Primary Myelofibrosis Patients Research Articles

Targeted Metabolomics Highlights Dramatic Antioxidant Depletion, Increased Oxidative/Nitrosative Stress and Altered Purine and Pyrimidine Concentrations in Serum of Primary Myelofibrosis Patients.

To date, little is known concerning the circulating levels of biochemically relevant metabolites (antioxidants, oxidative/nitrosative stress biomarkers, purines, and pyrimidines) in patients with primary myelofibrosis (PMF), a rare form of myeloproliferative tumor causing a dramatic decrease in erythropoiesis and angiogenesis. In this study, using a targeted metabolomic approach, serum samples of 22 PMF patients and of 22 control healthy donors were analyzed to quantify the circulating concentrations of hypoxanthine, xanthine, uric acid (as representative purines), uracil, β-pseudouridine, uridine (as representative pyrimidines), reduced glutathione (GSH), ascorbic acid (as two of the main water-soluble antioxidants), malondialdehyde, nitrite, nitrate (as oxidative/nitrosative stress biomarkers) and creatinine, using well-established HPLC method for their determination. Results showed that PMF patients have dramatic depletions of both ascorbic acid and GSH (37.3- and 3.81-times lower circulating concentrations, respectively, than those recorded in healthy controls, p < 0.0001), accompanied by significant increases in malondialdehyde (MDA) and nitrite + nitrate (4.73- and 1.66-times higher circulating concentrations, respectively, than those recorded in healthy controls, p < 0.0001). Additionally, PMF patients have remarkable alterations of circulating purines, pyrimidines, and creatinine, suggesting potential mitochondrial dysfunctions causing energy metabolism imbalance and consequent increases in these cell energy-related compounds. Overall, these results, besides evidencing previously unknown serum metabolic alterations in PMF patients, suggest that the determination of serum levels of the aforementioned compounds may be useful to evaluate PMF patients on hospital admission for adjunctive therapies aimed at recovering their correct antioxidant status, as well as to monitor patients' status and potential pharmacological treatments.

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.

Abstract 3628: Combined mek and jak inhibition in vitro is an effective strategy in overcoming jak inhibitor resistance in primary myelofibrosis (PMF)

Abstract Introduction Primary myelofibrosis (PMF) is a myeloproliferative neoplasm arising from various mutations in myeloid-committed hematopoietic progenitor cells. Treatment of PMF is challenging as Jak inhibitor resistance is common, making the expansion of molecular characterization and novel therapeutic target identification in PMF essential. The main goals of this study are to determine both the presence of Ras activity in PMF and the impact on Ras/MAP Kinase pathway targeting in the disease. Methods Integrated genomic-transcriptomic sequencing analyses were performed on peripheral blood mononuclear cells (PBMCs) purified from 27 patients with PMF at a single institution; protein was then obtained from whole cell lysates. Ras activity was then assessed by applying Ras binding domain (RBD) to sample in an ELISA and was compared between patients with PMF, essential thrombocythemia (ET), polycythemia vera (PV) and healthy donors. Statistical analyses were conducted using two-way unpaired t-tests. The next set of in vitro studies aimed to evaluate the effects of MEK inhibition in PMF. Cell viability studies were first performed on human erythroleukemia (HEL) cells to identify IC50 values. Colony forming assays in methylcellulose-enriched media formulated for the growth of CD34+ cells were then performed in 4 conditions: Ruxolitinib and Cobimetinib monotherapy, combined therapies and untreated sample. Results As previously reported in our initial study, Ras activity in 21 patients with MF was 1.74 fold higher than healthy donors (13 volunteers, p=0.0304) and 1.64 fold higher than in PV/ET (12 patients, p=0.0462). We also reported previously on Ras activity from differential enrichment of myeloid and lymphoid cells from PBMC samples, where a key observation was that myeloid contribution to Ras activity in PMF patients with RAS/MAP Kinase pathway mutations was 3.8 times higher in Ras/MAP Kinase WT patients (p=0.0012), potentially suggesting that RAS pathway mutations have a predominance in myeloid cells. In HEL cell viability assays, cobimetinib and ruxolitinib, when utilized in tandem, resulted in more cell growth inhibition than either agent individually. This same finding was recapitulated in colony forming assays in 4 different patients with varying levels of sensitivity to Jak inhibitors, in all of whom ruxolitinib and cobimetinib lead to a greater reduction in colony formation than ruxolitinib monotherapy. Discussion Data from colony forming assays of bone marrow from patients with PMF identifies at least an additive effect of combined Jak and Ras/MAP Kinase (specifically Mek) inhibition on CD34+ colony formation. The specific observation that Ras pathway inhibition may be utilized as a means of overcoming Jak inhibitor resistance in PMF is a novel finding in this field and may be a valuable avenue for future early-phase clinical trials. Citation Format: Samuel Benjamin Reynolds, Sho Matono, Malathi Kandarpa, Kristen Pettit, Mark Ribick, Moshe Talpaz, Qing Li. Combined mek and jak inhibition in vitro is an effective strategy in overcoming jak inhibitor resistance in primary myelofibrosis (PMF) [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 3628.

Newly identified roles for PIEZO1 mechanosensor in controlling normal megakaryocyte development and in primary myelofibrosis.

Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.

[Expression level of Wilms' tumor 1 gene and its correlation with clinical features in patients with myeloproliferative neoplasms].

