Degree Of Bone Marrow Fibrosis Research Articles

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<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>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<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<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>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.

Construction of a Mouse Model for Myeloproliferative Neoplasms and an Evaluation System

To construct a myeloproliferative neoplasms (MPN) transplanted mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation, and establish a systematic evaluation system to verify the success of model construction. The bone marrow c-kit+ cells of the mice were obtained by the following steps: The mice were killed by cervical dislocation, the femur, tibia and ilium were separated, and the bone marrow cells were collected. The c-kit+ cells were sorted after incubation with CD117 magnetic beads. The method of constructing mouse primary mutant cells is as follows: A gene mutation vector with a GFP tag was constructed by the retroviral system, and the retroviral vector was packaged into the Platinum-E cells to obtain the virus supernatant, and then used it to infect the c-kit+ cells of mice. The MPN mouse model was constructed as follows: the mouse primary c-kit+ cells containing the mutant genes were collected after infection, and then transplanted them via the tail vein into the female recipient mice of the same species which were irradiated with a lethal dose of gamma rays (8.0 Gy). The MPN mouse model was evaluated as follows: After transplantation, the peripheral blood of the mice was regularly collected from the tail vein to perform the complete blood count test, and the size of spleen and the degree of bone marrow fibrosis were estimated. The mouse c-kit+ cells with the mutant genes were successfully obtained from the bone marrow. MPN mouse model was successfully constructed: The peripheral blood cells of the MPN-transplanted mice carried exogenous implanted GFP-positive cells, and the white blood cells (WBC), platelet (PLT) and hematocrit (HCT) were all increased; the body weight loss, and the water and food intake were reduced in the transplanted mice; further pathological analysis showed that the transplanted mice displayed splenomegaly and bone marrow fibrosis. These results suggested that the MPN mouse model was successfully constructed. According to the common and different characteristics of the three MPN mouse model, a preliminary evaluation system for judging the success of MPN mouse model construction was summarized, which mainly included the following indicators, for example, the proportion of GFP-positive cells in the peripheral blood of mice; WBC, PLT and HCT; the degree of spleen enlargement and the bone marrow fibrosis. The MPN mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation is successfully established by retroviral system, which can provide an important experimental animal model for the research of MPN pathogenesis and drug-targeted therapy.

MYF3001: A Randomized Open Label, Phase 3 Study to Evaluate Imetelstat Versus Best Available Therapy in Patients with Intermediate-2 or High-Risk Myelofibrosis Relapsed/Refractory to Janus Kinase Inhibitor

Myelofibrosis (MF) is a life-threatening myeloproliferative neoplasm. Janus Kinase inhibitors (JAKi) ruxolitinib, pacritinib and fedratinib are the FDA-approved treatment options for MF. Despite benefits reported with ruxolitinib in the frontline setting, a high proportion of patients discontinue treatment (Abdelrahman 2015), and the median overall survival (OS) is 11-16 months (Newberry 2017; Kuykendall 2018; Palandri 2019; Schain 2019; Mascarenhas 2020), highlighting a great unmet need for patients whose disease has relapsed or is non-responsive to JAKi treatment. Imetelstat, a first-in-class telomerase inhibitor, has shown meaningful clinical improvement in IMbark, a Phase 2 study in patients with intermediate-2 (Int2) or high-risk (HR) MF whose disease has relapsed after or was refractory to JAKi (Mascarenhas JCO 2021; NCT02426086). Treatment with 9.4 mg/kg imetelstat resulted in 32.2% symptom response (total symptom score [TSS] reduction ≥50%) at Week 24 and median OS of 29.9 months with median overall study follow-up of 27.4 months (Figure 1). Dose-dependent inhibition of telomerase with imetelstat resulted in on-target activity that correlated with clinical benefits; including dose-dependent reduction in variant allele frequency (VAF) of MF driver mutations and improvement of bone marrow (BM) fibrosis. Furthermore, VAF reduction and BM fibrosis improvement correlated with OS. Imetelstat has an acceptable safety profile, with the most common Grade 3 or higher treatment-emergent adverse events being thrombocytopenia and neutropenia that were manageable and reversible, with minimal clinical consequences. These Phase 2 efficacy and safety results support continued study of imetelstat 9.4 mg/kg in a Phase 3 randomized controlled study. Study MYF3001 (IMpactMF; NCT04576156) is a Phase 3, randomized (2:1), open label, multicenter study of imetelstat compared with best available therapy (BAT) in ~320 adult patients with Int2 or HR MF whose disease has relapsed after or is refractory to JAKi treatment, who are not candidates for further JAKi treatment, and who are not eligible for allogeneic stem cell transplantation. Relapsed and refractory disease will be determined by predefined increases in spleen volume after a period of response and a lack of spleen and/or symptom responses after at least 3 months of JAKi therapy, respectively. Patients will be randomized to receive intravenous imetelstat 9.4 mg/kg every 21 days or investigator-selected BAT that may include hydroxyurea, thalidomide, interferon, danazol, hypomethylating agents, chemotherapy, or other non-JAKi-containing therapy as appropriate (Figure 2). Hematopoietic stem cell transplant and splenectomy are not permitted as BATs. Eligible patients will have an Eastern Cooperative Oncology Group Performance Status score of 0-2 and peripheral blood and marrow blast counts <10%. Patients cannot have clinically significant cardiovascular disease, active systemic hepatitis infection, or chronic liver disease unrelated to underlying MF. At randomization, patients will be stratified based on a) Int2 or HR per Dynamic International Prognostic Scoring System; and b) platelet count at entry (platelets ≥75 and <150 x 109/L vs ≥150 x 109/L). Patients who meet progressive disease criteria and discontinue BAT may be eligible to crossover to imetelstat if they exhibit a ≥25% increase in spleen volume from pretreatment baseline at any time during the study or a palpable increase in splenomegaly following 6 months of BAT, as described in the protocol. The primary endpoint of Study MYF3001 is OS and an interim analysis of safety and efficacy is planned when ~35% of patients planned to be enrolled have died. Secondary endpoints include symptom and spleen response rates at Week 24, progression-free survival, clinical response assessments per modified 2013 International Working Group - Myeloproliferative Neoplasms Research and Treatment criteria, time to and duration of response, reduction in degree of bone marrow fibrosis, safety, pharmacokinetics and patient-reported outcomes. Biomarkers and mutation analyses will be performed to evaluate the impact of imetelstat on reduction/depletion of malignant clones. Approximately 180 sites are planned in North and South America, Europe, Middle East, Australia and Asia. The study is actively enrolling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tracking fibrosis in myeloproliferative neoplasms by CCR2 expression on CD34+ cells.

