential protein and microbe levels between untreated patients with ET and pre-PMF. Laboratory and multi-omics differences were observed between ET and pre-PMF, encompassing diverse pathways, such as lipid metabolism and immune response. The pre-PMF group showed an increased neutrophil-to-lymphocyte ratio and decreased high-density lipoprotein and cholesterol levels. Protein analysis revealed significantly higher CXCR2, CXCR4, and MX1 levels in pre-PMF, while APOC3, APOA4, FABP4, C5, and CFB levels were elevated in ET, with diagnostic accuracy indicated by AUC values ranging from 0.786 to 0.881. Microbiome assessment identified increased levels of Mycobacterium, Xanthobacter, and L1I39 in pre-PMF, whereas Sphingomonas, Brevibacillus, and Pseudomonas_E were significantly decreased, with AUCs for these genera ranging from 0.833 to 0.929. Our study provides preliminary insights into the proteomic and microbiome variations in the bone marrow of patients with ET and pre-PMF, identifying specific proteins and bacterial genera that warrant further investigation as potential diagnostic indicators. These observations contribute to our evolving understanding of the multi-omics variations and possible mechanisms underlying ET and pre-PMF.

The characteristics of CALR mutations in myeloproliferative neoplasms: a clinical experience from a tertiary care center in Qatar and a literature review

ABSTRACT Background Myeloproliferative neoplasms (MPNs) are hematological disorders characterized by abnormal production of myeloid cells due to genetic mutations. Since 2013, researchers have identified somatic mutations in the Calreticulin (CALR) gene, primarily insertions or deletions, in two Philadelphia chromosome-negative MPNs; essential thrombocytosis (ET) and primary myelofibrosis (PMF), and occasionally in chronic myelomonocytic leukemia (CMML). This study aims to identify the various types of CALR mutations and their impact on CALR-positive MPN patients’ clinical manifestations and outcomes. Methods A single-center retrospective study was conducted. The data was collected from pre-existing records. The study was carried out on Philadelphia-negative MPN patients who were being followed up on at the NCCCR (National Center for Cancer Care and Research) to assess the clinical manifestation and outcome of disease treatment. All patients included, were followed in our center between January 1, 2008, and November 20, 2021. Results A total of 50 patients with CALR-positive MPN were reviewed with a median follow-up of three years (1–11). This cohort included 31 (62%) patients with ET, 10 (20%) patients with PMF, and 9 (18%) patients with prefibrotic myelofibrosis (pre-MF). The study involved 38 (76%) male and 12 (24%) female patients. There were 16 (32%) patients diagnosed before the age of 40, 24 (48%) patients diagnosed between the ages of 40 and 60; and 10 (20%) patients diagnosed after the age of 60. Molecular analysis showed 24 (48%) patients with CALR type 1, 21 (42%) patients with CALR type 2, and 5 (10%) patients with none Type 1, none Type 2 CALR mutations. Two patients have double mutations; 1(2%) with none Type 1, none Type 2 CALR and JAK2 mutations, and 1(2%) with CALR type 1 and MPL mutations. The thrombotic events were 3 (6%) venous thromboembolisms, 3 (6%) abdominal veins thromboses, 2 (4%) strokes, and 4 (8%) ischemic cardiac events. Only 4 (8%) patients progressed to Myelofibrosis and were carrying CALR 1 mutations, and 1 (2%) patient progressed to AML with CALR 2 mutation. Conclusion The data shows a significant rise in CALR-positive MPN diagnoses in younger people, emphasizing the need for a better assessment tool to improve disease management and reduce complications.

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

IntroductionThe 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 methodsWe 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).ResultsThe 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).DiscussionSystematic 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.

A comparative analysis of the clinical and genetic profiles of blast phaseBCR::ABL1-negative myeloproliferative neoplasm and acute myeloid leukemia, myelodysplasia-related.