Objective: To investigate the expression level of WT1 gene in patients with classical Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPN) and its correlation with clinical features. Methods: A retrospective study included 252 patients with newly diagnosed MPN in Zhongnan Hospital of Wuhan University from January 2015 to March 2023, including 128 males and 124 females, aged［M（Q1，Q3）］62 (53, 69) years. The WT1-positive group (n=93) and the WT1-negative group (n=159) were split based on the level of WT1 gene expression, and the variations in clinical indicators between the two groups were compared. Its levels of expression in each subtype and its relationships to thrombotic events and clinically significant variables were analyzed. As of March 31, 2023, the follow-up period [M (Q1, Q3)] was 12.0(6.5,21.0)months. The risk factors of thrombosis in MPN patients were analyzed by using the logistic regression analysis. Results: The WT1 gene expression level in the overall bone marrow samples of 252 patients with newly diagnosed MPN was 0.30% (0.10%, 1.10%). The expression level in primary myelofibrosis (PMF) patients was 1.45% (0.41%, 3.24%), which was higher than 0.15% (0.02%, 0.32%), 0.37% (0.16%, 1.09%) in essential thrombocythemia (ET) and polycythemia vera (PV) patients (both P<0.05). Positive correlations were found between WT1 gene expression levels and JAK2V617F gene mutation load, RDW, MPV (r=0.478, 0.346, 0.236, all P<0.01). While negative correlations between WT1 gene expression levels and PLT, LYM, PTTA, LDH were found (r=-0.339, -0.170, -0.206, -0.388, all P<0.01). Patients in the WT1-positive group exhibited a higher percentage of somatic symptoms, splenomegaly, positive JAK2V617F gene mutation, and higher levels of RDW, LDH, NEUT, and MPV compared to the WT1-negative group. In contrast, the proportion of triple-negative (negative for all three hot mutations of JAK2V617F, CALR and MPL) was lower, and the levels of PLT, LYM and PTTA were lower (all P<0.05). The thrombotic event rates of WT1-positive group and WT1-negative group were 32.3% (30/93) and 32.1% (51/159), respectively, and the difference was not statistically significant (P=0.883). Logistic regression analysis showed that male (OR=2.41,95%CI:1.02-5.71,P=0.046) and positive JAK2V617F gene mutation (OR=3.96,95%CI:1.50-10.42,P=0.005) were risk factors for thrombotic events in ET patients. Conclusions: WT1 gene expression is elevated in PMF patients and correlated with indicators of disease progression and transformation in MPN patients. It can be utilized as an auxiliary diagnostic indicator for classical MPN staging but is not correlated with the incidence of thrombotic events. Male and positive JAK2V617F gene mutation are risk factors for thrombotic events in ET patients.

[Efficacy and safety of allogeneic hematopoietic stem cell transplantation for the treatment of primary myelofibrosis].

Objective: To evaluate the efficacy and safety of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for the treatment of primary myelofibrosis (PMF) patients. Methods: A total of 14 cases of PMF who underwent allo-HSCT from December 2008 to December 2022 were analyzed retrospectively, including 8 males and 6 females with a median age [M(Q1, Q3)]of 36 (24, 42) years. Three-year overall survival (OS), disease free survival (DFS), cumulative incidence of relapse (CIR), transplantation-related mortality (TRM) were analyzed. Meanwhile, the complications were followed up by telephone and outpatient appointments for 49.6 (9.0,93.1) months. Results: All patients received myeloablative conditioning regimens (MAC). All patients had successful engraftment, and the median time of neutrophils and platelet engraftment were 13.5 (11.8, 18.0) days and 19.5 (13.5, 24.5) days, respectively. Ⅱ-Ⅳ acute graft versus host disease (GVHD) occurred in 3 cases, while chronic GVHD in 8 cases. The rate of 3-year OS,DFS,CIR and TRM were (92.9±6.9)%, (76.0±12.2)%, (38.6±2.7)% and (7.1±0.5)% respectively after a median follow-up time of 1 489.0 (270.3,2 794.8) days. Two patients died from treatment-related complications, one of which died 39 days after transplantation due to heart failure caused by severe anemia, the other patient died 6 years after relapse due to pulmonary infection. Conclusion: Allo-HSCT can be used as a safe and effective approach to treat PMF.

Lactate Transporters Modulate Stromal Cell Remodeling in Myeloproliferative Neoplasms (MPN)

Introduction. Philadelphia-negative myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The diagnostic approach proposed by the World Health Organization (WHO) uses clinical features, bone marrow (BM) morphology, karyotype and molecular genetic tests to classify MPN subtypes. The BM morphology includes cellularity, erythropoiesis, and neutrophil granulopoiesis in context with specific features of megakaryocytes as well as the BM fiber content, especially in early-stage myelofibrosis that present with thrombocytosis and clinically mimic ET. Since alteration of hematopoiesis is constantly associated with profound modifications of the bone marrow (BM), we hypothesize that CD34+ cells from PMF can actively produce lactate and thus rewiring metabolism of immune cells and mesenchymal stromal cells (MSCs). In turn, this could favor immune evasion and remodeling of BM niche. Methods.Cell proliferation was assessed in vitro on primary MSCs, HS-5 cell line, and PBMC cells. Protein expression was performed by Western blot and immunofluorescence assay. The NCBI GEO database was used to select transcriptome datasets and to analyze gene expression in PMF patients. Cytokine detections were performed by Multiplex immunobead assay technology. An adult TPO-mimetic-induced PMF Zebrafish model was used for in vivo evaluation. An immunohistology assay was performed in MPN BM biopsies. Results.CD34+ cells from PMF patients significantly upregulated the lactate monocarboxylate transporters (MCT-1 and -4) compared to healthy CD34+ cells. Consistently, lactate concentration was higher in PMF sera compared to healthy controls. Since PMF patients showed an increased percentage of circulating immunosuppressive cells such as G- and M-MDSC and Treg, we incubated healthy peripheral blood mononucleated cells (PBMNCs) with PMF sera in the presence or absence of MCT1inhibitor (AZD3965) to evaluate the role of lactate in the expansion of these immunological subsets. We found an expansion of both Treg and M-MDSCs, which was reverted after blocking of circulating lactate by AZD3965. In order to study the effect of lactate in BM remodeling, we treated in vitro healthy MSCs with lactate (20 mM) for 24h. Interestingly, lactate induced a modification of extracellular matrix organization, as demonstrated by increased reticulin and collagen deposition and metalloproteases (MMP9, MMP2). Moreover, lactate increased calcium deposit and soluble osteogenic molecular signals (i.e. osteoprotegerin, osteonectin). These results also were obtained after incubation with PMF sera and were reverted after AZD3965 treatment. Since it has been demonstrated the role of cancer-associated fibroblast (CAF) in cancer development through ECM remodeling, we next evaluated the expression of CAF markers in MSCs after lactate exposure. α-SMA, FAP1, and TGFβ were found upregulated and this effect was reverted by AZD3965. Finally, we further confirmed these results in a Zebrafish animal model of PMF. Interestingly, in Zebrafish model we observed a reduction of MCT4 expression that was confirmed in the PBMNCs of the PMF patients. Conclusions. Inhibition of lactate metabolism may represent a strategy to inhibit cancer cells and contribute to restoring the anti-cancer immune response. Therefore, lactate metabolism may represent a promising target to counteract inflammation, osteosclerosis, and fibrosis in PMF patients.