In myeloproliferative neoplasm (MPNs), bone marrow fibrosis - mainly driven by the neoplastic megakaryocytic clone - dictates a more severe disease stage with dismal prognosis and higher risk of leukemic evolution. Therefore, accurate patient allocation into different disease categories and timely identification of fibrotic transformation are mandatory for adequate treatment planning. Diagnostic strategy still mainly relies on clinical/laboratory assessment and bone marrow histopathology, which, however, requires an invasive procedure and frequently poses challenges also to expert hemopathologists. Here we tested the diagnostic accuracy of the detection, by flow cytometry, of CCR2+CD34+ cells to discriminate among MPN subtypes with different degrees of bone marrow fibrosis. We found that the detection of CCR2 on MPN CD34+ cells has a very good diagnostic accuracy for the differential diagnosis between “true” ET and prePMF (AUC 0.892, P<0.0001), and a good diagnostic accuracy for the differential diagnosis between prePMF and overtPMF (AUC 0.817, P=0.0089). Remarkably, in MPN population, the percentage of CCR2-expressing cells parallels the degree of bone marrow fibrosis. In ET/PV patients with a clinical picture suggestive for transition into spent phase, we demonstrated that only patients with confirmed secondary MF showed significantly higher levels of CCR2+CD34+ cells. Overall, flow cytometric CCR2+CD34+ cell detection can be envisioned in support of conventional bone marrow histopathology in compelling clinical scenarios, with the great advantage of being extremely rapid. For patients in follow-up, its role can be conceived as an initial patient screening for subsequent bone marrow biopsy when disease evolution is suspected.

P1048: MYF3001: A RANDOMIZED OPEN LABEL, PHASE 3 STUDY TO EVALUATE IMETELSTAT VERSUS BEST AVAILABLE THERAPY IN PATIENTS WITH INTERMEDIATE-2 OR HIGH-RISK MYELOFIBROSIS REFRACTORY TO JANUS KINASE INHIBITOR

Background: Myelofibrosis (MF) is a life-threatening myeloproliferative neoplasm. The Janus Kinase inhibitors (JAKi) ruxolitinib and fedratinib are the only FDA approved treatment options for MF. Despite benefits reported with ruxolitinib in the front-line setting, a high proportion of patients discontinue treatment (Abdelrahman 2015), and the median overall survival (OS) is 11-16 months (Kuykendall 2018; Newberry 2017; Schain 2019; Palandri 2019; Mascarenhas 2020), highlighting a great unmet need for pts non-responsive to a JAKi treatment. Imetelstat, a first-in-class telomerase inhibitor, has shown meaningful clinical improvement in IMbark, a Phase 2 study in patients with intermediate-2 or high-risk MF who have relapsed after or are refractory to JAKi (Mascarenhas JCO 2021). Treatment with 9.4 mg/kg imetelstat resulted in 32.2% symptom response (total symptom score [TSS] reduction ≥50%) at Week 24 and median overall survival (OS) of 29.9 months with overall study follow up of 27.4 months. Dose-dependent inhibition of telomerase with imetelstat resulted in on-target activity that correlated with clinical benefits; dose-dependent reduction in variant allele frequency of MF driver mutations indicated targeting of the underlying malignant clone. Aims: The Phase 2 results support continued study of imetelstat 9.4 mg/kg in a Phase 3 randomized controlled study, for which the study design is described here. Methods: Study MYF3001 (IMpactMF; NCT04576156) is an open label, randomized (2:1), multicenter, Phase 3 study of imetelstat compared with best available therapy (BAT) in ~320 patients with intermediate 2 or high-risk MF refractory to JAKi treatment. Patients will be randomized to receive imetelstat 9.4 mg/kg IV every 21 days or investigator selected BAT including hydroxyurea, thalidomide, interferon, danazol, hypomethylating agents, chemotherapy, or other non-JAKi containing therapy as appropriate). Eligible patients will be stratified based on a) Intermediate 2 or high-risk per Dynamic International Prognostic Scoring System; b) platelet count at entry (platelets ≥ 75 and < 150 x 109/L vs ≥ 150 x 109/L). Patients who meet progressive disease criteria and discontinue BAT may be eligible to crossover to imetelstat. Results: The primary endpoint is OS and one interim analysis is planned when approximately >71% of death events have occurred. Secondary endpoints include symptom and spleen response rates at week 24, progression-free survival, clinical response assessment per modified 2013 International Working Group - Myeloproliferative Neoplasms Research and Treatment criteria, time to and duration of response, reduction in degree of bone marrow fibrosis, safety, pharmacokinetics and patient-reported outcomes. Biomarkers and mutation analyses will be performed to evaluate the impact of imetelstat on reduction/depletion of malignant clones. (Figure 1) Image:Summary/Conclusion: Approximately 180 sites are planned in North and South America, Europe, Middle East, Australia and Asia. The study is open for enrollment.