The classic Philadelphia chromosome-negative myeloproliferative neoplasms (Ph (-) MPNs), have variable potential for progression to the blast phase (MPN-BP) of the disease. Except initiated by distinct driver mutations, MPN-BP frequently carry similar genetic abnormalities defining acute myeloid leukemia myelodysplasia-related (AML-MR). Because of dissimilar initial pathogenesis, MPN-BP and AML-MR are retained under different disease categories. To determine if separately classifying these entities is justified, we compare MPN-BP with AML-MR patients based on mutational landscape and clinical parameters. 104 MPN-BP patients and 145 AML-MR patients were identified with available clinical, cytogenetic, and genetic data. AML-MR patients presented with a higher blast count (median, 51% vs. 30%) while MPN-BP patients had higher WBC counts, platelet counts and bone marrow cellularity (all p<0.0001). Patients with MPN-BP showed similar genetic mutations with similar mutation pattern (functional domain, hotspot and locus involved by the mutations) but a different mutation rate from AML-MR, with more frequent JAK2, CALR, MPL, ASXL1, IDH2, SETBP1 and SRSF2 mutations and less frequent TP53 and DNMT3A mutations. The overall survival (OS) of MPN-BP (OS post-BP-progression) is comparable to that of AML-MR (median OS, 9.5 months vs. 13.1 months, p=0.20). In addition, the subgroups of MPN-BP show similar OS as AML-MR. When harboring certain mutation such as TP53, ASXL1, DNMT3A, TET2, RUNX1, IDH1, IDH2, EZH2, U2AF1, BCOR and SRSF2, MPN-BP and AML-MR patients carrying the same somatic mutation show no difference in OS. MPN-BP and AML-MR harbor similar somatic mutations and clinical outcomes, suggesting a unified clinical disease entity.

Conventional Cytogenetic Analysis and Array CGH + SNP Identify Essential Thrombocythemia and Prefibrotic Primary Myelofibrosis Patients Who Are at Risk for Disease Progression.

The Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPNs) are a heterogeneous group of clonal hematopoietic malignancies that include polycythemia vera (PV), essential thrombocythemia (ET), and the prefibrotic form of primary myelofibrosis (prePMF). In this study, we retrospectively reviewed the karyotypes from conventional cytogenetics (CC) and array Comparative Genomic Hybridization + Single Nucleotide Polymorphism (aCGH + SNP) in patients with ET or prePMF to determine whether the combined analysis of both methodologies can identify patients who may be at a higher risk of disease progression. We performed a comprehensive genomic review on 169 patients with a clinical diagnosis of ET (154 patients) or prePMF (15 patients). Genomic alterations detected by CC or array-CGH + SNP were detected in 36% of patients. In patients who progressed, 68% had an abnormal genomic finding by either technology. There was a shorter progression-free survival (PFS) among patients who were cytogenetically abnormal or who were cytogenetically normal but had an abnormal aCGH + SNP result. Leveraging the ability to detect submicroscopic copy number alterations and regions of copy neutral-loss of heterozygosity, we identified a higher number of patients harboring genomic abnormalities than previously reported. These results underscore the importance of genomic analysis in prognostication and provide valuable information for clinical management and treatment decisions.

TNF-α is a predictive marker in distinguishing myeloproliferative neoplasm and idiopathic erythrocytosis/thrombocytosis: development and validation of a non-invasive diagnostic model.

Philadelphia-chromosome negative myeloproliferative neoplasms (MPN) exhibit phenotypic similarities with JAK/STAT-unmutated idiopathic erythrocytosis and thrombocytosis (IE/IT). We aimed to develop a clinical diagnostic model to discern MPN and IE/IT. A retrospective study was performed on 77 MPN patients and 32 IE/IT patients in our center from January 2018 to December 2023. We investigated the role of hemogram, cytokine and spleen size in differentiating MPN and IE/IT among newly onset erythrocytosis and thrombocytosis patients. Independent influencing factors were integrated into a nomogram for individualized risk prediction. The calibration and discrimination ability of the model were evaluated by concordance index (C-index), calibration curve. MPN had significantly higher TNF-α level than IE/IT, and the TNF-α level is correlated with MF-grade. Multivariable analyses revealed that TNF-α, PLT count, age, size of spleen were independent diagnostic factors in differentiating MPN and IE/IT. Nomograms integrated the above 4 factors for differentiating MPN and IE/IT was internally validated and had good performance, the C-index of the model is 0.979. The elevation of serum TNF-α in MPN patients is of diagnostic significance and is correlated with the severity of myelofibrosis. The nomogram incorporating TNF-α with age, PLT count and spleen size presents a noteworthy tool in the preliminary discrimination of MPN patients and those with idiopathic erythrocytosis or thrombocytosis. This highlights the potential of cytokines as biomarkers in hematologic disorders.

Mutational Analysis of Driver and Non-driver Mutations of Philadelphia Chromosome-negative Myeloproliferative Neoplasms; Diagnosis and Recent Advances in Treatment

Mutational Analysis of Driver and Non-driver Mutations of Philadelphia Chromosome-negative Myeloproliferative Neoplasms; Diagnosis and Recent Advances in Treatment

Neutrophil to lymphocyte ratio in myelofibrosis patients treated with ruxolitinib may predict prognosis and rate of discontinuation.