Newly Identified Roles for PIEZO1 Mechanosensor in Controlling Normal Megakaryocyte Development and in Primary Myelofibrosis

Introduction: Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix (ECM) to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Primary Myelofibrosis (PMF) is a Chromosome Philadelphia-negative Myeloproliferative Neoplasm (MPN), characterized by altered stiffer ECM and megakaryocytosis, often driven by the gain-of-function mutation in JAK2 V617F. Here, we study a role for PIEZO1 in megakaryopoiesis and proplatelet formation under normal physiological conditions and in the context of PMF. Methods: Bone marrow Mks from C57BL/6J control and myelofibrotic mice carrying the human JAK2 V617F+ mutation, or Mks derived from stem cells of patients carrying the same mutation were analyzed at mRNA and protein levels for Piezo1 expression. Effects of Piezo1 activation or inhibition on Mk maturation and ploidy were assessed by flow cytometry, and proplatelet formation by fluorescent microscopy. Human peripheral blood samples from PMF patients, diagnosed according to established criteria, or healthy individuals were used to compare the two categories for Piezo1 relative expression on mRNA and protein levels and platelet biogenesis. Mk-specific double knockout mice of Piezo1/2 were generated to compare platelet counts under normal conditions or bone marrow ablation with 5-FU challenge. Culture of mouse and human Mks in different 3D microenvironments was used to demonstrate in vitro association of Piezo1 expression levels with different ECM stiffness substrates. Results: Mouse Mks express PIEZO1 and negligible levels of PIEZO2. Pharmacological activation of mouse PIEZO1 increases the number of immature CD41 + Mks, while the number of mature CD41 +CD42 + Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its pharmacological activation reduces Mk size, ploidy, maturation, and proplatelet development. Resulting effects of PIEZO1 activation on Mks resemble the profile in PMF. Intriguingly, Mks derived from PMF mice bearing the Jak2 V617F mutation show significantly elevated PIEZO1 expression, compared to wild-type controls. Pharmacological activation of PIEZO1 in these cells significantly augments the number of immature CD41 + Mks, and sharply reduces the number of mature CD41 +CD42 + cells and ploidy level. Accordingly, Mks isolated from bone marrow aspirates of JAK2 V617FPMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Additionally, PIEZO1 was overexpressed in 3D culture of human and mouse Mks in stiffer microenvironments as compared to softer ones and liquid medium. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2 V617FPMF patients are rescued upon PIEZO1 inhibition with GsMTx4. Discussion: Our data show that PIEZO1 might serve as a break of Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease. Our finding that inhibition of PIEZO1 activity partially rescues the aberrant Mk phenotype associated with PMF suggests that GsMTx4 holds potential as a therapeutic strategy to mitigate the pathological effects observed in Mk development in PMF patients.

Single-Cell RNA Sequencing Uncovers Abnormal Differentiation of Neutrophil and Megakaryocyte Might Mediated Ruxolitinib Resistance for Patinets with PMF

Background: Primary myelofibrosis (PMF ) is a heterogeneous disorder characterized by bone marrow fibrosis, megakaryocyte hyperplasia, systemic inflammation and extramedullary hematopoiesis. Ruxolitinib (Rux）have shown clinical benefit with reduction of splenomegaly and related symptoms. However, about 50% of patients discontinued ruxolitinib for resistance and/or intolerance，and the mechanism of resistance to ruxolitinib is still unclear. Recently studies found that immune microenvironment and megakaryocyte promote PMF progression, whether the abnormalities of immune microenvironment and megakaryocyte associated with Rux resistance needs to be further elucidated. M ethods: In this study, 8 participants (Newly Diagnosed PMF =3, Resistance to Rux PMF =3, Healthy Donors =2) were included. Peripheral blood was taken and single-cell transcriptome sequencing technology was used. Combined with pseudo-temporal analysis, SCENIC analysis, Single Cell Lineage Inference Using Cell Expression Similarity and Entropy (SLICE), cellular interaction analysis, gene ontology (GO) and Kyoto Gene and Genome Encyclopedia (KEGG) pathway analysis, Gene Set Variation Analysis (GSVA), L-R receptor coexpression analysis and GEPIA online tool for data analysis, Use R language or Python for data processing. Results: In PMF patients, the proportion of neutrophils and monocytes was significantly increased, while the proportion of T cells was significantly decreased in Resistance to Rux group (P&amp;lt;0.05). Further subdivision of neutrophils showed that neutrophils were divided into 6 subgroups. Compared with healthy control group, neutrophils increased in Resistance to Rux group and decreased in Neutrophil_1, among which Neutrophil_6 was only enriched in PMF patients. Neutrophil_6 significantly up-regulated tumor necrosis factor (TNF) superfamily product pathway, IL-1 signaling pathway, Toll-like Receptor 4 (TLR4) signaling pathway, peroxide-producing NADPH oxidase activator activity pathway, MAPK pathway and IL-8 product signaling pathway. Neutrophil_4-6 significantly up-regulates MYB, EZH2, SUZ12, YBX1, POLR3G, E2F7, E2F8 and other transcription factors. L-R up-regulation between Neutrophil_4 and Neutrophil_6 and T cells mediated T cell exhaustion by LGALS9/Tim3 relate to resistance to Rux. We have identified a subset of HSCs in PMF that exhibits a bias towards megakaryocytic/platelet differentiation (HSCs_2) in Rux resistant group. This subset is characterized by the upregulation of specific genes, such as GATA1, PRKAR2B, ITGA2B, PLEK, and KLF1.CellPhoneDB revealed that the regulation of platelets-platelets involves CD40L-CD40 ligand receptors. Notably, the heightened reactivity of platelets may be closely associated with the risk of PMF-related thrombosis. Additionally, we observed an upregulation of the MIF-CD74 cytokine ligand receptor between the immune platelet subsets and HSC subsets in Rux resistant group. Conclusion: In Rux resistant group, Up-regulation of inflammatory and tumor-related signaling pathways promotes the occurrence of PMF inflammation and amplification of malignant clones; Up-regulation of LGALS9 in neutrophil band to receptor TIM3 mediates T cell depletion; the regulation of platelets-platelets in PMF is mediated by CD40L-CD40 ligand receptors; MIF-CD74 might be the relationship between PMF-HSC and PMF-PLT in the context of primary myelofibrosis.