Relationship between first-line treatment response and bone marrow fibrosis in newly diagnosed multiple myeloma

Objectives: Multiple myeloma (MM) occurs with uncontrolled and clonal increase proliferation of plasma cells in the bone marrow. Myelofibrosis can be primary or can be secondary when associated with other malignant or non-malignant diseases. MM is a malignant disease in which both collagen and reticulin fibrosis can be detected together at the time of diagnosis.The aim of this study is to investigate the relationship between bone marrow fibrosis at diagnosis and response to after first-line treatment in newly diagnosed MM. Material and Methods: In this study, 95 newly diagnosed MM patients were analyzed retrospectively. Demographic characteristics, complete blood count, biochemical examinations, bone marrow fibrosis grades, and first-line treatment response of the patients were retrieved from records as it is a retrospective study. Patients were divided into 2 groups according to their response to first- line treatment. Patients who have a strict complete response (sCR), complete response (CR) or a very good partial response (VGPR) responses to first-line therapy were included in the first group. Patients who gave partial response (PR), minimal response (MR) or progressive disease (PD) responses to the first-line therapy were included in the second group. Results: There were 72 patients in the Group I (good response group) and 23 patients in Group II (poor response group). Between the good response group and poor response group myeloma type, platelet count at diagnosis, β2 microglobulin, lactate dehydrogenase, erythrocyte sedimentation rate, bone marrow plasma cell ratio, R-ISS, and first-line treatment were not statistically significant (P &gt; 0.05 ). Age was statistically significantly lower in the good response group (P = 0.04). In male gender, a better response was obtained (P = 0.02). At the time of diagnosis, hemoglobin levels in the good response group were found high compared to the poor response group (P = 0.02). Bone marrow fibrosis was found to be lower at the time of diagnosis in the group that responded good response to first-line treatment (P = 0.01). Conclusion: In this study, it was shown that bone marrow fibrosis at diagnosis is an important factor affecting the response to first-line treatment.The degree of bone marrow fibrosis detected at the time of diagnosis in MM may guide the selection of targeted therapy in first-line treatment.

Immunoblotting-assisted assessment of JAK/STAT and PI3K/Akt/mTOR signaling in myeloproliferative neoplasms CD34+ stem cells.

Philadelphia-negative myeloproliferative neoplasms (pH-MPNs) origin from the clonal expansion of hematopoietic stem cells with acquired mutations leading to uncontrolled proliferation of differentiated myeloid cells. The main entities of Ph-MPNs are represented by Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) that are characterized by microvascular disorders, thrombosis and bleeding, splenomegaly secondary to extramedullary hematopoiesis, various degree of bone marrow fibrosis and a progressive risk of leukemic transformation. Somatic mutations in myeloid genes including JAK2, CALR, and MPL cause the constitutive activation of the Janus Kinase 2 (JAK)/signal transducer and activator of transcription (STAT) pathway that confers proliferative and differentiative advantage to mutated hematopoietic progenitors and ultimately drives the development of a Ph-MPNs phenotype. Beyond the JAK/STAT axis, a wide number of intracellular signaling pathways were found deregulated in Ph-MPNs including the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) constitutive activation. In this chapter, we provide a detailed protocol for the immunoblotting assisted assessment of Ph-MPNs pathways activation. This protocol can be easily adapted to study protein expression and phosphorylation of hematopoietic stem progenitors and differentiated cell lineages.

