Clonal Myeloproliferative Neoplasm Research Articles

Assessment of Molecular Response to Treatment of Chronic Myeloid Leukemia at Kigali University Teaching Hospital of Kigali

Abstract Introduction/Objective Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative neoplasm that gives proliferation advantage to the granulocytes, it is one of the commonest chronic hematological malignancies and it is a burden to the world especially Rwanda. Since the introduction of Tyrosine Kinase Inhibitors (TKIs) especially the first line Imatinib in 2001, the outcomes of CML have been much improved and currently, in developed world, the life expectancy of patients with CML is almost the same as general population. However, it is not the case in developing world especially Rwanda due to limitation on access of all TKIs. The aim of this study is to assess molecular response on Imatinib of patients with CML. Methods/Case Report This was a one arm open label prospective cohort study that recruited 31 patients with CML and on Imatinib for at least three months and followed for 11 months. The clinical features at diagnosis and recruitment were captured from the hospitals filing systems and investigator’s own assessment at the time of recruitment respectively. Information was filled to a well-structured questionnaire. Lab results at the time of diagnosis was obtained from laboratories reporting systems. Samples for full blood count and BCR-ABL1/ABL1 were withdrawn from the patients at the time of diagnosis and they were run at University Teaching Hospital of Kigali (CHUK). Data analysis was performed using Statistical Package for the Social Sciences (SPSS 28) and South Texas Art Therapy Association (STATA 15.0). Results (if a Case Study enter NA) Better response according to European LeukemiaNet (ELN) defined as optimal response was found among only 38.7% and resistance to imatinib defined by ELN as Failure and warning was found in 45.2% and 16.1% respectively. The eleven months survival rate was 93.5% in which it is 85.7% among those in treatment failure group. Late presentation showed by presence of very high White Blood Cells above 100x109/L and massive splenomegaly at diagnosis, and poor adherence to Imatinib according to Morsiky Medication Adherence Scale (MMAS-8) are associated with poor molecular response to Imatinib. Conclusion CML is a serious disease especially to those who suffer from it; on time diagnosis, access to all available lines of TKIs and better follow up are crucial for patients with CML to access the same life expectancy as general population as it is in developed world.

Unveiling aging dynamics in the hematopoietic system insights from single-cell technologies.

As the demographic structure shifts towards an aging society, strategies aimed at slowing down or reversing the aging process become increasingly essential. Aging is a major predisposing factor for many chronic diseases in humans. The hematopoietic system, comprising blood cells and their associated bone marrow microenvironment, intricately participates in hematopoiesis, coagulation, immune regulation and other physiological phenomena. The aging process triggers various alterations within the hematopoietic system, serving as a spectrum of risk factors for hematopoietic disorders, including clonal hematopoiesis, immune senescence, myeloproliferative neoplasms and leukemia. The emerging single-cell technologies provide novel insights into age-related changes in the hematopoietic system. In this review, we summarize recent studies dissecting hematopoietic system aging using single-cell technologies. We discuss cellular changes occurring during aging in the hematopoietic system at the levels of the genomics, transcriptomics, epigenomics, proteomics, metabolomics and spatial multi-omics. Finally, we contemplate the future prospects of single-cell technologies, emphasizing the impact they may bring to the field of hematopoietic system aging research.

Flow Cytometric Assessment of CD26-Positive Leukemic Stem Cells: A Rapid and Valuable Tool in the Diagnosis and Follow-Up of Chronic Myeloid Leukemia.

Context Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm. Recent studies have suggested that CD26-positive leukemic stem cells (LSCs) circulating in peripheral blood are specific for CML. Objective This study was undertaken to determine the proportion of CD26-positive LSCsat diagnosis and its change during tyrosine kinase inhibitor therapy. Design This prospective study was conducted on 43 cases of CML at diagnosis. For flow cytometry, peripheral blood cells were stained with CD45, CD34, CD38, CD3, and CD26. A sequential gating strategy with CD45/SSC (side scatter), CD34/SSC, and CD34/CD38 was applied to identify CD45+/34+/38- populations, from which CD26-positive stem cells were identified and compared with controls. Data analysis was done with Kaluza software. Results All patients diagnosed with CML were detected with CD26-positive LSCs. The median percentage of CD26-positive CML LSCs was 0.02 with a range of 0.001 to 1.77. None of the control samples showed CD26 positivity. The percentage and absolute count of CD26-positive CML LSCs were reduced after six months of tyrosine kinase therapy in patients with complete hematological remission. Conclusion Flow cytometric analysis of circulating CD26-positive CML LSCs is a non-invasive, rapid, and useful tool in the diagnosis and follow-up of CML.

Abstract 263: RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms for dynamic detection of clonal myeloproliferative neoplasm stem cell fitness

Abstract Introduction Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) derived disorders that can progress to acute myeloid leukemia (AML) at variable rates depending on macroenvironmental stressors and microenvironmental inflammatory cytokines. The total symptom score (TSS) and variant allele frequency (VAF) of mutations have proven valuable predictors of therapeutic response. Recently, we discovered inflammatory-cytokine-responsive ADAR1 RNA editing enzyme overexpression during MPN progression. However, dynamic prediction of clonal MPN stem cell fitness and progression to AML remains challenging. Methods We performed longitudinal 150-gene next-generation sequencing (NGS) analyses for 129 MPN patients. We developed single-cell RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms to quantify ADAR1 activity with a novel ADAR1 nanoluc-GFP reporter assay, whole transcriptome RNA editome analyses, and MPN stem cell replating assays. To identify novel RNA editing sites, we performed whole genome sequencing on CD34+ cells from 43 MPN patients and whole transcriptome sequencing on FACS-purified HSC and progenitor cells from 32 MPN patients and 24 non-MPN controls. Results Sequential NGS analysis was performed with a median follow-up of 958 days (range 0-4214; interquartile range (IQR) 314-1350). On average, 6 NGS analyses (range 1-19; IQR 3-9) were performed for each patient. In our cohort, 58.9% of patients were JAK2 V617F+, while mutations in CALR and ASXL1 were observed in 11.6% and 14.0% of cases, respectively. In total, 44.2% (N=57) of patients in our cohort received Inrebic, with 56% reaching the maximum dose of 400mg. Notably, most patients on Inrebic were JAK2 V617F+ (N=38, 67.7%). In JAK2 V617F+ patients on Inrebic, 50% (n=19) experienced a reduction in the JAK2 V617F VAF from their peak value with a median drop of 16% (range 4-57). Our analyses using READAR platforms revealed increased RNA editing levels in JAK2 V617F+ patients from whole transcriptome sequencing analyses compared with JAK2 wild-type patients. Transduction of our ADAR1 nanoluc-GFP reporter into CD34+ cells from MPN patients (N=7), enabled us to quantify ADAR1 activity in an ex vivo setting. Moreover, CD34+ cells from MPN patient samples (n=7) treated in stromal co-cultures with ADAR1 inhibitor, Rebecsinib, demonstrated inhibition of self-renewal in replating assays. A significant reduction in ADAR1-GFP reporter activity was observed in MPN CD34+ cells following Rebecsinib treatment of stromal co-cultures. Also, humanized ADAR1-GFP-luciferase reporter AML mouse models showed a significant reduction in ADAR1 activity and leukemia stem cell self-renewal following Rebecsinib treatment. Conclusion These READAR platform results suggest that ADAR1 activity can be utilized to predict clonal MPN stem cell fitness and progression. Citation Format: Inge van der Werf, Larisa Balaian, Jessica Pham, Wenxue Ma, Athena Mohebbi, Emma Klacking, Antonio Ruiz, Karla Mack, John Mascarenhas, Thomas Whisenant, Ludmil Alexandrov, Sheldon Morris, Catriona Jamieson. RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms for dynamic detection of clonal myeloproliferative neoplasm stem cell fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 263.