Neoplastic Monocyte-Derived Fibrocytes Induce Bone Marrow Fibrosis in Patients with Primary Myelofibrosis

Progressive bone marrow (BM) fibrosis is a defining feature of primary myelofibrosis (PMF). The extent of BM fibrosis has been closely associated with the clinical manifestations of advanced disease, including anemia and thrombocytopenia, rendering it an important prognostic indicator. Mesenchymal stromal cells (MSCs) have long been regarded as the exclusive inducer of BM fibrosis. However, using xenograft and transgenic animal models, we and other investigators demonstrated that neoplastic monocyte-derived fibrocytes independently contribute to the induction of BM fibrosis in PMF. Notably, our data revealed that PMF fibrocytes but not MSCs, carry pro-fibrotic signaling pathways that are absent in healthy individuals' fibrocytes or MSCs. Despite these findings, the precise contribution of BM fibrocytes and MSCs to the induction of BM fibrosis in patients with PMF remains elusive. To determine which of two subsets of collagen-producing cells are the main contributors to the induction of BM fibrosis in PMF, we analyzed BM biopsies of patients with PMF and assessed whether their distribution spatially correlated with areas of fibrosis. We analyzed formalin-fixed paraffin-embedded BM trephine biopsies obtained from previously untreated patients with PMF (n = 85) and age-matched healthy donors (n = 6). Using a 7-plexed fluorescence immunohistochemistry (IHC) in conjunction with hematoxylin/eosin, Masson's trichrome, and Gomorri's silver stains we analyzed those BM biopsy specimens. To ensure comprehensive assessment, we imaged serial whole-tissue sections from each patient at 400x resolution, meticulously aligning and carefully correcting for bone trabeculae and any artifacts. To quantitate the spatial distribution of individual cells, collagen fibers, and reticulin fibers, we developed a set of analytical algorithms based on a U-Net neural network in Visiopharm software. Our analysis revealed a 6-fold higher density of fibrocytes, defined as CD45 +/CD68 +/procollagen type I + cells, in the BM of patients with PMF ( P=0.0003) compared to normal controls. By contrast, the number of MSCs, defined as CD45 -/CD90 +/CD105 + cells, in the PMF BM was not different from the number of MSCs in normal BM. After dividing the area of each BM biopsy into a 100 μm regular hexagonal grid, the observed spatial distribution of fibrocytes strongly and positively correlated with the areas covered by reticulin and collagen fibers ( P&amp;lt;0.0001 in both). Furthermore, spatial analysis showed that increased densities of fibrocytes were associated with an increased number of fibrotic cellular zones in the BM tissue of PMF patients. To investigate the clonality of PMF BM fibrocytes, we conducted DNA in situ hybridization (ISH) analysis, employing two sets of locus-specific probe pairs. The DNA-ISH analysis provided valuable insights, revealing that fibrocytes obtained from PMF patients with trisomy 9 or 20q deletion carried the same corresponding cytogenetic abnormalities, indicating that PMF fibrocytes indeed originate from the neoplastic PMF clone. Because increased peripheral blood monocyte counts, commonly detected in patients with PMF, are associated with a poor prognosis, we sought to determine the potential correlation between BM fibrocyte and circulating monocyte counts. As expected, we found a significant positive correlation between BM fibrocyte counts and the percentage of circulating monocytes ( P=0.001). Moreover, we also found that fibrocyte numbers correlated with the levels of the plasma cytokines pentraxin 3, known to induce the differentiation of monocytes into fibrocytes, and matrix metalloproteinase 2, as assessed by using magnetic immunoassay, suggesting that neoplastic fibrocytes induce BM fibrosis in PMF and that fibrocyte quantification could serve as a valuable indicator of disease severity and prognosis. Taken together, our quantitative imaging analysis of BM biopsy tissue from treatment naïve PMF patients revealed that neoplastic fibrocytes, rather than MSCs, are the main contributors to the induction of BM fibrosis in PMF. Our findings underscore the importance of BM fibrocytes in the pathogenesis of PMF and warrant further investigation to identify novel therapeutic agents that will either inhibit fibrocyte differentiation or block their pro-fibrotic function.

Conventional Cytogenetic Analysis and Array CGH+SNP Identify Essential Thrombocythemia (ET)/Prefibrotic Primary Myelofibrosis (pre-PMF) Patients Who Are at Risk for Disease Progression

ET and pre-PMF are myeloproliferative neoplasms (MPNs) that are each characterized by thrombocytosis but are difficult to distinguish by clinical and histopathological criteria. Moreover, each has the propensity to progress at variable rates to more advanced forms of the disease. The cause of progression to more overt forms of myelofibrosis (MF) or accelerated /blast phase (AP/BP) has yet to be fully understood, making prognostication difficult. To identify patients (pts) who are at risk for disease progression, we retrospectively reviewed the karyotypes from conventional cytogenetics (CC) and array Comparative Genomic Hybridization + Single Nucleotide Polymorphism (aCGH+SNP) of 158 pts with a diagnosis of ET or pre-PMF from 2017 to 2022 at our institution. Among these pts, 58% were female (92 pts) and 42% were male (66 pts), and the median age was 61 years (range 13-92). Mutational analysis was performed in 155 pts, and JAK2 mutations were present in 63% of cases, CALR in 19%, and MPL in 7% of cases. The remaining 11% of pts were triple negative. An abnormal karyotype was observed in 15% (24/158), and the remaining 85% (134/158) were either normal or had non-diagnostic results due to failed or limited peripheral blood chromosome analysis. Of the 111 pts that had aCGH+SNP testing, 6% (7/111) had prior abnormalities as detected by conventional cytogenetic analyses, while abnormalities were found in 30% (33/111) with aCGH+SNP exclusively. In this cohort, 29% (46/158) of pts underwent disease progression. Among these cases, 65% (30/46) had progression to either MF or chronic myeloid leukemia or myelodysplasia, and 22% (10/46) progressed to AP/BP. In addition, 13% (6/46) evolved to polycythemia vera. Disease progression, irrespective of its form, was associated with genomic alterations detected by aCGH+SNP, cytogenetic analysis, or both at the time of first encounter. Of the 46 pts undergoing progression during their clinical course, 65% (30/46) had an abnormal genomic finding by either technology compared to 35% (16/46) who were normal (p&amp;lt;0.001). Strikingly, among 24 pts who were abnormal by classical cytogenetics, 79% (19/24) progressed to either AP/BP or MF compared to only 20% (26/125) who were cytogenetically normal (p&amp;lt;0.001). As shown in Figure 1, there was a shorter progression-free survival among pts who were cytogenetically abnormal as compared to normal pts (p&amp;lt;0.001). Among 134 pts with ET/pre-PMF who were normal or had non-diagnostic results by classical cytogenetic studies, aCGH+SNP testing was performed in 104 cases and additional genomic aberrations were detected in 32% (33/104). Within this group, 30% (10/33) progressed to MF with a trend towards a shorter progression-free survival. In those patients that progressed to MF, the most frequent abnormality was 9p CNLOH occurring in 6 pts. In the remaining 71 pts that were cytogenetically and aCGH+SNP normal, only 11% (8/71) had disease progression. Overall, the combination of both conventional cytogenetics and aCGH+SNP increased the sensitivity of detecting genomic abnormalities from 15% (cytogenetics alone) to 37% when incorporating aCGH+SNP. These data indicate that genomic alterations detected by conventional cytogenetic analyses or CGH+SNP array identify a subset of ET/pre-PMF pts that are at a higher risk of disease progression.