Assessment of Bone Marrow Biopsy and Cytogenetic Findings in Patients with Multiple Myeloma

Objective:Multiple myeloma (MM) is a malignant condition characterized by the accumulation of malignant plasma cells. Although MM remains incurable, the survival of MM patients has improved considerably due to the application of autologous stem cell transplantation, novel agents, and advanced treatment strategies. This study aimed to determine the cytogenetic characterization and bone marrow (BM) features of Turkish patients with MM.Materials and Methods:Eighty-five MM patients were admitted to Dokuz Eylül University Hospital in Turkey. BM samples of these MM patients were subjected to cytogenetic analyses at diagnosis and during therapy as a part of therapeutical and clinical evaluation. A complete cytogenetic study was performed using the G-banding technique. Fluorescence in situ hybridization (FISH) analysis was performed using cytoplasmic immunoglobulin. The degree of BM fibrosis was determined using reticulin histochemical staining. We determined the percentage of BM plasma cells based on the extent of CD38 staining.Results:Eighty-five MM patients were retrospectively identified between 2015 and 2021. The median age was 63 (38-90) years. Of the 85 patients, 60 (70.6%) were male and 25 (29.4%) were female. Seventy-two (84.7%) cases had BM fibrosis at the time of diagnosis. The most common was grade 2 fibrosis, recorded in 35 cases (41.2%). About 72.9% of the patients showed more than 50% plasma cells. FISH analysis indicated the presence of abnormal chromosomes in 37% (32/85) of the patients. The most frequent abnormality was Immunoglobulin heavy-chain (IGH) translocation (21.3%).Conclusion:Subgroup analysis of IGH mutations is crucial in the identification of high-risk MM patients. We believe that our study will contribute to the determination of BM biopsy and cytogenetic features of MM patients in our country.

Spleen Stiffness By Magnetic Resonance Elastography or Ultrasound Elastography As a Surrogate Marker of Bone Marrow Fibrosis in Myelofibrosis Patients

▪IntroductionPrimary myelofibrosis (PM) is a myeloproliferative neoplasm characterised by bone marrow fibrosis and extramedullary hematopoiesis. Both clinical findings and laboratory parameters are used for prognostic scores in Myelofibrosis patients. In addition, the degree of bone marrow fibrosis has an important prognostic value and has correlation with overall survival. Recently, bone marrow fibrosis was correlated with degree of splenic stiffness (SS) measured by imaging elastography techniques. 1,2 Despite these findings, there were patients with insignificant measures that could not be classified according to marrow fibrosis. In order to advance knowledge in this field, we studied splenic and hepatic stiffness (HS) in patients with myelofibrosis using elastography by two methods, ultrasonography (EUS) and magnetic resonance elastography (MRE), and its correlation with prognostic scores and bone marrow fibrosis.Study Design and MethodsThis is a prospective, cross-sectional, observational study in patients from the outpatient clinic for myeloproliferative neoplasms who had given informed consent according to procedures approved by institution´s ethical committee. Patients with PM, as well as post-essential thrombocythemia (ET) or post-polycythemia vera (PV) myelofibrosis, were included in this study. Myelofibrosis patients with diagnosis of other associated pathologies that may alter SS, as portal hypertension or cirrhosis, were excluded from the study. Patients were assessed for splenic stiffness measured by ultrasound conducted by two examiners, with more than 10 years of experience. EUS was performed in US Epiq 7 equipment - Philips - with ARFI elastometry methodology. The SS measurements was reported in m/s. In addition, they were also evaluated for splenic and liver stiffness by MRI technique. All exams were performed in 1.5 T MR equipment (Magneton Aera, Siemens Healthineers, Erlangen, Germany) and the MRI protocol included T2-weighted and gradient-echo MRE sequences using steady-state 60-Hz excitation and an external driver placed on the right side and, on the left side of the abdomen. The measures of SS were also obtained by two experienced examinersResultsAt this moment we present the results of 16 patients with myelofibrosis (PM: 8 cases; post-PV myelofibrosis: 2 cases; post-ET myelofibrosis: 6 cases). The median age was 69y (41-88y) and 62,5% of participants were male. The JAK2 V617F mutation was detected in 9 cases; three cases were CALR positive, and three cases were triple negative. The CBC showed: Hb: 10.9 g/dL (6.5-18.7); WBC (x10 9/L): 9.17 (1.8-44.5) and platelets (x10 9/L): 393 (10-957). Our preliminary results show that bone marrow fibrosis increased according to splenic stiffness by EUS and MRE (Figure 1a; table 1). Patients with osteosclerosis also presented a higher SS by MRE (Figure 1b). We could not find correlation of splenic stiffness with prognostic score DIPSS plus, although Int-2/High risk patients presented a trend to be associated with higher liver stiffness.ConclusionTo the moment, our preliminary findings suggest a correlation between SS and degree of bone marrow fibrosis and osteosclerosis, though the correlation between both measures and prognostic scores is still to be determined. We expect to have a better definition for all correlations, as we progress through the assessment of the other patients in our service. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Randomized Open-Label, Phase 3 Study to Evaluate Imetelstat Versus Best Available Therapy (BAT) in Patients with Intermediate-2 (Int-2) or High-Risk Myelofibrosis (MF) Refractory to Janus Kinase Inhibitor (JAKi)

A Randomized Open-Label, Phase 3 Study to Evaluate Imetelstat Versus Best Available Therapy (BAT) in Patients with Intermediate-2 (Int-2) or High-Risk Myelofibrosis (MF) Refractory to Janus Kinase Inhibitor (JAKi)