Myelofibrosis (MF) is a clonal Philadelphia chromosome negative myeloproliferative neoplasm (Ph-MPN). MF is featured by an inflammatory condition that can also drive the progression of disease. Ruxolitinib (ruxo) is the-first-in-class Jak1/2 inhibitor approved for treatment ofMF, proved to reduce spleen volume and decrease symptom burden. In various malignancies neutrophil-to-lymphocyte ratio (NLR) has been indicated as predictor of progression free survival (PFS) and overall survival (OS). NLR might reflect the balance between systemic inflammation and immunity and is emerging as a prognostic biomarker in several neoplasms, including the hematological ones. We analyzed a cohort of 140 MF patients treated with ruxo to validate baseline NLR (as a continuous variable and as a cut-off 2) as predictor of OS and of ruxo treatment discontinuation. We found that both baseline NLR as a continuous variable [HR 0.8 (95% CI: 0.7-0.9) (p = .006)] and NLR (<2 vs. ≥2) [HR 3.4 (95% CI: 1.6-7.0) (p = .001)] were significantly associated with OS. Censoring for patients undergone allotransplant, baseline NLR <2 was predictive of an earlier ruxo any-other-cause discontinuation [HR 3.7 (95%CI 1.7-8.3) (p < .001)]. NLR before starting ruxo treatment may be used as a simple and early predictor of OS and earlier ruxo discontinuation in clinical practice.

Health-Related Quality of Life and Symptom Burden Profile of Patients with Pre-Fibrotic and Overt Myelofibrosis: A Preliminary Report from the Gimema-Prophecy Observational Study

Background Patients with overt myelofibrosis (MF) suffer a significant symptom burden and impairment of health-related quality of life (HRQoL). However, very little evidence-based data is available on HRQoL and the symptom burden of patients with pre-fibrotic MF. Aim The primary objective of this study was to describe the HRQoL profile of patients with pre-fibrotic MF relative to patients with overt MF. The secondary objective was to examine disease-specific symptom prevalence in both groups. Methods. Baseline data from an ongoing prospective multicenter observational study by the GIMEMA Group were analyzed. The inclusion criteria of this study were: adult patients diagnosed with Philadelphia chromosome-negative myeloproliferative neoplasm (MPN), according to the 2016 WHO classification, within one year before the registration date. For the purpose of this analysis, only patients with pre-fibrotic and overt MF were considered. HRQoL profile was assessed with the EORTC QLQ-C30, which consists of 30 items and includes five functional scales (physical, role, emotional, social, and cognitive), three symptoms (fatigue, nausea, vomiting, and pain), a global health status/QoL scale and six single items (dyspnea, insomnia, appetite loss, constipation, diarrhea, and financial difficulties). Well-established evidence-based guidelines for interpreting the QLQ-C30 were used to determine clinically meaningful differences between groups (i.e., pre-fibrotic vs overt MF). Given the exploratory nature of the study, n o statistical testing was performed on mean score differences. Symptom prevalence was assessed by items of the MPN-SAF TSS. The MPN-SAF TSS is a comprehensive and brief 10-item instrument that concisely assesses the prevalence and severity of debilitating symptoms of MF. The percentage of patients who reported symptoms on the MPN-SAF TSS was based on the number of patients who answered at least 1 on a given item. Results. Overall, 104 patients with a median age of 68 years (IQR: 59-76) were enrolled across 20 community and university-based hospitals. More than half of the patients were male (n=58; 56%), and 55 (53%) revealed splenomegaly. Thirty-five patients (34%) reported at least one comorbidity. The median time since diagnosis for the overall group was 4 months (IQR: 1-7), and there were 69 and 35 patients diagnosed with overt and pre-fibrotic MF, respectively. The median age of patients with overt MF and pre-fibrotic MF was 69 (IQR: 61-76) and 63 (IQR: 57-75), respectively. At study entry, no statistically significant differences were observed between the two groups regarding age, sex, prevalence of comorbidities, WBC, and PLT counts. However, patients with overt MF had lower Hb levels (p=.003). Inspection of the HRQoL profile, according to the EORTC QLQ-C30, showed that patients with overt MF had a statistically and clinically meaningful worse global HRQoL compared to patients' pre-fibrotic MF (Δ=11.4). The following functioning scales were also mostly impaired in patients with overt MF: role functioning (Δ=14.2), cognitive functioning (Δ=4.7), and social functioning (Δ=8.1) (Figure 1). With regard to symptoms severity, patients with overt MF reported clinically meaningful higher severity for fatigue (Δ=6), pain (Δ=7.2), insomnia (Δ=6), constipation (Δ=5.4), and appetite loss (Δ=6.8). However, patients with pre-fibrotic MF reported clinically meaningful higher severity for dyspnea (Δ=5.5). The prevalence of symptoms according to the MPN-SAF TSS, overall and by disease group, is reported in Table 1. The prevalence of symptoms in the overall group of patients was high, with the top three most prevalent symptoms being fatigue (78%), concentration problems (53%), and inactivity (50%). Notably, the prevalence of symptoms was also high in patients with pre-fibrotic MF, with some one-third of them reporting: fatigue, concentrations problem, inactivity, early satiety, abdominal discomfort, night sweats, itching, and bone pain. Conclusions. To the best of our knowledge, this is one of the first evidence describing HRQoL and the symptom burden of patients with pre-fibrotic MF. Although our preliminary data suggest that the HRQoL profile of patients with pre-fibrotic MF was generally worse than those with overt MF, our findings also indicate a high symptom prevalence in patients with pre-fibrotic MF.