Preclinical Evaluation of INCB160058 - a Novel and Potentially Disease-Modifying Therapy for JAK2V617F Mutant Myeloproliferative Neoplasms

The Janus kinase 2 ( JAK2) mutation, JAK2V617F, is the most common oncogenic driver in myeloproliferative neoplasms (MPNs), with nearly all cases of polycythemia vera (PV) and over half of primary myelofibrosis (MF) and essential thrombocythemia (ET) patients positive for the somatic mutation. Approved therapies for MPNs such as ruxolitinib, which act by directly inhibiting activity of the kinase domain (JH1) of JAK2, have demonstrated impressive clinical efficacy and safety in patients with MPNs; however, they do not address JAK2V617F allelic burden or achieve molecular remission of disease. A targeted JAK2V617F selective agent sparing wild-type (WT) JAK2 activity has potential to eliminate mutant cells, induce molecular remission, and theoretically lead to functional cure of MPNs. We report herein the preclinical development of pseudokinase (JH2)-targeting INCB160058, a first-in-class, orally bioavailable small molecule with the ability to selectively target JAK2V617F + cell populations derived from patients with JAK-mutant MPNs. Using structure- and function-guided molecular design, INCB160058 was designed to bind with picomolar affinity to the JH2 domain of JAK2V617F at the canonical ATP-binding site, with high specificity (&amp;gt;2500-fold) relative to binding at the active kinase domain (JH1) targeted by currently approved JAK inhibitors. Live cell single-molecule fluorescence microscopy showed that INCB160058 binding to JAK2V617F blocked ligand-independent thrombopoietin receptor dimerization induced by the mutation, and consequently led to loss of JH1 domain kinase activity. X-ray crystallography analysis indicates that the observed inhibition is likely driven by conformational disruption of the ⍺C helix motif at Phe 594 and Phe 595 in conjunction with a shift of the upstream region from Leu 583 to Asn 589 upon INCB160058 binding to the JH2 domain of JAK2V617F. We utilized both CD34 + human multipotent hematopoietic stem cells derived from patients with JAK2-mutant MF, engineered JAK2-mutant human hematopoietic cancer cell lines (eg, SET2 and UKE-1), and murine BA/F3 cell lines to explore the selective effects of INCB160058 on JAK2V617F compared with WT JAK2. INCB160058 treatment selectively reduced pathogenic phospho-STAT5 levels, decreased abnormal megakaryopoiesis, and suppressed colony formation only in JAK2V617F + CD34 + cells but not in CD34 + cells from healthy volunteers. Importantly, continuous exposure of mutant and WT JAK2 cells to INCB160058 in co-cultures at concentrations below IC 50 resulted in progressive elimination of JAK2V617F + cells without affecting WT cells. At the end of the testing period, the JAK2V617F-harboring population was no longer detectable in the co-culture assay. In NSG mice subcutaneously inoculated with JAK2V617F-expressing SET2 cells, INCB160058 was tolerated and exhibited significant antitumor activity. In addition, following INCB160058 treatment, a significant reduction in the engraftment of total human cells, particularly human erythroid progenitors (hCD45 − mCD45 − Ter119 − hCD71 + hCD235a +), was observed in NSGS mice xenotransplanted with JAK2V617F + CD34 + cells. Moreover, INCB160058 treatment also led to the normalization of various pathogenic cytokines, such as interleukin (IL)-6 and IL-8. Importantly, these observations were absent in NSGS mice engrafted with CD34 + cells from healthy volunteers following INCB160058 treatment, further demonstrating the selectivity of INCB160058 for JAK2V617F. In summary, our results indicate a novel mechanism of action of INCB160058, a high-affinity pseudokinase (JH2) binding inhibitor of JAK2V617F that blocks cytokine-independent activity of JAK2V617F while preserving cytokine-dependent signaling. Extended treatment with INCB160058 at low therapeutic doses results in the specific elimination of mutant JAK2V617F-harboring cells in mouse models and human cancer cells with minimal impact on WT counterparts. Clinical testing of INCB160058 may allow patients with MPNs to achieve molecular remission by eliminating cells with the main genetic aberration and afford an opportunity to overcome the disease.

Acquired Von Willebrand Syndrome, Epidemiology, Laboratory Findings and Molecular Alterations on Classic Myeloproliferative Neoplasms in Mexican Population