IL13 Contributes to Fibrotic Progression of the Myeloproliferative Neoplasms

Pre-fibrotic-primary myelofibrosis PMF (Pre-PMF) is an indolent form of PMF that frequently progresses to overt-PMF. While both stages of the disease are characterized by the presence of dysplastic megakaryocytes, progression to fulminant disease is associated with significantly increased fibrosis in the bone marrow. We have previously shown that megakaryocyte maturation in overt-PMF is impaired due to a GATA1 deficiency. However, in Pre-PMF patients most megakaryocytes express GATA1. This raises the possibility that alterations in megakaryocyte development occur during the progression of the disease and may contribute to fibrosis. This progression in megakaryocyte defects is likely driven not only by the aberrant JAK/STAT signaling but also microenvironmental factors. To identify such factors, we performed studies in two mouse models of PMF (driven by JAK2V617 and MPLW515L mutations) before and after development of fibrosis. Using an unbiased approach, we measured the levels of different cytokines in the bone marrow, plasma, and spleen. In addition, we performed single cell RNAseq in bone marrow populations. We observed extensive changes in the level of cytokines in the bone marrow of the MPLW515L mouse model compared to the JAK2V617F model. We initially focused on those cytokines that are elevated in the bone marrow of both murine models, including IL13 (Figure 1A), because previous studies have shown that IL13 is elevated in PMF patients and that JAK2 inhibitors do not decrease IL13 (1-3). Moreover, elevated IL13 has been identified in patients who progress to secondary AML (2). How IL13 may contribute to the progression of the disease has not been investigated.We assayed the effect of IL13 on megakaryocytes in vitro and discovered that it promoted megakaryocyte differentiation in the absence of thrombopoietin (TPO) and potentiated the effect of TPO. This effect was observed in cultures of both wild-type and MPLW515L megakaryocytes. Next, we assayed for expression of the IL13 receptor (Il13ra1) in the bone marrow of JAK2V617F and MPLW515L mutant mice and found that it was highly upregulated compared to wild-type animals. IL13ra1 expression was particularly intense in the megakaryocyte lineage, and its expression increased with disease progression (Figure 1B). Next, we asked whether IL13 is essential for myeloproliferative neoplasm (MPN) development in vivo. To study this, we transplanted bone marrow cells from Il4/13 f/f Mx1-Cre mice expressing MPLW515L to irradiated recipients, waited until MPN developed, and then excised by pIpC injection. This experiment revealed that loss of IL13 and IL4 led to a profound reduction in disease burden (Figure 1C), decreased splenomegaly, and diminished degree of bone marrow fibrosis. Moreover, loss of IL13 and IL4 decreased the levels of pro-inflammatory cytokines in the bone marrow and spleen (Figure 1D). We attribute this effect to deletion of IL13 because IL4 was only moderately increased in the bone marrow of the MPLW515L mouse model, and because IL4 has been reported to not be altered in the MPNs.Finally, we performed single cell RNA-seq on bone marrow cells from mice transplanted with JAK2V617F or control progenitor cells early and late in the disease process (Figure 1E). Our results revealed that there was decreased myeloid progenitors but an enhancement in the mast cell lineage that tracked with the degree of fibrosis. We confirmed the presence of elevated numbers of mast cells in the bone marrow by immunohistochemistry (Figure 1F). Mast cells produce IL13, and therefore they are the likely source for the increased IL13. Finally, consistent with the observation that IL13 signaling is primarily mediated through STAT6, we found enrichment of STAT6 target genes in megakaryocyte progenitors from the late timepoint in our scRNAseq data (Figure 1G).In summary, our data demonstrate that IL13 is involved in the progression of PMF and that inhibition of the IL13 signaling pathway should be investigated as a therapeutic option in PMF.1. Tefferi A, et al. J Clin Oncol (2011)2. Fisher DAC, et al. Leukemia (2019)3. Chen P, et al. Front Med (2021) [Display omitted] DisclosuresCrispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy.

SPLEEN STIFFNESS BY MAGNETIC RESONANCE ELASTOGRAPHY OR ULTRASOUND ELASTOGRAPHY AS A SURROGATE MARKER OF BONE MARROW FIBROSIS IN MYELOFIBROSIS PATIENTS