MPN Transformation Is Characterized By Heterogeneous Shifts in Lineage Character

Introduction: The Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are characterized by the clonal proliferation of differentiated hematologic cells. However, there is a limited understanding of how perturbations at the transcriptional level may contribute to accelerated or blast-phase disease, together referred to here as transformation. Here we characterize transcriptomic characteristics and cell type composition of MPNs along the transition to transformed disease. Methods: RNAseq and clinical data were collected on 261 patients who were part of BeatAML, including those with MPN, de-novo AML, and transformed MPN. This was combined with data from 56 transformed MPN patients seen at MSK, 11 of whom also had paired chronic-phase data available. Differential expression analysis was performed with DESeq2; both GSEA and GSVA were used to evaluate gene-set enrichment and pathways of biological relevance. A custom RNA-seq lineage deconvolution algorithm was used to estimate hematopoietic stage proportions and was used for unsupervised hierarchical clustering of cases. Kaplan-Meier methods were used to estimate survival among transformed MPN lineage subtypes. Results: The initial analysis included 207 de novo AML cases and 75 MPN-BP cases from BeatAML and MSK on which RNAseq and molecular data were integrated across cohorts. Unsupervised analysis was performed using hierarchical clustering applied to batch corrected, variance stabilized RNA-seq count data. Transformed MPN cases were enriched in 2 of 6 clusters (N = 10, 33.3%; N = 36, 54.5%). Notably, the molecular characteristics in these clusters are varied, suggesting that multiple genomic perturbations may lead to similar pathways of transformation. Gene set variation analysis (GSVA) demonstrated a coalescence of transcriptomic perturbation into specific pathways for transformed MPNs, such as hematopoietic stem cell gene sets (q-value = 0.00018). On the contrary, AML cases typically demonstrated upregulation in later stages of hematopoiesis, such as monocytes (q-value =0.00016). In evaluating specific genes differentially expressed between transformed MPNs compared to de novo AML and tAML patients, microRNA MIR29B1 was particularly upregulated in MPN-BP (logFC = 9.52, q &amp;lt; 0.01), while the proto-oncogene CT45A1 had decreased expression in transformed MPN compared to other AMLs (logFC = -7.47, q &amp;lt; 0.01). Paired GSEA of the 11 patients with myelofibrosis MF and transformed disease showed that transformed disease was enriched for CD34-positive HSCs (NES = 2.64, q-value &amp;lt; 0.0001) and E2F transcription factors (NES 2.66, q-value &amp;lt; 0.0001), as well as doxorubicin resistance (NES 2.40, q-value &amp;lt; 0.0001). Given enrichment of the CD34 positive HSC geneset, we performed our RNA-seq based lineage deconvolution on each of the paired samples ( Figure 1). MPNs appeared to transform in a heterogenous fashion reminiscent of the biological diversity in AML. In aggregate, transformation was associated with decreased monocytic (p = 0.002), natural killer (NK) (p = 0.042) character and expansion of common lymphoid progenitor character (p = 0.022). Lineage composition changes varied, with certain samples showing HSC expansion (ID: 54910, ID: 46567) and others exhibiting LMPP expansion (ID: 29485, 2456). Figure 2 defines seven distinct clusters of transformed MPN patients, driven by their lineage components. While these clusters are transcriptionally distinct, we again see that they are not defined by their genomic or cytogenetic alterations. We evaluated for a difference in overall survival (OS) between each cluster and intermediate-risk de novo AML. We found significantly worse OS among patients in three MPN-BP clusters - MPP (p =0.008), LMPP (p = 0.038), and GMP (p &amp;lt; 0.001) - represented as clusters 2, 5, and 7 in Figure 2. Compared to intermediate risk AML, transformed MPNs had significantly worse OS (HR: 1.79 95% CI: 1.18, 2.71, p = 0.0057). Discussion: To our knowledge, this is the first ever description of the transcriptional characteristics of MPN-BP, its comparison to de novo AML, and the first paired comparison of transcriptional and lineage characteristics for patients with chronic-phase MPNs that subsequently transformed. Our study provides valuable insights into the transcriptomic landscape of transformed MPN including both accelerated and blast phase disease, and draws novel contrasts to de novo AML.