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.

Expression of HOTAIR and PTGS2 as potential biomarkers in chronic myeloid leukemia patients in Brazil.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm in which all the patients has the translocation (9;22) that generates de BCR::ABL1 tyrosine kinase. Despite this disease possessing a good biomarker (BCR::ABL1 transcripts level) for diagnosis and prognosis, many studies has been performed to investigate other molecules, such as the long noncoding RNAs (lncRNAs) and mRNAs, as potential biomarkers with the aim of predicting a change in BCR::ABL1 levels and as an associated biomarker. A RNAseq was performed comparing 6 CML patients with high BCR::ABL1 expression with 6 healthy control individuals, comprising the investigation cohort to investigate these molecules. To validate the results obtained by RNAseq, samples of 87 CML patients and 42 healthy controls were used in the validation cohort by RT-qPCR assays. The results showed lower expression of HOTAIR and PTGS2 in CML patients. The HOTAIR expression is inversely associated with BCR::ABL1 expression in imatinib-treated CML patients, and to PTGS2 showing that CML patients with high BCR::ABL1 expression showed reduced PTGS2 expression.

Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms

The role of hematopoietic Hedgehog signaling in myeloproliferative neoplasms (MPNs) remains incompletely understood despite data suggesting that Hedgehog (Hh) pathway inhibitors have therapeutic activity in patients. We aim to systematically interrogate the role of canonical vs. non-canonical Hh signaling in MPNs. We show that Gli1 protein levels in patient peripheral blood mononuclear cells (PBMCs) mark fibrotic progression and that, in murine MPN models, absence of hematopoietic Gli1, but not Gli2 or Smo, significantly reduces MPN phenotype and fibrosis, indicating that GLI1 in the MPN clone can be activated in a non-canonical fashion. Additionally, we establish that hematopoietic Gli1 has a significant effect on stromal cells, mediated through a druggable MIF-CD74 axis. These data highlight the complex interplay between alterations in the MPN clone and activation of stromal cells and indicate that Gli1 represents a promising therapeutic target in MPNs, particularly that Hh signaling is dispensable for normal hematopoiesis.

Predictive Biomarkers of Response to Luspatercept in Patients with Myelofibrosis- (MF) Associated Anemia: Biomarker Analysis from the ACE-536-MF-001 Study

Introduction: MF is a clonal myeloproliferative neoplasm that results in fibrosis of the bone marrow and impaired hematopoiesis. Among other clinical manifestations, ineffective erythropoiesis leads to anemia resulting in poor quality of life and reduced overall survival (Passamonti et al., Blood 2010). Current treatment options for MF-associated anemia with most agents lack optimal efficacy and durability of response (Cervantes et al., Expert Rev Hematol., 2016). Luspatercept is a transforming growth factor-β (TBF-β) family ligand trap that inhibits the TGF-β-Smad signaling axis and promotes late-stage erythropoiesis. Luspatercept is approved in low-risk MDS with ring sideroblasts and β-thalassemia requiring red blood cell (RBC) transfusion. The ACE-536-MF-001 study investigates the safety and efficacy of luspatercept in the treatment of MF-related anemia. Here, through mutational and cytokine analysis of ACE-536-MF-001 biomarker samples, we sought to identify mutational and cytokine profiles associated with response. Methods: Eligible patients were divided into 4 cohorts based on transfusion dependence (TD) and treatment with ruxolitinib (RUX). Cohorts 1 and 3A were non transfusion dependent (NTD) while cohorts 2 and 3B were TD patients receiving 4-12 RBC units/84 days up to C1D1. Cohorts 3A and 3B received a stable dose of RUX. All patients received 1.0 mg/kg of luspatercept (1.33 mg/kg for cohort 3B) with titration up to 1.75 mg/kg once every 21 days. Primary endpoint was anemia response (defined as an increase of ≥ 1.5 g/dL hemoglobin (Hgb) from baseline for cohorts 1/3A or RBC transfusion free for ≥ 12 consecutive weeks for cohorts 2/3B). Secondary endpoint was mean Hgb increase ≥ 1.5 g/dL from baseline for ≥ 12 consecutive weeks for cohorts 1/3A, or ≥ 50% decrease in RBC transfusions from baseline for ≥ 12 consecutive weeks and duration of RBC transfusion-free period for cohorts 2 and 3B. Mutational analysis was performed in DNA from peripheral blood mononuclear cells using a panel targeting 74 genes. Rules-Based Medicine's Human Inflammation panel was used to measure changes in 54 serum cytokines. Nexelis custom panel was used for detection of GDF8, GDF15, erythroferrone, soluble transferrin receptor 1 (sTfR1) and hepcidin. Changes in soluble factors from cycle 1 to end of week 24 (day 169) were examined for correlation with clinical response. Results: All 4 cohorts (N = 95) displayed improvement in anemia and TD patients receiving RUX had the greatest benefit (Gerds et al. EHA 2023 S167). In the biomarker cohort (n = 63 across all 4 cohorts), mutational analysis revealed that most patients carried the JAK2 V617F mutation, and patients with SF3B1 or SF3B1 with co-occurring DTA mutations ( DNMT3A, TET2 or ASXL1) had a favorable prognosis related to anemia response (p= 0.0260, 0.0495; OR= 3.54, 3.86). In addition, JAK2 V617F patients in cohort 3B showed clinical benefit with luspatercept (OR= 6.75, p= 0.0445). Across cohorts and endpoints, the general trend of responders was low baseline (2-3-fold lower relative to nonresponders) levels of key inflammatory cytokines. Low levels of IL-10 (TD patients) and VCAM-1, TNFR2, CRP and IL-18 (NTD patients) were significant ( P &amp;lt; 0.01) predictors of response. Paired analysis between cycle 1 and week 24 revealed increases in alpha-2-macroglobulin, haptoglobin, TIMP1, and decrease in C3, vitamin D-binding protein, hepcidin and sTfR1 associated with anemia response. When transfusion burden reduction was used as a response criterion, ferritin, hepcidin, thrombopoietin, von Willebrand Factor, sTfR1, and beta-2-microglobulin were significantly ( P &amp;lt; 0.05) associated with response (Figure). In addition, Hgb increase was associated with increased TGF-β1 and VEGF levels and decreased GDF-15 and ERFE. These cytokines are implicated in iron metabolism (ferritin, hepcidin, alpha-2-macroglobulin), Hgb catabolism (haptoglobin), anemia (vitamin D-binding protein, C3), and luspatercept-target engagement (hepcidin, sTfR1). Conclusions: Our analysis identifies unique predictors of response to luspatercept treatment in patients with MF. While the data describe our findings across cohorts, cohort 3B (TD, receiving RUX), which had the highest response rate, also had higher levels of RANTES and TPO at baseline in responders. The ongoing phase 3 study, INDEPENDENCE (NCT04717414), would validate these observations in a larger cohort of patients