Primary myelofibrosis (PM) is a myeloproliferative neoplasm characterised by bone marrow fibrosis and extramedullary hematopoiesis. Both clinical findings and laboratory parameters are used for prognostic scores in Myelofibrosis patients. In addition, the degree of bone marrow fibrosis has an important prognostic value and has correlation with overall survival. Recently, bone marrow fibrosis was correlated with degree of splenic stiffness (SS) measured by imaging elastography techniques. Despite these findings, there were patients with insignificant measures that could not be classified according to marrow fibrosis. In order to advance knowledge in this field, we studied splenic and hepatic stiffness (HS) in patients with myelofibrosis using elastography by two methods, ultrasonography (EUS) and magnetic resonance elastography (MRE), and its correlation with prognostic scores and bone marrow fibrosis. This is a prospective, cross-sectional, observational study in patients from the outpatientclinic for myeloproliferative neoplasms who had given informed consent according to procedures approved by institutions ethical committee. Patients with PM, as well as post-essential thrombocythemia or post-polycythemia vera myelofibrosis, were included in this study. Myelofibrosis patients with diagnosis of other associated pathologies that may alter SS, as portal hypertension or cirrhosis, were excluded from the study. Patients were assessed for splenic stiffness measured by ultrasound conducted by two examiners, with more than 10 years of experience. EUS was performed in US Epiq 7 equipment - Philips – with ARFI elastometry methodology. The SS measurements was reported in m/s. In addition, they were also evaluated for splenic and liver stiffness by MRI technique. All exams were performed in 1.5 T MR equipment (Magneton Aera, Siemens Healthineers, Erlangen, Germany) and the MRI protocol included T2-weighted and gradient-echo MRE sequences using steady-state 60-Hzexcitation and an external driver placed on the right side and, on the left side of theabdomen. The measures of SS were also obtained by two experienced examiners. At this moment we present the results of 16 patients with myelofibrosis (PM: 8 cases; post-PV myelofibrosis: 2 cases; post-ET myelofibrosis: 6 cases). The median age was 69y (41-88y) and 62,5% of participants were male. The JAK2 V617F mutation was detected in 9 cases; three cases were CALR positive, and three cases were triple negative. The CBC showed: Hb: 10.9 g/dL (6.5-18.7); WBC (x10/L): 9.17 (1.8-44.5) and platelets (x10/L): 393 (10-957). Our preliminary results show that bone marrow fibrosis increased according to splenic stiffness by EUS and MRE. Patients with osteosclerosis also presented a higher SS by MRE. We could not find correlation of splenic stiffness with prognostic score DIPSS plus, although Int-2/High risk patients presented a trend to be associated with higher liver stiffness. To the moment, our preliminary findings suggest a correlation between SS and degree of bone marrow fibrosis and osteosclerosis, though the correlation between both measures and prognostic scores is still to be determined. We expect to have a better definition forall correlations, as we progress through the assessment of the other patients in our service.

Heterogeneity of Bone Marrow Morphology in Patients with Polycythemia Vera Resistant to First and Second Line Therapies - Impact of Hydroxyurea Dosage on MDS-like Progression

Background: First-line therapy in polycythemia vera (PV) generally consists of low-dose aspirin, phlebotomy, cytoreductive agents, and interferon. Hydroxyurea (HU) is the most frequently used cytoreductive agent, however, about 15-20% of patients (pts) become therapy resistant or intolerant. Resistance to HU is frequently associated with an aggressive disease course by increasing the risk of progression to post-PV myelofibrosis (PPMF). The clinical phenotype at this disease stage is very heterogeneous and often triggers a switch to second-line treatment options like JAK-inhibitor therapy.Design: A series of more than 500 consecutively collected cases with PV was screened in this observational study and analyzed with regard to inadequately controlled disease and resistance, intolerance or both to first-line cytoreductive agents. Only pts with available bone marrow (BM) trephine biopsies at this disease stage were finally selected. PPMF was defined according to the WHO 2016 criteria and HU resistance was assessed according to the European LeukaemiaNet (ELN). BM trephines were evaluated for grade of BM fibrosis (reticulin/collagen), cellularity, amount of CD34+ progenitors and morphology of megakaryocytes including the occurrence of myelodysplastic features associated with disease progression. Hematological and clinical data were collected at corresponding time points.Results: A total of 56 cases (31 males, 25 females, median age 67.5 years) with a representative BM trephine and uncontrolled disease or resistance to first-line cytoreductive agents was analyzed. Median time after diagnosis was 96.5 months (range 4 - 381 months), median treatment time with first-line cytoreductive agents (HU in 87.5%) was 48 months (range 12 - 240 months). Only 62.5% (35/56) of cases presented at this time point with a BM fibrosis grade ≥ 2 and thus fulfilled the WHO criteria for classical PPMF. 14 cases (25.0%) revealed a BM fibrosis grade of 1, whereas an uncontrolled myeloproliferation defined by increased platelet and/or leukocyte counts was found in 7 cases (12.5%). In 2 patients (3.5%) a blast phase was diagnosed at the time of treatment failure which was not associated with progressive fibrosis. In classical PPMF BM cellularity was heterogeneous but generally increased. Cases with grade 1 fibrosis or uncontrolled myeloproliferation had higher hemoglobin levels, lower leukocytes count, and higher platelet counts at time of treatment failure as compared to the PPMF group. On the other site, classical post-PV MF more often was presenting with relevant cytopenia and erythrocyte transfusion dependency. There was no difference in the amount of CD34+ progenitors between classical PPMF and cases presenting with either grade 1 fibrosis or uncontrolled myeloproliferation. MDS-like progression characterized by highly atypical megakaryocytes was observed in 12 pts (21.4%), all pts presented with classical PPMF. For all pts. treated with HU the total cumulative dosage, the average dosage per years, as well as the highest used dosage to control disease was calculated. Pts presenting with MDS-like BM features received a significantly higher total HU dosage (6,230 vs 721 gram, p=0.011) and showed a longer median treatment time with HU (72 vs 48 months), whereas the average annual dosage showed no significant difference.Conclusions: Progression of inadequately controlled PV represents a biological spectrum and is not restricted by degree of BM fibrosis as in classical WHO defined PPMF, but can also be defined by resistance to cytoreductive agents like HU. In clinical practice it is important to recognize that high dosages of HU and long treatment duration are more often seen in the important subgroup presenting with MDS-like features. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Randomized Open-Label, Phase 3 Study to Evaluate Imetelstat Versus Best Available Therapy (BAT) in Patients with Intermediate-2 or High-Risk Myelofibrosis (MF) Refractory to Janus Kinase (JAK) Inhibitor

A Randomized Open-Label, Phase 3 Study to Evaluate Imetelstat Versus Best Available Therapy (BAT) in Patients with Intermediate-2 or High-Risk Myelofibrosis (MF) Refractory to Janus Kinase (JAK) Inhibitor

Reduced renal function strongly affects survival and thrombosis in patients with myelofibrosis.