Outcomes of Patients with Accelerated/Blast-Phase MPNs That Received Allogeneic Stem-Cell Transplant in the Current Era of Myeloid Therapies

Background Patients (pts) with Philadelphia chromosome-negative (Ph-neg) myeloproliferative neoplasms (MPNs) that progress to an accelerated phase (AP) or blast phase (BP) have a median overall survival (mOS) of less than 1 year (Odenike, Blood 2018; Tam et al, JCO 2009). Allogeneic hematopoietic stem cell transplant (allo-HCT) is the only curative approach for MPN-AP/BP, but mOS remains about a year for pts with MPN-BP that undergo allo-HCT (2005-2019) (Orti et al, AJH 2023). We previously reported on outcomes of pts with MPN-AP/BP treated in the current era of myeloid therapies (2017 onward) and found a mOS of 0.73 years in a multi-center cohort of 80 pts (Patel et al, ASH 2022). We aim to characterize treatment patterns and outcomes of pts with MPN-AP/BP that underwent allo-HCT via a multi-center retrospective analysis. Methods Retrospective chart review was performed at 8 institutions to identify pts with pathology-confirmed Ph-neg MPN-AP/BP diagnosed in 2017 or later. All patients that met inclusion criteria were included. Response was assessed using 2017 European Leukemia Net AML (2017 ELN) criteria (Dohner et al, Blood 2017). OS from diagnosis of MPN-AP/BP and from time of allo-HCT was calculated utilizing Kaplan-Meier analysis. Results The full MPN-AP/BP cohort consists of 180 pts. This analysis includes 58 pts that underwent allo-HCT for MPN-AP/BP across 8 institutions. Median age at MPN-AP/BP diagnosiswas 64 years. The most common therapies for chronic-phase MPN included hydroxyurea (55%), JAK inhibitor (JAKi) (26%), interferon (9%), and hypomethylating agent (HMA, 4%). No patient previously received allo-HCT for chronic-phase MPN. Driver mutations included JAK2 (60%), CALR (22%), MPL (10%), but some pts had triple-negative disease (7%). Sixty-two percent of pts had high-risk disease by 2017 ELN criteria. Forty-four pts had next-generation sequencing performed at time of MPN-AP/BP diagnosis. The most frequently co-occurring mutations detected at time of MPN-AP/BP diagnosis were ASXL1 (30%), SRSF2 (23%), RUNX1 (23%), TP53 (18%), TET2 (16%), IDH2 (11%), U2AF1 (7%), EZH2 (7%), and IDH1 (9%). Additional characteristics are in Table 1. One pt proceeded directly to allo-HCT at time of MPN-AP/BP diagnosis. Frontline therapies (1L) for MPN-AP included intensive chemotherapy (IC) (n=26), hypomethylating agent (HMA) + venetoclax (VEN) (n=16), HMA monotherapy (n=14), and low-dose cytarabine (LDAC) + VEN + JAKi (n=1). Responses are summarized in Table 2A. Thirty-seven pts proceeded to allo-HCT after 1L therapy (17 after IC, 10 after HMA+VEN, 9 after HMA, 1 after LDAC+VEN+JAKi). Twenty pts proceeded to allo-HCT after 2 nd-line and beyond (2L+) therapies. Therapies utilized and responses are summarized in Table 2B. Transplant-specific characteristics include 15 pts receiving myeloablative conditioning while 43 received reduced-intensity conditioning. Donor source was matched unrelated donor for 33 pts, haploidentical donor for 10 pts, matched related donor for 9 patients, mismatched unrelated donor for 5 patients, and cord blood for 1 patient. Twenty-two pts developed acute GVHD while 15 developed chronic GVHD. Amongst the 58 pts that underwent allo-HCT, 19 had relapse of disease and 8 patients received treatment after relapse. Amongst the full cohort of 180 patients, mOS was 0.72 years from time of MPN-AP/BP diagnosis. The mOS for the 58 pts that underwent allo-HCT was 1.82 years from time of MPN-AP/BP diagnosis and 1.30 years from time of allo-HCT. Survival analysis by initial MPN-AP/BP therapy demonstrated a mOS from time of MPN-AP/BP diagnosis of 1.9 years for HMA-based therapy, 1.9 years for HMA + VEN, and 1.5 years for IC ( p=0.006). We also analyzed mOS by disease status prior to transplant. Thirty-four pts had a complete response (CR) or CR with incomplete count recovery (CRi) with mOS of 1.7 years from time of allo-HCT, 12 pts with a partial response (PR) or morphologic leukemia-free state (MLFS) had mOS of 1.8 years, and 11 pts with treatment failure (TF) had mOS of 0.47 years (p=0.82). Conclusions Our cohort of 180 pts with MPN-AP/BP treated in the current era of myeloid therapies had limited survival with a mOS of 0.72 years from MPN-AP/BP diagnosis. Amongst the 58 pts that underwent allo-HCT the mOS was 1.82 years with longer mOS seen in pts that received HMA or HMA + VEN as their 1L therapy when compared to IC. This underscores the need for novel management strategies even in pts eligible for allo-HCT.