A Dual-Specific Inhibitor of Rock/Aurk, RR-1752, for Primary Myelofibrosis

Myelofibrosis (MF) is a clonal myeloproliferative neoplasm (MPN) characterized by driver mutations in JAK2, CALR, and MPL resulting in constitutive activation of JAK-STAT signaling. Abnormalities in megakaryocyte (MK) numbers and morphology are a common feature of MF where abnormal MKs are associated with bone marrow fibrosis and pro-inflammatory cytokine release. Therapies that target constitutive JAK-STAT signaling and atypical megakaryocytes could provide clinical benefit for patients with MF. ROCK and Aurora kinase (AURK) are overexpressed in MPN and linked to dysregulated MKs of patients with MPN. We therefore tested the activity of RR-1752, a dual-specific inhibitor of ROCK and AURK, in preclinical models of MF. RR-1752 acted as an inhibitor of pSTAT3 in IL-2 stimulated human PBMC 1h post stimulation and significantly inhibited phosphorylated myosin light chain 2 (pMLC2) in two human MPN cell lines, HEL-92.1.7 and UKE-1, confirming an on-target action. RR-1752 inhibited colony formation by HEL-92.1.7 and UKE-1, (IC 50 = 25.2 nM and 54.6 nM respectively). RR-1752 also induced cell cycle arrest in HEL-92.1.7 and UKE-1 specifically at the G2/M checkpoint. RR-1752 was also tested in a hematopoietic colony formation assays of MF patient derived CD34 + cells. RR-1752 led to concentration specific inhibition of MF patient derived CD34 + cell colony formation between 100-190 nM demonstrating relevant activity against patient-derived MF samples (51% inhibition at 190 nM as compared to vehicle control). These data were in line with flow cytometrically determined cell viability data of MF CD34 + cells cultured in the presence of varying concentrations of RR-1752 in liquid culture. The in vivo activity of RR-1752, was examined using the human HEL- 92.1.7 cell line, homozygous for both JAK2 V617F and p53 M133K in a mouse xenograft model. The effects of RR-1752 alone and in combination with ruxolitinib were compared to single agent ruxolitinib. The administration of RR-1752, 25 mg/kg PO daily resulted in a significant (p&amp;lt;0.01) improvement in median survival (31 days) compared to ruxolitinib alone, 45 mg/kg PO daily (21 days). RR-1752 5.0 mg/kg PO QD plus ruxolitinib 45 mg/kg PO QD also resulted in a significant improvement in survival (p&amp;lt;0.05) compared to single agent ruxolitinib but was inferior to single agent RR-1752 at 25mg/kg. The demonstrated activity in MF patient derived CD34 + cells combined with the significant improvement in survival compared to the approved drug ruxolitinib in an aggressive, JAK2/p53 mutant MF xenograft model provides a compelling case for its evaluation as a therapeutic modality in MF patients. Dose optimization of RR-1752 alone and in combination with ruxolitinib in the HEL-92.1.7 IV dissemination model as well as testing in the hMPL-W515L-evoked myelofibrosis model is ongoing. Conventional treatments for MF have largely focused on symptom management but RR-1752 treatment could result not only in beneficial symptom improvement but also important disease modifying potential by inhibiting JAK-STAT signaling, cell cycle progression and targeting dysregulated MKs.

Dysfunctional Stromal Pathways Mark Early Changes within the Bone Marrow Microenvironment in JAK2 V617F-Driven Model of Myeloproliferative Neoplasm