We retrospectively investigated a cohort of 176 myelofibrosis patients (128 primary-PMF; 48 secondary-SMF) from five hematology centers. The presence of chronic kidney disease (CKD) was determined in addition to other clinical characteristics. CKD was present in 26.1% of MF patients and was significantly associated with older age (P < 0.001), higher WBC (P = 0.015), and its subsets (neutrophil, monocyte, and basophil counts), higher platelets (P = 0.001), lower albumin (P = 0.018), higher serum uric acid (P = 0.001), higher LDH (P = 0.022), and the presence of CV risk factors (P = 0.011). There was no significant association with driver mutations, degree of bone marrow fibrosis, PMF/SMF, or DIPSS risk categories (P > 0.05 for all analyses). The presence of CKD was significantly associated with shorter time to arterial (HR = 3.49; P = 0.041) and venous thrombosis (HR = 7.08; P = 0.030) as well as with shorter overall survival (HR 2.08; P = 0.009). In multivariate analyses, CKD (HR = 1.8; P = 0.014) was associated with shorter survival independently of the DIPSS (HR = 2.7; P < 0.001); its effect being more pronounced in lower (HR = 3.56; P = 0.036) than higher DIPSS categories (HR = 2.07; P = 0.023). MF patients with CKD should be candidates for active management aimed at the improvement of renal function. Prospective studies defining the optimal therapeutic approach are highly needed.

Chronic myelomonocytic leukemia genomic signature correlates with the degree of bone marrow fibrosis: A single-institutional retrospective study.

7559 Background: Chronic myelomonocytic leukemia (CMML) has features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. The median overall survival (OS) in most series is 20-40 months. CMML is a relatively rare entity, and there is limited understanding of prognostic molecular markers. CMML associated with bone marrow fibrosis grade 1, appear to have shorter progression free survival. In this study we investigated the correlation of mutations with bone marrow reticulin fibrosis in patients with CMML. Methods: We investigated a cohort of 41 consecutive patients diagnosed with CMML 0, 1, 2, and CMML-AML from 2014 to 2019 at our institute. The median age of 41 patients was 68 years (range, 34-82). 27% were females and 73% were males. This cohort consists of 8 (20%) patients with CMML0, 19 (46%) with CMML1, 8 (20%) with CMML2 and 6 (15%) with CMML-AML. Genomic DNA was extracted from the bone marrow aspirates and targeted mutation NGS libraries were prepared from 200 ng of genomic DNA using the SureSelect target enrichment system (Agilent Technologies Inc.). The gene panel consists of 73 genes focused on myeloid neoplasms. The data were curated on the basis of our molecular pathology and national databases. Additionally, clinical data and bone marrow (BM) reticulin fibrosis grades (n = 27 available) were retrieved from the patient’s medical record. Results: The mutational profile frequency in our cohort showed that the most common mutations were TET2 (31%), ASXL1 (31%), and SRSF2 (23%), with frequencies very similar to those reported in the literature. Of the 27 cases with an available reticulin stain, only low grade fibrosis (MF-0, n = 18. MF-1, n = 9) were identified in our cohort. The frequencies of mutations in ASXL1, U2AF1, TP53, JAK2 and RUNX1, positively correlated with low grade fibrosis (MF-1). Additionally, patients with higher frequencies of SRSF2, TET2, and SETBP1 mutations showed no fibrosis (MF-0). Conclusions: This study is the first to correlate the degree of fibrosis with the frequency of mutations in CMML. We found similar mutations spectrum reported in the literature in patients with CMML. The mutational profile associated with CMML cases appears to affect the degree of the bone marrow fibrosis at the time of diagnosis.

Significance of cytogenetic-risk categories and refined international prognostic scoring system for overall survival in primary myelofibrosis: A single-center experience

Abstract Background/Aim. Primary myelofibrosis (PMF) is a chronic, malignant hematological disease characterized by a leucoerythroblastic blood picture, anisopoikilocytosis teardrop- shaped erythrocytes, different degrees of bone marrow fibrosis and hepatosplenomegaly due to extramedullary hematopoiesis. Among genetic specificities of the disease, those that stand out are chromosomal aberrations in pathological, myeloid blood cells. The aim of this study was to examine the prognostic significance of clinical, hematologic and cytogenetic parameters in PMF. Methods. A retrospective study included 144 patients with PMF. Karyotypes were analyzed using conventional cytogenetic methods. Results. The chromosome examinations were successful in 126 (88%) patients and failed in the remainder ones (12%). Karyotype was abnormal in 36/126 (29%) subjects at presentation. The most frequent changes included +9, 13q- and 20q- (28%). Other abnormalities were: aberrations of chromosome 18 and 16, deletions (9q-, 12p-, 7q-, 5q-, 6q-, 8q-), trisomies (+1q, +8, +10, +21), monosomies (-7, -11), 3q inversion and loss of Y chromosome. We detected four novel balanced translocations in PMF: t(17;22)(q11;q13), t(15;17)(q22;q25), t(9;12)(q22;q24) and t(2;4)(q21;p16), one constitutional translocation-rob(13;14)(q10;q10) and some new karyotype anomalies ? deletion of both homologues, hyperdiploidy and the coexistence of unrelated pathological clones. Conclusion. Chromosomal aberrations had a significant influence on overall survival of patients with PMF according to the refined cytogenetic-risk of the International Prognostic Scoring System (Refined CIPSS) (p = 0.004). Our patients matched the pattern of chromosome aberrations usually observed in PMF but some newly registered, balanced translocations and other rare karyotype anomalies were recorded as well.