Introduction: Acquired von Willebrand syndrome (AvWS) has been associated with Philadelphia-negative chronic myeloproliferative neoplasms (MPN) since 1984. However, the epidemiology of these disorders is not precise and has been underestimated worldwide but prevalence of 10-20% has been described in other series. Determinations of the antigenic von Willebrand factor (VWF:Ag) and its activity (VWF:RCo) are only performed when patients present thrombocytosis over 1,000 x10 3/µL. Essential Thrombocythemia (ET) is the most commonly associated MPN, found in 5-30%. Some cases have been described in patients with polycythemia vera (PV) and primary myelofibrosis (PMF) who develop AvWS even with platelet counts below 1,000 x10 3/µL. The epidemiology of AvWS in MPN and its association between laboratory and molecular parameters among the Mexican population have not been described. Objectives - Describe the clinical, biochemical and molecular characteristics of patients who develop AvWS with the aim of establishing the difference between cut-off scores of platelets to suspect AvWS in Mexican patients. - Establish the association between mutations in JAK2, CALR and MPL, and the appearance of AvWS. Material and methods: Retrospective, descriptive, observational, and single-center analysis. Patients over 18 years of age with a diagnosis of MPN from the INCMNSZ were recruited. The mutational analysis in peripheral blood (PB) for JAK2, CALR and MPL was assessed, with at least one follow-up between 2012 and 2022, excluded whose medical record were incomplete. Information was consecutively collected from 184 patients with MPN diagnosis between 01/01/2000 to 05/31/2022. Thirty patients were identified as having AvWS with a VWF:RCo/VWF:Ag diagnostic ratio of &amp;lt; 0.7. The epidemiology, clinical, laboratory and mutational alterations of the patients who developed AvWS by was analyzed using descriptive statistics with SPSS. This study was approved by the institutional bioethics committee. Results: AvWS developed in 16.4% (n = 30) of patients with MPN with an annual incidence of 2.4 cases per year was identified. Most patients were women in 66% (n = 20), with a median age of 57 years (IQR 20-72 years). Of the thirty cases with AvWS, 46.7% (n =14) corresponded to PMF, 40% (n =12) to PV, and 13.3% to ET (n = 4). The most frequently found mutation was JAK2 exon 14 with 76.7%, followed by CALR in 13.3% and triple negatives in 10%, no patient presented MPL mutations. The global median of VWF:RCo/VWF:Ag was 0.47 [PV 0.47 (IQR 0.31-0.64), ET 0.53 (IQR 0.5-0.64) and PMF 0.48 (0.29-0.65)] (Figure 1) and the median of the VWF:Ag 118 U/dL with FVW:RCo of 44.6 UI/dL (Table 1). The median platelet count for patients with the JAK2 mutation was 987.9 x103/µL (IQR 218 - 2307 x10 3/µL), while those with CALR and triple negatives were 1,033 x10 3/µL (IQR 713 - 1339 x10 3/µL), and 1,325 x10 3/µL (IQR 760 - 2059 x10 3/µL), respectively. Regarding hemoglobin (Hb) and hematocrit (Hto) levels, patients with PV and JAK2 mutation with AvWS presented medians of 17.45 g/d and 54.4% respectively, highlighting that AvWS was generated in some patients with platelet counts below 1,000 x10 3/µL. At diagnosis, bleeding episodes occurred in 13.3%. Lastly, 96.7% of the patients were treated with hydroxyurea, which corrected cellular alterations and laboratory abnormalities found in AvWS. Conclusions: AvWS can be found in patients with platelet counts below 1,000 x10 3/µL, especially among those with PV with elevated Hb and Hct levels. This may deem it necessary to carry out diagnostic tests in patients with MPN and any degree of thrombocytosis. To our knowledge, this is the most comprehensive study among latin american patients that describes the coexistence of PMF and AvWS; even though the mechanism by which some PMF patients develop AvWS is not perfectly understood. In our study, patients with PMF presented AvWS more frequently; this is a contrasting result from other international reports where ET was most attributed. We attribute this finding to changes made to the WHO criteria between 2008 and 2016.

Atrial Fibrillation in Patients with Myelofibrosis

Background: Atrial fibrillation (AF) is the most common arrythmia (1-2% of the general population) and is associated with high risk of arterial thrombotic events. AF is not well characterized in the context of myeloproliferative neoplasms (MPN). Recent studies imply that thrombotic risk stratification among MPN AF patients may be suboptimal using the standard CHA2DS2VASC score, and true predictor of thrombotic risk may be presence of myelofibrosis instead. Clinical context in which AF occurs in myelofibrosis patients had not been investigated so far, but such analysis is needed to better understand why is the presence of myelofibrosis in particular the main predictor of thrombotic events among AF MPN patients. We aimed to evaluate prevalence and clinical associations of AF among patients with myelofibrosis. Methods: We retrospectively investigated a cohort of 154 patients with overt myelofibrosis treated in 6 Croatian hematology centers in period 2004-2022. Diagnoses were reassessed according to the 2016 WHO criteria for primary myelofibrosis (PMF) and the 2008 IWGMRT criteria for secondary myelofibrosis (SMF). Presence of AF documented in medical records and clinical correlations of AF status with main disease related characteristics were analyzed. Results: Among a total of 154 patients, 86 had PMF, 35 had post polycythemia vera (PV) and 33 had post essential thrombocythemia (ET) SMF. Median age was 69 years. There were 91 (59%) males, 111 (75%) patients were JAK2, 9 (7.3%) CALR and 4 (3.3%) MPL mutated. AF was present in 15 (9.7%) patients with myelofibrosis which was significantly more frequent than anticipated in the general population of similar age (P&amp;lt;0.001). There were no significant associations of AF with neither etiology of myelofibrosis, age, sex, mutational status, degree of bone marrow fibrosis, spleen size, history of thrombosis, estimated glomerular filtration rate, hemoglobin, WBC nor platelet count (P&amp;gt;0.05 for all analyses). DIPSS score did not differ between AF and non-AF PMF patients, but MYSEC-PM score was significantly higher among AF SMF patients (P=0.029). AF in comparison to non-AF patients more frequently had constitutional symptoms (87% vs 54%, P=0.015), positive bleeding history (27% vs 6%, P=0.004), heart failure (47% vs 12%, P&amp;lt;0.001), obesity (21% vs 4%, P=0.010), lower MCHC (313 vs 318 g/L, P=0.031), higher urea (median 8.5 vs 6.8 g/L, P=0.009) and higher CHA2DS2VASC score (4 vs 3 points, P=0.003). AF more frequently received anticoagulants and diuretics than non-AF patients (P&amp;lt;0.05), whereas similar rates of cytoreductive therapy and aspirin were utilized. Despite all myelofibrosis patients with AF patients having CHA2DS2VASC score ≥2 points, only 60% of them received anticoagulant therapy, 27% received aspirin and 73% received cytoreductive therapy. No significant difference in survival and thrombosis was observed between AF and non-AF myelofibrosis patients (P&amp;gt;0.05 for all analyses) although study may be underpowered for these analyses. Summary/Conclusion: AF is frequent in patients with myelofibrosis and might be associated with higher frequency of constitutional symptoms, particular cardiovascular comorbidities and consistently high predetermined CHAD2DS2VASC risk. Nevertheless, unexpectedly low proportion of patients receives anticoagulant therapy, possibly due to anticipated risk of bleeding and cytopenias common in myelofibrosis patients. It remains a question whether suboptimal treatment or presence myelofibrosis itself might be a risk factor for unfavorable outcomes among AF MPN patients.