Background: JAK2V617F (JAK2VF) is the primary driver mutation in classical myeloproliferative neoplasms (MPN). JAK2VF is present decades prior to diagnosis (Mitchell et.al. Nature 2022), and the JAK2VF+ clone may remodel the BM microenvironment further supporting its expansion (Curto-Garcia, et. al. Haematologica 2020). Understanding this relationship, particularly during early disease development may aid identification of new therapeutic targets to stop or revert these early changes. Human BM procured before MPN diagnosis is rarely available and most murine MPN models use myeloablative conditioning (eg., irradiation), permanently altering the BM stroma (Costa &amp; Reagan Frontiers 2019), diminishing the clinical relevance of resultant findings. We developed an innovative translatable model of MPN with sustainable low-level disease engraftment and no requirement for recipient conditioning. By preserving the normal microenvironment, our model enabled detection of early changes within the BM stroma induced by the JAK2VF+ clone. Methods: Whole BM transplantation (BMT) was performed via a single tail vein injection into unconditioned C57BL/6 Ptprc a (JAX#002014, CD45.1+) recipient mice. Donor animals consisted of either wildtype ( wt) C57BL/6 (JAX#000664) mice or our PolyI:C inducible MPN-like model created by crossing a floxed-JAK2VF (JAX#031658) with Mx1-Cre (JAX#003556) animals (JAK2VF Parental). For all BMTs, donors and recipients were age- (10wks) and sex-matched. BMT cell concentrations ranged from 1e6 to 15e6per injection, n≥10 mice, both sexes were used per group. A cohort of non-BMT age- and sex-matched recipients were maintained as naïve controls. Recipient serial submandibular blood collections were used to examine complete blood cell counts and donor chimerism. Bulk RNA-seq transcriptomic analysis was performed on digested and lineage-negative sorted whole BM from matched naïve and 5e6 BMT recipients (n=7 and 6, respectively) to delineate stromal alterations driven by JAK2VF+ MPN clone. Qualitative x-ray analysis was performed on femurs. Results: 15e6 wt donor CD45.2 BM cells can be transplanted sustainably into unconditioned CD45.1 recipients with ~3.0% donor chimerism, maintained up to 200-days post-BMT (Fig A). Follow up secondary BMT into conditioned recipients using as few as 25e4CD45.2+ cells sorted from primary BMT recipients displayed 83% average engraftment by 150-day post BMT, confirming functional wt donor CD45.2 HSC. This successful strategy was used to perform BMT using JAK2VF parental (1e6, 5e6, and 15e6) cells. Detected chimerism followed a dose response, not exceeding an average of 10% up to &amp;gt;225 days. Surprisingly, with the engraftment at &amp;lt;10%, we observed MPN-like phenotype (elevated hemoglobin and monocytosis) except for no thrombocytosis (Fig A). To identify alterations in the BM niche, stromal cells were isolated from BMT recipients and subjected to RNA-seq. Enrichment of stromal cells was validated by confirming that 23% of the top 100 highest read coverage transcripts were encoding stromal genes (i.e. Sparc, Dcn, Col2a1, and Cxcl12). Gene set enrichment analysis (GSEA) of stromal fraction showed upregulation of osteoclastic differentiation and bone remodeling. GSEA against hallmark gene sets indicated upregulation of mesenchymal phenotype and inflammatory response suggesting an underlying effect on the stroma driven by the presence of the JAK2VF clone. Qualitative x-ray of femurs indicated loss of bone density, suggesting disease-driven effect on the stroma (Fig B). Discussion: The development of our translatable model of MPN, where a small subset of cells drives the disease within unconditioned recipients represents an important milestone towards studying early disease pathology in MPNs. Using wt BMT, we demonstrated the successful establishment of long-term, low-level chimerism in unconditioned recipients. We found the clone-dependent impact on the stroma without myeloablative conditioning. Bulk transcriptomics and femur x-rays indicate aberrant bone morphology in BMT mice, despite the low level of engraftment (&amp;lt;5%) in the recipient system. Our data suggests that even low VAF of JAK2VF may support pathologic remodeling of the BM environment, warranting further exploration of patient BM niche early and late in the MPN course.

Glycated Hemoglobin (HbA1c) and Thrombotic Risk in Polycythemia Vera and Essential Thrombocythemia

BACKGROUND: Essential thrombocythemia (ET) and polycythemia vera (PV) are BCR::ABL1 negative myeloproliferative neoplasms (MPNs) characterized by overproduction of one or more mature myeloid cell lineages and high thrombotic risk. Diabetes mellitus (DM) is a rare cardiovascular (CV) comorbidity in MPNs and the optimal treatment target levels of glycated hemoglobin (HbA1c) in MPNs remain unknown. OBJECTIVE&amp;METHODS: This multicenter study was conducted in Croatia and Serbia in the period between 01/1997 and 01/23 and investigated clinical correlations of HbA1c in ET and PV patients whose diagnoses were reassessed according to 2016 WHO criteria. We retrospectively collected data regarding diabetic ET and PV patients with available HbA1c at disease diagnosis, stratified them according to HbA1c levels, and compared their clinical outcomes to a control group of 93 non-diabetic ET and PV patients. Outcomes of interest were time to thrombosis (TTT; death as a censoring event) and overall survival (OS). Survival analyses were based on Kaplan-Meier method and performed with log-rank test and the Cox-regression analysis. The study was approved by Ethics Committees from all participating centers. RESULTS: We included 109 MPN patients (33 ET and 76 PV) with DM (all type II); median age was 69 years (range 37-86), 60 (55%) were females, 81 (74.3%) were JAK2- and 10 (9.2%) CALR-positive. Ninety-two (84.4%) patients were classified as high-risk (age&amp;gt;60 years or prior thrombosis), 68 (62.4%) received hydroxyurea (HU), 91 (83.5%) had arterial hypertension, 44 (40.4%) hyperlipidemia, and 36 (33%) were smokers. The median follow-up time was 38 months; 16 (14.7%) thrombotic (13 arterial and 3 venous) events and 25 deaths (22.9%) occured during this time. Median HbA1c in diabetic MPN patients was 6.8% (range 4.8-16.2%). There were no associations of HbA1c with disease phenotype, age, sex, mutational status, prior thrombosis, presence of individual CV risk factors, or any of the blood cell count components (p&amp;gt;0.050 for all analyses). ROC curve defined the optimal HbA1c cut-off level of ≥7.2% for the best discrimination of thrombotic events in MPN patients with DM. Diabetic MPN patients with low HbA1c (&amp;lt;7.2%) had similar TTT (median 182 months) as non-diabetic MPN patients (median 197 months) whereas those with higher HbA1c (≥7.2%) had significantly worse TTT (median 124 months; HR 3.76, p=0.012), as presented in Figure 1A. The detrimental effect of high HbA1c was evident in high-risk (HR 3.2, p=0.031) but not in low-risk diabetic MPN patients (p=0.115). Also, JAK2-negative diabetic MPN patients with higher HbA1c had inferior TTT when compared to their counterparts (HR 28.17, p&amp;lt;0.001), with no difference among JAK2-positive patients (Figure 1B). There were significant interactions between high HbA1c, thrombotic risk, and advanced age (p=0.026), higher leukocytes (p=0.028), higher erythrocytes (p=0.007), higher platelets (p=0.034), higher hematocrit (p=0.007) and higher hemoglobin (p=0.013), with older age and higher blood counts favoring higher thrombotic risk associated with high HbA1c. There was no difference in OS when comparing non-diabetic to diabetic MPN patients with respect to HbA1c (p=0.209). In the multivariate Cox regression model, high HbA1c (HR 5.04, p=0.013) remained as the only variable associated with inferior TTT among diabetic MPN patients when additionally controlling for age, sex, history of thrombosis, presence of JAK2 mutation, HU treatment, baseline leukocyte counts, and the presence of at least one other CV risk factor. DISCUSSION: This study demonstrated that diabetic MPN patients with low (&amp;lt;7.2%) HbA1c had similar TTT as non-diabetic patients suggesting that stringent control of DM in MPNs is extremely important; this effect was most evident in JAK2-negative diabetic MPN patients. Also, significant interactions with high trombotic risk were found between high HbA1c, older age, and different myeloid cell lineages, suggesting that uncontrolled DM acts synergistically with MPN clone and underlies CV development. However, no correlations between HbA1c and blood cell counts were detected, indicating that HbA1c measurements may not be accurrate in MPNs for DM diagnosis. The lack of association of high HbA1c with OS is probably related to optimal post-thrombotic management. Future studies are needed to elucidate optimal HbA1c values for the diagnosis and treatment of DM in MPNs.