Mutation Clearance of Driver Mutations after Transplantation for Myelofibrosis Predicts Outcome

Myelofibrosis is one of the Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) characterized by clonal myeloproliferation. Allogeneic hematopoietic stem-cell transplantation is the only potentially curative treatment for patients with myelofibrosis. With that, it can resolve fibrosis and lead to complete loss of the clonal burden. More than 90% of myelofibrosis patients harbor driver mutations in one of the 3 genes JAK2, CALR, and MPL, with JAK2 mutations being associated with worse outcomes. We herein report a study to determine whether the persistence of cells with MPN-associated driver mutations in the early period after allogeneic hematopoietic stem-cell transplantation was associated with outcomes. Patient samples for molecular analysis were prospectively collected from peripheral blood at 5 different time points: at start of conditioning prior to stem cell infusion, at days 30, 100, 180, and 1 year after transplantation. We applied quantitative PCR technology to detect individual driver mutations in JAK2, CALR, and MPL (with a high sensitivity of 0.01%). Simulation was applied to explain variances at different time points. Multivariable modelling was employed to determine independent effects. A total of 312 patients receiving first reduced-intensity conditioning transplantation were included. Distribution of driver mutation genotype at time of transplantation was as follows: 67% with JAK2, 19% with CALR, 4% with MPL, and 8% were triple-negative. Conditioning regimen comprised busulfan and fludarabine. Donor platform for transplantation was: matched related (18%), matched unrelated (58%), haploidentical (1%), and mismatched unrelated (23%). Disease risks at time of transplantation were low (1%), intermediate-1 (22%), intermediate-2 (55%), and high (22%). Median follow-up of the total cohort was 6 years. The median allele burden at time of transplantation was significantly (P&amp;lt;0.001) different between driver mutations: 20% (range, 0.1-100%) for JAK2, 44% (range, 3-54%) for CALR, 60% (range, 43-99%) for MPL. CALR and MPL showed earlier mutation clearance after transplantation compared with JAK2 ( Table). Mutation clearance was achieved at day 30 after transplantation in 46% with JAK2, 67% for CALR, and 30% for MPL (P=0.02). At day 100 after transplantation, 66% of JAK2, 75% of CALR, and 100% of MPL showed complete mutation clearance (P=0.09). Complete mutation clearance at day 30 after transplantation was predictive of post-transplantation relapse at 1 year (P=0.009), showing cumulative incidence of relapse of 6% versus 16%. Similar results were seen for complete mutation clearance at day 100 after transplantation, demonstrating cumulative incidence of relapse of 4% versus 24% (P&amp;lt;0.001). Mutation clearance at day 30 and 100 after transplantation explained 80% and 87% of variance of later time points. The effect of mutation clearance on post-transplant relapse was irrespective of driver mutation status ( Figure). 6-year disease-free survival according to mutation type was 52% for JAK2, 63% for CALR, and 80% for MPL (P=0.045). Mutation clearance at day 30 after transplantation was associated with better disease-free survival, irrespective of driver mutation status ( Figure). Moreover, day-30 complete mutation clearance predicted disease-free survival in JAK2, showing rates of 60% for negative versus 47% in JAK2 positive patients (P=0.05). Importantly, achievement of mutation clearance at day 30 after transplantation could also distinguish different risk groups in patients with onset better prognosis harboring CALR/MPL mutations. The 6-year disease-free survival was 76% for patients with mutation clearance versus 59% for CALR/MPL-positive patients(P=0.02) . Multivariate analysis (adjusting for donor type, diagnosis of primary or secondary myelofibrosis, allele burden at time of transplantation, and age) confirmed that patients who had mutation clearance at day 30 after transplantation had lower risk of relapse, better disease-free and overall survival. In conclusion, this is the first study to show that CALR and MPL are associated with earlier mutation clearance after transplantation compared with JAK2. Mutation clearance at day 30 and 100 after transplantation was the best predictor of relapse and disease-free survival, independent of driver mutation status.