Preclinical Evaluation of JAK2 Specific Investigational Oligonucleotide for the Treatment of MPNs

Background: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell malignancies with cytokine independency or hypersensitivity. Polycythemia vera (PV), an acquired MPN characterized by increased blood cell mass and hematocrit and leukocyte count, is associated with incidental myelofibrosis (MF). Myelofibrosis (MF) is a chronic and progressive disorder characterized by bone marrow fibrosis, extramedullary hematopoiesis, leukoerythroblastosis and splenomegaly. Essential Thrombocythemia (ET) is another type of MPN associated with increased number and size of circulating platelets. All MPNs leave patients at risk for progression to leukemia. Abnormal cytokine signaling due to an aberrant JAK2-STAT pathway has a vital role in pathogenesis of various MPNs. JAK2 mutations can result in hematologic malignancies, where hyperactive signaling of the JAK2-STAT pathway promotes tumor cell proliferation, invasion, and angiogenesis. Increased JAK2 kinase activity is observed in hematologic malignancies; somatic JAK2 V617F gain-of-function mutations are found in large numbers between different subtypes of MPNs patients (40-95%) and are also implicated in MDS-MPN cases. We hypothesize that preventing JAK2 transcriptionby antisense oligonucleotide (ASO) mediated exon masking of the JAK2 intron-exon junction will result in reduced JAK2 mRNA, and thus JAK2 protein, by providing nonsense mediated decay (NMD) in the reading frame. This may alleviate disease manifestations of many patients suffering from different forms of MPNs. Methods: This study utilized a HEL92.1.7 cell line harboring the V617F JAK2 gain-of-function mutation. Antisense Oligonucleotides (ASOs) are small fragments of DNA/RNA that have the ability to reduce production of target protein via interaction with RNA. We designed a series ASOs targeting JAK2 sequence and tested these at a range of concentrations. The ASOs were designed and prioritized based on in silico binding affinity and limited off-target activity. HEL92.1.7 cells underwent treatment with several ASO-prototypes and functional readout at the end of 72h of incubation. Results: We observed significant JAK2 protein decrease (~50%) in ASO-treated samples compared to untreated samples. JAK2 qPCR results confirmed reduction of target transcript in 40-60% range. STAT5 phosphorylation status further confirmed this effect, and we report a ~35% pSTAT5 reduction. Furthermore, this ASO showed limited off-target effects in silico. siRNA and CRISPR knockout protein lysates were used as internal controls. Our preclinical data support our ASO as a highly specific JAK2 agent, affecting direct levels of JAK2 as well as downstream STAT signaling. Conclusions: Around 50-60% of primary MF patients harbor the JAK2 V617F gain-of-function mutation. ASOs offer the capability to directly target JAK2-driven neoplasms with highest precision and effectively reduce JAK2 protein production, without off-target effects linked with small molecules targeting various kinase families. The ability to reduce JAK2 protein may alleviate the disease burden that patients with hematologic malignancies face, resulting in a higher quality of life through prevention and treatment.

Clinical and Genetic Features By Next-Generation Sequencing and RNA Sequencing in Pre-Fibrotic Primary Myelofibrosis Patients

The 2016 WHO classification recognized pre-fibrotic primary myelofibrosis (pre-PMF) as a distinct entity. Such knowledge is essential to depict the landscape of pre-PMF and develop mechanism-based approaches to slow the progression of pre-PMF. Here, we conducted a retrospective study of 234 pre-PMF patients defined by 2016 WHO diagnostic criteria in 13 hematooncology centers between 01 January 2015 and 31 July 2023 (ChiCTR2200061208). We evaluated clinical characteristics, risk factors and genetic features of progression to overt-PMF in pre-PMF patients. Among 234 pre-PMF patients, 135 (57.7%) harbored JAK2V617F, 36 (15.4%) CALR and 3 (1.3%) MPL mutations; 60 (25.6%) patients were negative for all three mutations. JAK2V617F-mutated patients were older ( P=0.036), had more constitutional symptoms ( P=0.027) and cardiovascular risk factors ( P=0.045), higher white blood cell count (WBC) ( P&amp;lt;0.001), hemoglobin level (HB) ( P&amp;lt;0.001), neutrophile granulocyte (NEU) ( P&amp;lt;0.001), eosinophilic granulocyte (EOS) ( P&amp;lt;0.001), basophilic granulocyte (BAS) ( P&amp;lt;0.001), hematocrit (HCT) ( P=0.002) and red blood cell distribution width (RDW) ( P&amp;lt;0.001) (Figure A). CALR-mutated cases had higher platelet hematocrit (PCT) ( P=0.043) and lactic dehydrogenase (LDH) ( P=0.008). Besides, CALR group had a higher proportion of 25.0% to progress to overt-PMF than JAK2V617F group (9.6%) and TN group (6.7%) ( P=0.025) (Figure A). As for risk factors of progression to overt-PMF, univariable analysis found it was associated with mean platelet volume (MPV) ( P=0.019), HB ( P=0.027), WBC ( P=0.008), LDH ( P=0.004) and CALR mutations ( P=0.007). Multivariable analysis revealed that lower HB value ( P=0.041; HR 0.981, 95% CI 0.962-0.999) and CALR mutation ( P=0.001; HR 3.716, 95% CI 1.331-10.069) had negative impact on the progression to overt-PMF (Figure B). When divided by HB with cut-off point of 128 g/L, patients with HB value ≤128 g/L were more likely to progress to overt-PMF compared to &amp;gt;128 g/L groups ( P=0.007) (Figure C). And patients carried CALR mutation were also more likely to progress to overt-PMF ( P&amp;lt;0.001) (Figure D). We performed next-generation sequencing (NGS) in 18 pre-PMF patients, including 9 JAK2V617F, 3 CALR and 6 TN, 3 of which progressed to overt-PMF and 15 stayed in pre-PMF until the end of the follow-up. JAK2V617F was the most frequently mutated gene (9/38, 23.7%), followed by TET2 (7/38, 18.4%), RELN (4/38, 10.5%) and ASXL1 (3/38, 7.9%). JAK2V617F-mutated patients possessed more mutations related to activation signaling pathway ( P=0.025) and DNA methylation ( P=0.033) (Figure F). TN patients possessed more mutations related to transcription factor ( P=0.048) (Figure F). Patients who progressed to overt-PMF possessed more mutations associated with myeloid related transcription factor ( P=0.025) (Figure H). We further performed RNA-Seq analysis in 6 pre-PMF samples and 14 overt-PMF samples. Compared to pre-PMF, overt-PMF showed 49 up-regulated and 13 down-regulated differentially expressed genes (DEGs), of which CSCF3 and CXCL13 were significantly up-regulated (Figure I,J). Gene ontology (GO) functional enrichment analysis showed that DEGs were enriched in cell-cell signaling, cytokine activity, monocyte chemotaxis and chemokine activity (Figure K). KEGG biofunctional analysis showed that DEGs were significantly enriched in cytokine-cytokine receptor interaction, chemokine signaling pathway, IL-17 signaling pathway and NF-kappa B signaling pathway (Figure L). In conclusion, CALR-mutated pre-PMF patients with HB value ≤128 g/L were more likely to progress to overt-PMF. Genetic analysis found patients with high proportion of progress to overt-PMF tended to possess mutations associated with myeloid related transcription factor. And the upregulation of CSCF3 and CXCL13 may potentially drive the progression of overt-PMF in pre-PMF patients through inflammatory and cytokine related pathways. Acknowledgement: This research was funded by the Key R&amp;D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. *Correspondence to: Jian Huang, M.D., Ph.D., Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang, China. E-mail: househuang@zju.edu.cn