Fedratinib Overcomes Ruxolitinib Resistance through Inhibition of the Interferon Signaling Pathway

Background: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm characterized by hyperactive JAK-STAT signaling, bone marrow fibrosis, extramedullary hematopoiesis and splenomegaly. Ruxolitinib (RUX) and Fedratinib are both JAK inhibitors used for the treatment of patients with intermediate or higher risk MF. While RUX is the first line treatment for patients with MF, about 50% of the patients discontinue treatment due to lack of efficacy or intolerance (Harrison et al., Ann. Hematol. 2020) after 3 years. Fedratinib is used as a second line therapy in MF based on efficacy from the JAKARTA2 study. The recently concluded FREEDOM and FREEDOM2 studies further support this. In this work, using in vitro approaches, we have explored the mechanistic basis of fedratinib's ability to overcome RUX resistance. Pharmacologically, Fedratinib binds with high affinity to the substrate-binding site and with low affinity to the ATP-binding site of JAK2(Kesarwani etal., Sci. Rep. 2015) while RUX binds only to the ATP-binding site. Previous studies on RUX resistance have shown acquired mutations driving resistance to RUX but not to Fedratinib. Here, we use RUX sensitive and resistant BaF3 (expressing human JAK2 WT or V617F) cell lines to examine the mechanism of resistance to RUX and the mechanism of efficacy of Fedratinib in RUX resistance. Methods: BaF3 cell lines overexpressing JAK2 WT or JAK2 activating mutation V617F were incrementally exposed to RUX over 3 weeks (up to IC 90) to induce resistance. Cell proliferation and phosphorylation of STAT5 in Fedratinib and RUX-treated cells were measured using celltiter glo and immunoblotting, respectively. RNAseq and ATAC-seq analyses were performed in triplicates by treating the relevant cell lines with DMSO, RUX or Fedratinib at two timepoints (6 and 15 hours). Pathway analysis was performed on RNAseq data using Metacore®. Results and Discussion: Ruxolitinib resistant BaF3 JAK2V617F cells showed minimal inhibition of proliferation (&amp;lt;10%) when treated with RUX but maintained sensitivity towards Fedratinib (IC 50 650nM vs 1552nM in RUX sensitive and resistant cells respectively) in 24-hour proliferation assays. Biochemical analysis revealed that Fedratinib inhibited JAK-STAT signaling in the RUX resistant cell line while RUX was unable to do so as measured by p-STAT5. Similar results were observed in BaF3-JAK2-WT cells as well. To further understand the mechanism of RUX-resistance and differential activity of RUX and Fedratinib, we performed RNAseq analysis on our RUX sensitive and resistant models before and after RUX and Fedratinib treatment. Myelofibrosis is associated with aberrant cytokine production and pro-inflammatory signaling that are mediated through activation of TNF-α and NF-κB signaling (Dunbar et al., Blood 2023). Consistent with this, RNAseq analysis of BaF3 JAK2 V617F cells treated with RUX or Fedratinib showed effective inhibition of these pathways by both agents as early as 6 hours. In the RUX resistant cells, while RUX did not have any significant pathway level changes, the top enriched pathways upon treatment with Fedratinib were predominantly interferon signaling pathways which includes JAK-STAT signaling suggesting that Fedratinib's efficacy in MF is not impacted by RUX resistance. To understand the mechanism of resistance, comparison of gene expression data from RUX senstive and RUX resistant cells without any drug treatment revealed minimal gene expression/pathway level differences suggesting that the mechanism of resistance could be driven by genetic (Downes et al. NPJ Precis Oncol, 2021) or epigenetic changes(Dunbar et al., Blood 2023). Ongoing analyses includes whole genome sequencing and ATAC-seq to investigate potential mutations and chromatin accessibility changes during resistance acquisition to RUX and how Fedratinib may overcome these. Conclusion: Our stable RUX resistant preclinical model demonstrates that Fedratinib is effective in overcoming RUX resistance by continuing to target key pathways in MPN biology including interferon signaling. Resistance to RUX on the other hand seems to be driven through potential genetic or epigenetic changes based on the minimal changes observed in gene expression profiles. Our ongoing analyses will reveal these potential genetic and epigenetic modifications associated with RUX resistance and explain the differential activity of RUX versus Fedratinib.

Alternatively Spliced ATP6V1H induces Inflammation in Polycythemia Vera and Essential Thrombocythemia