Inflammation Mediated By TNFα Disrupts the Autophagic Flux and Oxidative Phosphorylation of Megakaryocytes and Platelets through the Downregulation of STX17

Background. JAK2 V617F-positive polycythemia vera (PV) is the most common Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Elevated levels of TNFα, and a significantly higher risk of thrombosis and bleeding (thrombohemorrhage) are hallmarks of JAK2 V617F PV. Platelets from patients with PV are characterized by hyperreactivity (fast activation of the αIIbβ3 integrin), higher P-selectin exposure (thromboinflammation), and phosphatidylserine (procoagulant potential). We have also found that in addition to these phenotypes, platelets from PV patients have dysfunctional autophagy promoting mitochondrial dysfunction, accumulation of dysmorphic mitochondria, and decreased clot retraction. Therefore, we hypothesized that inflammation mediated by TNFα (like in some cancers) dysregulates autophagy in JAK2 V617F PV by reprogramming (immunometabolism) metabolism of MKs and platelets. Methods. Platelets from sex and age-matched healthy subjects and patients with PV were analyzed. Platelets were treated with chloroquine (CQ) to analyze the autophagic flux by western blot and electron microscopy. Platelet bioenergetics was analyzed using the Seahorse extracellular flux analyzer. Semi-quantitative metabolomics was performed using the Vanquish UHPLC system. Thrombus formation was measured by microfluidics using T-TAS and ATLAS equipment. Clot retraction was assessed by measuring the weight of serum extruded from the clot. Platelets for clot retraction were treated with CQ or EACC. The MEG-01 cell line was treated with TNFα to analyze its impact on the autophagic flux, mitochondrial metabolism, and transcription (RNAseq). Murine studies using platelets from wild type (WT), DARE (transgenic mice with constitutive expression of TNFα or a neutralizing antibody against TNFα were performed. Results. Platelets from patients with PV have significantly impaired autophagic flux (p&amp;lt;0.01), mitochondrial dysfunction (p&amp;lt;0.002), and accumulation of dysmorphic mitochondria (p&amp;lt;0.007). Platelets from PV patients have significantly lower levels of STX17 (p&amp;lt;0.008), a key regulatory molecule of autophagosome and lysosome fusion (Figure 1). The inhibition of STX17 from healthy donor platelets decreased clot stability (p&amp;lt;0.004). Moreover, platelets from chronically (DARE) and acutely inflamed mice (WT mice treated with TNFα) exhibited the same autophagic defect (p&amp;lt;0.03 and 0.02, respectively) secondary to the downregulation of STX17 (p&amp;lt;0.01 and 0.02, respectively). In vitro experiments showed that megakaryocytes downregulate the transcription and translation STX17 (p&amp;lt;0.006 and p&amp;lt;0.03, respectively) in response to TNFα. Finally, in vivo treatment of inflamed mice with a neutralizing anti-TNFa antibody restored the expression of STX17 (p&amp;lt;0.0001) and the autophagic flux (p&amp;lt;0.002). Conclusion. Using several in vitro and in vivo approaches, we demonstrated for the first time the immunometabolic effect of TNFα in platelets and megakaryocytes. TNFα is a key regulator of the autophagic flux in platelets inducing platelet hyperreactivity and decreasing clot stability. Our findings may explain why patients with JAK2 V617F PV are at increased risk for both thrombo-hemorrhagic events and suggest that targeting autophagy might lower this risk.

Splanchnic Vein Thrombosis in Myelofibrosis-An Underappreciated Hallmark of Disease Phenotype.

Splanchnic vein thrombosis (SVT) encompasses thrombosis in the vessels of the splanchnic basin and has a relatively rare occurrence with a reported frequency in the general population of 1-2%. An episode of seemingly unprovoked SVT almost always triggers a diagnostic work-up for a Philadelphia chromosome-negative myeloproliferative neoplasm (MPN), since atypical site thrombosis is a hallmark of MPN-associated thrombophilia. Primary myelofibrosis (PMF) is a rare MPN with an estimated incidence between 0.1 and 1/100,000 per year. Although prothrombotic tendency in PMF is not envisioned as a subject of specific therapeutic management, unlike other MPNs, such as polycythemia vera (PV) and essential thrombocythemia (ET), thrombotic risk and SVT prevalence in PMF may be comparably high. Additionally, unlike PV and ET, SVT development in PMF may depend more on procoagulant mechanisms involving endothelium than on blood cell activation. Emerging results from registry data also suggest that PMF patients with SVT may exhibit lower risk and better prognosis, thus highlighting the need for better thrombotic risk stratification and identifying a subset of patients with potential benefit from antithrombotic prophylaxis. This review highlights specific epidemiological, pathogenetic, and clinical features pertinent to SVT in myelofibrosis.