Positive-DAT and autoimmune manifestations in patients with JAK2V617F mutation

Objective: Myeloproliferative neoplasms (MPN) are occasionally associated with autoimmune manifestations. The prevalence of positive direct antiglobulin test (DAT) in patients with JAK2V617F mutation is not yet known. Methods: We conducted a cohort study of all consecutive patients with JAK2V617F mutation who underwent DAT at presentation from 2010 to 2022. We also looked for the prevalence of autoimmune diseases (AIDs) and relation to thrombosis. Results: Fifty nine patients formed the cohort, 22 females and 37 males, with a mean age of 65 years. The median follow-up time was 90 months. A positive-DAT was found in 6 to 7% of essential thrombocythemia (ET) and polycythemia vera (PV) patients, 60% of primary myelofibrosis (PMF) patients and 50% of myelodysplasia/myeloproliferation (MDS/MPN) patients with or without anemia. Autoimmune manifestations were present in 13% of patients with PV and 50% of those with MDS/MPN features. Overall, 25% of patients had autoimmune phenomena including the 15% with positive-DAT. Conclusion: In patients with JAK2V617F mutation, those with PMF and MDS/MPN subtypes are most likely to display a positive-DAT results and autoimmune manifestations (≥ 50%). Patients with PV and ET are less likely to exhibit autoimmune manifestations (≤ 10%). No correlation with the occurrence of thrombosis was seen.

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.

The chaperone protein GRP78 released from MPN cells increases the expression of lysyl oxidase in a human stromal cell line

Impaired function of the endoplasmic stress (ER) response causes numerous pathological conditions, including tissue fibrosis. In the present study, we aimed to determine the pathological role of ER stress response systems in myeloproliferative neoplasms (MPNs). We found increased expression of the chaperone protein glucose-regulated protein (GRP) 78, a central regulator of ER stress, in megakaryocytes from primary myelofibrosis or postessential thrombocythemia myelofibrosis patients. GRP78 was overexpressed in JAK2V617F-harboring cell lines; however, inhibitors of ER stress did not affect the expression levels of GRP78. In contrast, ruxolitinib, a well-known inhibitor of JAK2V617F, clearly blocked GRP78 expression in these cells through downregulation of transcription factor 4 (ATF4). Interestingly, GRP78 was secreted from HEL and SET-2 cells into culture media. Coculture of these cells with HS-5 cells, a human bone marrow stroma-derived cell line, induced enhanced expression of lysyl oxidase (LOX), which mediates cross-linking of collagen fibers and induces tissue fibrosis, in HS-5 cells. An anti-GRP78 neutralizing antibody abrogated LOX elevation; in contrast, recombinant GRP78 protein induced LOX protein expression in HS-5 cells. Our observations suggest that the oncogenic protein JAK2V617F induces overexpression and release of GRP78, which may induce a fibrotic phenotype in surrounding bone marrow stromal cells.

Detection of primary myelofibrosis in blood serum via Raman spectroscopy assisted by machine learning approaches; correlation with clinical diagnosis

Primary myelofibrosis (PM) is one of the myeloproliferative neoplasm, where stem cell-derived clonal neoplasms was noticed. Diagnosis of this disease is based on: physical examination, peripheral blood findings, bone marrow morphology, cytogenetics, and molecular markers. However, the molecular marker of PM, which is a mutation in the JAK2V617F gene, was observed also in other myeloproliferative neoplasms such as polycythemia vera and essential thrombocythemia. Therefore, there is a need to find methods that provide a marker unique to PM and allow for higher accuracy of PM diagnosis and consequently the treatment of the disease. Continuing, in this study, we used Raman spectroscopy, Principal Components Analysis (PCA), and Partial Least Squares (PLS) analysis as helpful diagnostic tools for PM. Consequently, we used serum collected from PM patients, which were classified using clinical parameters of PM such as the dynamic international prognostic scoring system (DIPSS) for primary myelofibrosis plus score, the JAK2V617F mutation, spleen size, bone marrow reticulin fibrosis degree and use of hydroxyurea drug features. Raman spectra showed higher amounts of C-H, C-C and C-C/C-N and amide II and lower amounts of amide I and vibrations of CH3 groups in PM patients than in healthy ones. Furthermore, shifts of amides II and I vibrations in PM patients were noticed. Machine learning methods were used to analyze Raman regions: (i) 800 cm−1 and 1800 cm−1, (ii) 1600 cm−1–1700 cm−1, and (iii) 2700 cm−1–3000 cm−1 showed 100 % accuracy, sensitivity, and specificity. Differences in the spectral dynamic showed that differences in the amide II and amide I regions were the most significant in distinguishing between PM and healthy subjects. Importantly, until now, the efficacy of Raman spectroscopy has not been established in clinical diagnostics of PM disease using the correlation between Raman spectra and PM clinical prognostic scoring. Continuing, our results showed the correlation between Raman signals and bone marrow fibrosis, as well as JAKV617F. Consequently, the results revealed that Raman spectroscopy has a high potential for use in medical laboratory diagnostics to quantify multiple biomarkers simultaneously, especially in the selected Raman regions.