Constitutively Activated STAT3 Induces the Production of PTX3 That Contributes to the Induction of Bone Marrow Reticulin Fibrosis in Patient with CLL

Constitutively Activated STAT3 Induces the Production of PTX3 That Contributes to the Induction of Bone Marrow Reticulin Fibrosis in Patient with CLL

Myelofibrosis in Patients of Chronic Myeloid Leukemia in Chronic Phase at Presentation.

To determine the frequency and grading of myelofibrosis in patients of chronic myeloid leukaemia in chronic phase, and the association of cytopenias/cytosis with the degree of fibrosis. Descriptive study. King Edward Medical University, Lahore, from March 2015 to April 2017. Patients of both genders and all age groups with Philadelphia chromosome/BCR-ABL positivity were included in the study. Detailed medical history and examination findings were recorded. Sample for CBC was taken in EDTA and run on automated haematology analyser. Bone marrow aspirate and trephine biopsy was done from posterior iliac crest. Bone marrow aspirate slides were stained with Giemsa stain. The trephine biopsy was processed and stained with haematoxilyn and eosin. Silver (reticulin) and trichrome staining was done on trephine biopsy to assess the degree of fibrosis. Grading of fibrosis was done according to WHO revised classification 2016. Among the 82 cases, 65% (n=53) were having WHO Grade MF 1 myelofibrosis, 27% (n=22) WHO Grade MF 2 myelofibrosis; whereas, 08% (n=07) WHO Grade MF 3 myelofibrosis. CBC counts were analysed to see any association between anemia, thrombocytocytosis and leucocytosis with advanced fibrosis (p=0.148, 0.413 and 0.174, respectively). Some degree of bone marrow fibrosis was present in all patients of chronic myeloid leukemia, while advance fibrosis (WHO Grade MF 2 and 3) is also common. Peripheral blood counts are not predictor of increased fibrosis. Therefore, bone marrow biopsy with special staining should be done in all cases of chronic phase of CML.

PS1452 GLI‐1, EFFECTOR OF CANONICAL HEDGEHOG SIGNALING PATHWAY, DOES NOT SEEM TO BE DYSREGULATED IN PATIENTS WITH MYELOFIBROSIS

Background:Although activation of Hedgehog signaling pathway was reported to be present in murine bone marrow transplant model of primary myelofibrosis (PMF) and increased expression of Hedgehog target genes was reported in granulocytes isolated from unselected patients with myeloproliferative neoplasms, therapeutic Hedgehog inhibition showed only limited results in myelofibrosis so far. Also, inconsistent effects of such therapy on Gli1 (effector of hedgehog signaling) were observed.Aims:We aimed to investigate Gli‐1 expression in bone‐marrow tissues of patients with primary and secondary myelofibrosis (SMF) and to assess its clinical correlations.Methods:We retrospectively investigated Gli‐1 mRNA expression in 32 diseased (28 PMF, 4 SMF) and 16 healthy (limited stage agressive Non‐Hodgkin lymphoma) bone marrow samples using real‐time‐polymerase‐chain‐reaction (GLI1 Hs01110766_m1 Thermo Fischer Scientific TaqMan assay). Expression was normalized to Abl and expressed as a ΔCT value. Correlations with clinical parameters were made. Study was approved by the institutional review board. All procedures were in accordance with the Helsinki Declaration. All patients provided written informed consent.Results:Median age of our patients was 70 years, there were 21 (66%) males and 11 (34%) females. Gli‐1 expression did not significantly differ between PMF, SMF and controls (median ΔCT 7.2, 7.3 and 6.9 respectively; P = 0.864).Higher Gli‐1 bone marrow expression was not significantly associated with neither age, gender, JAK2, CALR, MPL mutational status nor degree of bone marrow fibrosis (P&gt;0.05 for all analyses). Only statistically significant associations were present between higher Gli‐1 expression and higher transferrin saturation (P = 0.039; Rho = 0.4) and higher absolute lymphocyte count (P = 0.032; Rho = 0.4).Gli‐1 expression separated at median (ΔCT 7.25) had neutral effect on overall survival in our cohort of patients (P = 0.651) as shown in a Figure.Summary/Conclusion:Our findings indicate Gli‐1 mRNA expression might not be dysregulated in patients with myelofibrosis and has neutral impact on prognosis of these patients. Although limited by small number of patients, retrospective study design and single center experience, our results are in line with previous observations of modest clinical effects of Hedgehog inhibition in patients with myelofibrosis.image