Philadelphia chromosome-negative myeloproliferative chronic neoplasms: is clonal hematopoiesis the main determinant of autoimmune and cardio-vascular manifestations?

In this article, we reviewed the possible mechanisms linking the clonal hematopoiesis of indeterminate potential (CHIP) to chronic myeloproliferative neoplasms (MPNs), autoimmune diseases (ADs), and cardiovascular diseases (CADs). CHIP is characterized by the presence of clonal mutations with an allelic frequency >2% in the peripheral blood without dysplasia, overt hematological neoplasms, or abnormalities in blood cell count. The prevalence may reach 20% of elderly healthy individuals and is considered a risk factor for myelodysplastic neoplasms and acute leukemia. In MPNs, CHIP is often associated with mutations such as JAK2V617F or DNMT3A, TET2, or ASXL1, which exhibit a 12.1- and 1.7-2-fold increase in CADs. Specifically, JAK2-mutated cells produce excessive cytokines and reactive oxygen species, leading to proinflammatory modifications in the bone marrow microenvironment. Consequently, the likelihood of experiencing thrombosis is influenced by the variant allele frequency (VAF) of the JAK2V617F mutation, which also appears to be correlated with anti-endothelial cell antibodies that sustain thrombosis. However, DNMT3A mutations induce pro-inflammatory T-cell polarization and activate the inflammasome complex, while TET2 downregulation leads to endothelial cell autophagy and inflammatory factor upregulation. As a result, in patients with TET2 and DNMT3A-related CHIP, the inflammasome hyperactivation represents a potential cause of CADs. CHIP also occurs in patients with large and small vessel vasculitis, while ADs are more frequently associated with MPNs. In these diseases, monocytes and neutrophils play a key role in the formation of neutrophil extracellular trap (NET) as well as anti-endothelial cell antibodies, resulting in a final procoagulant effect. ADs, such as systemic lupus erythematosus, psoriasis, and arthritis, are also characterized by an overexpression of the Rho-associated coiled-coil containing protein kinase 2 (ROCK2), a serine/threonine kinase that can hyperactivate the JAK-STAT pathway. Interestingly, hyperactivation of ROCK2 has also been observed in myeloid malignancies, where it promotes the growth and survival of leukemic cells. In summary, the presence of CHIP, with or without neoplasia, can be associated with autoimmune manifestations and thrombosis. In the presence of these manifestations, it is necessary to consider a "disease-modifying therapy" that may either reduce the clonal burden or inhibit the clonally activated JAK pathway.

Management of classical Philadelphia chromosome-negative myeloproliferative neoplasms in Asia: consensus of the Asian Myeloid Working Group.

Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized clinically by the proliferation of one or more hematopoietic lineage(s). The classical Philadelphia-chromosome (Ph)-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The Asian Myeloid Working Group (AMWG) comprises representatives from fifteen Asian centers experienced in the management of MPN. This consensus from the AMWG aims to review the current evidence in the risk stratification and treatment of Ph-negative MPN, to identify management gaps for future improvement, and to offer pragmatic approaches for treatment commensurate with different levels of resources, drug availabilities and reimbursement policies in its constituent regions. The management of MPN should be patient-specific and based on accurate diagnostic and prognostic tools. In patients with PV, ET and early/prefibrotic PMF, symptoms and risk stratification will guide the need for early cytoreduction. In younger patients requiring cytoreduction and in those experiencing resistance or intolerance to hydroxyurea, recombinant interferon-α preparations (pegylated interferon-α 2A or ropeginterferon-α 2b) should be considered. In myelofibrosis, continuous risk assessment and symptom burden assessment are essential in guiding treatment selection. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in MF should always be based on accurate risk stratification for disease-risk and post-HSCT outcome. Management of classical Ph-negative MPN entails accurate diagnosis, cytogenetic and molecular evaluation, risk stratification, and treatment strategies that are outcome-oriented (curative, disease modification, improvement of quality-of-life).

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.

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

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. 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. 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. 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). 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.