Polycythemia vera (PV) and essential thrombocythemia (ET) are clonal myeloproliferative neoplasm originating from a hematopoietic stem cell. PV is characterized by increased hemoglobin; ET is characterized by high platelets due to gain-of-function mutations of the tyrosine kinase JAK2 gene, with JAK2V617F being the most common phenotype defining mutation. Thrombosis remains a major cause of PV and ET morbidity and mortality. Clonal myelopoiesis in PV is further enhanced by inflammation, contributing to PV pathogenesis. The molecular mechanisms underlying the augmented inflammation in PV remain incompletely characterized but neutrophils play a major role in inflammatory and prothrombotic complications in PV and ET. Our whole transcriptome analysis using 46 PV neutrophils and 10 healthy controls showed that 1833 genes in PV granulocytes (adjusted p-value &amp;lt;0.05 and log2 fold changes &amp;gt;0.5) were dysregulated. Gene set enrichment analysis showed that genes involved in inflammatory response were upregulated in PV (FDR: 0.047, enrichment score: 0.578). We then interrogated in our transcriptome set differential exon usage and identified a differentially spliced exon of ATP6V1H (AS-ATP6V1H) in PV. ATP6V1H encodes ATPase H+ transporting V1 (v-ATPase) subunit H. Inhibition of v-ATPase augments the production, release, and expression of inflammatory genes. We found higher expression levels of AS-ATP6V1H (ENST00000522849) in PV neutrophil transcriptome (Figure B). The exon in the black box in Figure A is only present in AS-ATP6V1H (ENST00000522849) and its expression was higher in PV. This transcript undergoes nonsense-mediated decay, resulting in decreased ATP6V1H expression in PV (Figure C). This finding was then verified using 46 patients with PV, 24 with ET and 29 healthy controls. AS-ATP6V1H was upregulated both in PV and ET neutrophils (Figures D). However, these expression differences were not detected in platelets (Figure E). AS-ATP6V1H transcript levels demonstrated a positive correlation with genes that are upregulated in inflammation - CXCL8, CD55, IRAK1, and IL1RAP, while showing an inverse correlation with genes downregulated in inflammation, including IGHG1, CD40LG, and TNFRSF4.These transcript levels showed a positive correlation with white blood cell (r=0.4677, p&amp;lt;0001) and neutrophil counts (r=0.5268, p&amp;lt;0001), indicating an association with heightened inflammation in PV and ET. PV and ET patients with a history of thrombosis had higher AS-ATP6V1H transcript levels compared to those without thromboses (p=0.0427). AS-ATP6V1H transcript levels positively correlated with the levels of thrombotic genes tissue factor ( F3) and p-selectin ( SELP) and inversely correlated with an anti-thrombotic gene, KLF2 (Song, Blood Advances, 2023). These results suggest that elevated AS-ATP6V1H may also be associated with an increased risk of thrombosis. The expression of inflammatory and thrombotic genes is influenced by hypoxia inducible factors (HIFs) (Gangaraju, Blood Advances, 2020). Reduced ATP6V1H increases HIF-1a protein levels and its transcriptional activities (Yambire, elife, 2019). We also measured the expression levels of HIF target genes in neutrophils ( EDN1, LDHA, SLC29A1, CKB, VEGFA, and SLC2A1), and these levels positively correlated with AS-ATP6V1H transcript levels. Since inflammation is also associated with increasing JAK2V617F variant allele frequency (VAF), we also tested the correlation with JAK2V617F VAF. We observed a positive correlation of JAK2V617F VAF with AS-ATP6V1H transcript levels (r=0.74, p&amp;lt;0001), suggesting that hyperactive JAK2 activity might induce an mRNA splicing event in ATP6V1H. In conclusion, our study showed a relationship between AS-ATP6V1H and inflammation in PV and ET. Through whole transcriptome analysis, we identified AS-ATP6V1H as a candidate gene, and its upregulation in PV and ET suggests its potential involvement in increased inflammation and clonal myelopoiesis. We show that AS-ATP6V1H transcript levels correlated with the expression levels of genes involved in inflammation and thrombosis and that AS-ATP6V1H may be regulated by HIFs and JAK2 activity. Further investigations should demonstrate the molecular mechanisms underlying AS-ATP6V1H's role in inflammation and thrombosis, which could lead to novel therapeutic approaches for managing PV and ET.

Fedratinib Treatment Reduces the Inflammatory Cytokine Profile and Decreases Exhausted T Cells Correlating with Clinical Response in Patients with Myelofibrosis: Biomarker Analysis from the Phase 3 FREEDOM2 Trial

Introduction: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm (MPN) characterized by a hyperactive JAK-STAT pathway, splenomegaly, and constitutional symptoms. JAK inhibitors (JAKi) have been the mainstay in MF treatment over the past decade. Pro-inflammatory cytokines, the levels of which have prognostic value (Tefferi et al. J Clin Oncol 2011), have been shown to be upregulated in MF. However, there is paucity of data on chronic immune dysregulation in pathogenesis of MF. Here, we report the multiplatform biomarker analysis including immune changes from phase 3 (FREEDOM2) trial in patients with MF previously exposed to ruxolitinib (Rux) and treated with fedratinib vs. best available therapy (BAT) (primary efficacy: 35.8% vs 6% responders at the end of cycle 6 (EOC6) in fedratinib vs BAT, respectively). Of note, 78% of the patients on BAT continued RUX therapy until EOC6, allowing direct comparison between RUX and fedratinib. Methods: In the FREEDOM2 trial, patients (N=201) were randomized into two arms, fedratinib (400 mg/day) or BAT in a 2:1 ratio, respectively. The primary endpoint was the proportion of patients with ≥35% reduction of spleen volume (SVR35) on MRI at the end of cycle 6 (EOC6). In the FREEDOM2 biomarker cohort, mutational analysis was performed in enriched CD66b+ cells using the MLL myeloid panel targeting 74 genes. Rules-Based Medicine's (RBM) HumanMAP v2.0 panel targeting 85 cytokines was used to measure changes in serum cytokine levels from C1D1 to EOC6. Custom Q2 immune panels to detect myeloid and lymphoid subsets were used for immunophenotyping of peripheral blood samples. Results: Baseline mutations in FREEDOM2 were commonly found MPN mutations that include JAK2 V617F (71%), ASXL1 (43%), CALR (22%), TET2 (22%) and MPL (11%). However, baseline mutation status was not a predictor of response. Patients receiving fedratinib demonstrated significant down-regulation of several pro-inflammatory cytokines including CRP, ENRAGE, IL-18, IL-16, TIMP-1, and increase of anti-inflammatory cytokines including adiponectin, MMP2, and CEA (Wilcoxon signed rank test, BH adjusted p &amp;lt;0.001) at EOC6 vs C1D1. In addition, the change in levels of many cytokines correlated with the clinical endpoint (SVR35) demonstrating that the therapeutically relevant changes were mediated by fedratinib (Pearson rho P&amp;lt;0.05) (Figure 1). Importantly, fedratinib was superior to the BAT in its ability to alter levels of key cytokines (p &amp;lt;0.05) at EOC6. PD-1 expression has been shown to be upregulated in CD4+ and CD8+ T cells in MF patients and is associated with poor overall survival. However, RUX did not have a significant effect on PD1+ T cells (Veletic et al. Haematologica 2021). Interestingly, flow cytometric analysis revealed that fedratinib decreased exhausted CD8+ T cells (PD1+) up to 40% (∆ median change -11.4%,p&amp;lt;0.001)) (figure 2). Importantly, the reduction in exhausted T cells correlated with the reduction in spleen volume with fedratinib treatment ( R= -0.51, P=0.00011; Figure 2). Consistent with this, we also observed median increases in NK (+3%, p&amp;lt;0.001) and NK-T (+13.8%, p&amp;lt;0.001) populations when compared to the BAT arm. Conclusions: To our knowledge, this is the first comprehensive study detailing cytokine and immune changes in patients with MF in a clinical trial. In addition, our data for the first time demonstrates the impact of fedratinib on the immune system and its ability to reduce exhausted T cells, highlighting the immune modulatory mechanism of fedratinib. Notably, the cytokine and immune changes correlated with the primary endpoint and were not observed in the BAT arm demonstrating superior efficacy of fedratinib in previously RUX exposed patients. Furthermore, since majority of the patients in the BAT arm continued to receive RUX, these findings highlight the mechanistic differences between fedratinib and RUX on clinical response in MF (SVR35). Overall, our data highlight multiple novel avenues to achieve therapeutic efficacy in MF and positions fedratinib as an effective therapy with a distinctive mechanism of action. Additional analyses on the effect of fedratinib treatment on myeloid immune subsets and transcriptomic changes are underway to gain comprehensive mechanistic understanding.

Diagnostic Performance of Red Blood Cell Indices, Serum Erythropoietin, and JAK2 Mutation Testing for the Evaluation of Polycythemia Vera at High Altitude

Introduction: Polycythemia vera (PV) is a clonal myeloproliferative neoplasm associated with mutation of Janus kinase 2 ( JAK2) in virtually all patients. Patients with PV typically present with constitutional symptoms and thrombotic complications related to myeloproliferation but can present with only mild or no symptoms. In 2016, the World Health Organization (WHO) diagnostic criteria for PV were revised to use lower hemoglobin and hematocrit cut-off levels in order to avoid missing masked polycythemia. Accordingly, a hemoglobin concentration of more than 16 g/dL (HCT&amp;gt;48%) for females and 16.5 g/dL (HCT&amp;gt;49%) for males can be considered for PV evaluation (previously &amp;gt;18.5 g/dL for men and &amp;gt;16.5 g/dL for women). In conjunction, concerns arise about the unnecessary evaluation of individuals with otherwise high-normal hemoglobin levels, more so for those living at high altitude. Altitude exposure, amongst others, decreases plasma volume to increase [Hb] which may accordingly muddle the diagnosis of PV at altitude. Nonetheless, Red Cell Mass (RCM) is seldom assessed in clinical practice. In addition, red blood cell count, related indices, and serum erythropoietin may also be affected by high altitude, and their diagnostic performance in the evaluation of PV at altitude has not been prospectively assessed. Method: We collected lab records for all JAK2 requests over three years (2020-2022) in two referral hospitals serving a population residing at an altitude of 2,270-3,000 meters above sea level. Indications for requests, as well as laboratory data and imaging, were gathered. Complete blood count (CBC) and other laboratory values were collected when ordered within one week of JAK2 testing. For CBC, EDTA anticoagulated samples were processed using Sysmex XN-3100. For JAK2 testing, RNA isolated from peripheral blood samples (using the MagNA Pure System) was reverse-transcribed, amplified, and sequenced for any mutations involving exons 12, 13, 14, or 15 using an ABI 3730 XL genetic analyzer. For serum EPO, frozen serum samples were outsourced to Bio Scientia Labs, Germany. Reference values for serum erythropoietin were lab generated at 4.3-29 mIU/ml and were not validated for our altitude. . Results:Of the 208 JAK2 tests performed during the reviewed period, 132 (63.4%) were for polycythemia (Table 1). The JAK2 V617F mutation was found in 22 (16.7%) patients, with 10 (45.5%) of them being men. No JAK2 mutation other than the exon 14 V617F substitution was found. JAK2-positive patients had a median age of 61 years, while JAK2-unmutated patients had a median age of 47 years. Overt polycythemia, according to the WHO 2016 classification, was identified in 15 (68.2%) , while the remainder had masked PV ( defined as increased RBCs with normal hemoglobin/ HCT). Patients with PV had significantly higher RBCs and lower MCV/MCH/MCHC compared to those with JAK2-unmutated polycythemia. They also exhibited significant elevation of RDW-CW and significant neutrophilia, basophilia, thrombocytosis, and splenomegaly. All PV patients with available serum EPO tests had low values (median 2 mIU/ml). Conclusion: At altitude, patients with PV show the same age and sex characteristics as those living at low elevations. Patients with PV have higher RBCs and lower MCV/MCH/MCHC compared to those with JAK-unmutated (secondary) polycythemia. RDW is significantly increased in patients with PV, reflecting an associated iron deficiency. Serum EPO performed well at altitude to suggest the presence of PV. The low rate of positive JAK2 mutation in our high-altitude cohort may reflect poor selection of subjects and over-workup of otherwise altitude-associated erythrocytosis. Isolated mild polycythemia at altitude remains common. Using hemoglobin/hematocrit alone to select patients for PV evaluation has a low positive predictive value and may be associated with unnecessary workup. RBC indices and RDW, along with an evaluation for iron status, might help select those more likely to have PV and could decrease unnecessary workups.

Exploring the Molecular Landscape of Myelofibrosis, with a Focus on Ras and Mitogen-Activated Protein (MAP) Kinase Signaling.

Myelofibrosis (MF) is a clonal myeloproliferative neoplasm (MPN) characterized clinically by cytopenias, fatigue, and splenomegaly stemming from extramedullary hematopoiesis. MF commonly arises from mutations in JAK2, MPL, and CALR, which manifests as hyperactive Jak/Stat signaling. Triple-negative MF is diagnosed in the absence of JAK2, MPL, and CALR but when clinical, morphologic criteria are met and other mutation(s) is/are present, including ASXL1, EZH2, and SRSF2. While the clinical and classic molecular features of MF are well-established, emerging evidence indicates that additional mutations, specifically within the Ras/MAP Kinase signaling pathway, are present and may play important role in disease pathogenesis and treatment response. KRAS and NRAS mutations alone are reportedly present in up to 15 and 14% of patients with MF (respectively), and other mutations predicted to activate Ras signaling, such as CBL, NF1, BRAF, and PTPN11, collectively exist in as much as 21% of patients. Investigations into the prevalence of RAS and related pathway mutations in MF and the mechanisms by which they contribute to its pathogenesis are critical in better understanding this condition and ultimately in the identification of novel therapeutic targets.

ß-catenin expression in myelodysplastic syndromes and myeloproliferative neoplasms in bone marrow, in relation to CD34 and CD117 status

Aims: The activation of the Wnt/ ß-catenin signaling pathway has been demonstrated to play a crucial role in the development of myeloid neoplasms. In addition to CD34, which has been used until now in the diagnosis and staging of clonal hematopoietic diseases, Myelodysplastic Syndromes (MDS) and Myeloproliferative Neoplasms (MPN), CD117, which has found its place in hematopoietic diseases, also provides significant benefits in these respects. In this study, we evaluated the immunohistochemical presence and utility of ß-catenin in blasts relative to other markers, as the inhibition of ß-catenin activity may be an attractive therapeutic approach Methods: By retrospectively analyzing bone marrow samples with ß-catenin immune marker, we determined the staining rates, intensities, and patterns of 30 MDS, 29 MPN cases and 30 normal bone marrow controls, in comparison to the efficacy of the the well-known CD34 and CD117. We statistically interpreted the correlation between them. Results: Based on the findings, ß-catenin, which has recently been used in hematopoietic diseases and is said to have a high efficacy in acute myeloid leukemia (AML) cases, was not immunohistochemically detectable in our study. As expected, CD34 and CD117 immun markers exhibited significant blast staining. MPN cases were more prone to staining with CD117. Conclusion: CD34 continues to be the most reliable marker for identifying blasts for diagnosing and grading bone marrow neoplasms while CD117 may have a supportive role in this process. Further investigation is required to ascertain the true effectiveness of ß-catenin, a molecule that has demonstrated encouraging potential in the context of AML.

Modulation of human thrombopoietin receptor conformations uncouples JAK2 V617F-driven activation from cytokine-induced stimulation

AbstractThe thrombopoietin receptor (TpoR) plays a central role in myeloproliferative neoplasms (MPNs). Mutations in JAK2, calreticulin, or TpoR itself drive the constitutive activation of TpoR and uncontrolled proliferation and differentiation of hematopoietic stem cells and progenitors. The JAK2 V617F mutation is responsible for most MPNs, and all driver mutants induce pathologic TpoR activation. Existing therapeutic strategies have focused on JAK2 kinase inhibitors that are unable to differentiate between the mutated MPN clone and healthy cells. Surprisingly, the targeting of TpoR itself has remained poorly explored despite its central role in pathology. Here, we performed a comprehensive characterization of human TpoR activation under physiological and pathological conditions, focusing on the JAK2 V617F mutant. Using a system of controlled dimerization of the transmembrane and cytosolic domains of TpoR, we discovered that human TpoR (hTpoR) adopts different dimeric conformations upon Tpo-induced vs JAK2 V617F–mediated activation. We identified the amino acids and specific dimeric conformation of hTpoR responsible for activation in complex with JAK2 V617F and confirmed our findings in the full-length receptor context in hematopoietic cell lines and primary bone marrow cells. Remarkably, we found that the modulation of hTpoR conformations by point mutations allowed for specific inhibition of JAK2 V617F–driven activation without affecting Tpo-induced signaling. Our results demonstrate that modulation of the hTpoR conformation is a viable therapeutic strategy for JAK2 V617F–positive MPNs and set the path for novel drug development by identifying precise residues of hTpoR involved in JAK2 V617F–specific activation.

Evaluation of Imatinib Adherence in Chronic Myeloid Leukemia Patients in Babylon Province, Iraq

Background: Chronic myeloid leukemia is a clonal myeloproliferative neoplasm that results in proliferation, mainly of granulocytic components. Resulting from t(9;22) with fusion oncogen BCR-ABL1 which has uninhibited tyrosine kinase activity. The introduction of tyrosine kinase inhibitors, such as Imatinib, has significantly altered the management and prognosis of this condition, but poor adherence is still a significant factor in therapy failure. Objective: To measure chronic myeloid leukemia (CML) patients’ adherence to Imatinib treatment and to analyze potential reasons for poor adherence. Materials and Methods: The study included 52 CML patients in the chronic phase treated with Imatinib for at least 12 months; 30 of them were women and 22 were men, with a median age of 45 years. Patient adherence to Imatinib was assessed using the Morisky Medication adherence scale. Response to treatment was assessed by the evaluation of the level of BCR-ABL1 mutation in peripheral blood. Results: Out of 52 patients, 17 were adherent to Imatinib while 35 of them were nonadherent. The patient’s adherence status to Imatinib was found to have a significant effect on the response to treatment. The most common cause of nonadherence was forgetfulness. Age, sex, occupation, educational level, marital status, residence, duration of treatment, relation to meal time, taking other medications, and drug side effects have no significant effect on adherence. Conclusions: Adherence to Imatinib was poor and this has a negative impact on patients’ response to therapy. Patients’ forgetfulness was the most frequent reason for nonadherence, followed by drug-related patient disturbance.

Predictors of survival in the Egyptian patients with primary myelofibrosis: a single-center prospective cohort study

Abstract Background Primary myelofibrosis (PMF) is a clonal hematological myeloproliferative neoplasm distinguished by poor prognosis and reduced life expectancy. This study aimed to detect the overall survival (OS) in patients with PMF and assess the factors affecting clinical outcomes. Aim To assess factors affecting OS in patients with PMF. Design and Methods A prospective cohort study included 73 patients with PMF. The dynamic international prognostic scoring system (DIPSS) score was calculated for risk stratification. Age, DIPSS score, excess circulating blast, JAK2 V617F mutation, bleeding, and thrombosis complications were correlated with OS. Results The median age of our participants at diagnosis was 55 years. The JAK2 V617F mutation was positive in 26 (35%) patients. Our patients were classified according to the DIPSS score into intermediate-1, with 21 (28.77%); intermediate-2, with 43 (58.9%) patients; and high, with nine (12.33%) patients. The Kaplan–Meier curve was used to calculate time-to-death curves, which were compared by the log-rank tests. The Cox regression test was done to recognize risk factors for the OS. Age and excess peripheral blast cells were negative predictors of survival outcome (P=0.012 and 0.005, respectively). Conclusion Advanced age and excess peripheral blast cells are predictors of inferior survival outcomes in patients with PMF.