Megakaryocyte Morphology Research Articles

Morphology, Morphometry, and Immunohistochemical Profile of Megakaryocytes and Bone Marrow Microenvironment in Disease Progression and Therapy Resistance in Chronic Myeloid Leukemia.

Background Tyrosine kinase inhibitors have revolutionized the treatment of chronic myeloid leukemia (CML) since the beginning of the century. However, resistance to therapy and the progression of disease tend to occur in certain patients. The bone marrow microenvironment may play a role in the disease outcome. Megakaryocytes have multiple roles in the regulation and maintenance of the hematopoietic stem cell microenvironment. In the current study, we evaluated the association of megakaryocyte morphology, morphometry, and microenvironment with disease progression and therapy resistance in CML. Methodology Megakaryocyte morphology and morphometry were analyzed and compared between the different phases (chronic and advanced) at diagnosis in 150 cases of BCR-ABL-positive CML. All CML-CP patients (n = 119) were followed up on tyrosine kinase inhibitor therapy for a minimum of 15 months and classified based on their treatment outcome as a response, resistance to therapy, or progression of disease based on standard criteria. Immunohistochemistry on a bone marrow trephine biopsy was done for vascular endothelial growth factor (VEGF), FOXP3, CD150, CD48, CD44, osteopontin, CXCL12, N-cadherin, PDL-1, and IL-7, and their expression on megakaryocytes and their association with treatment outcome was evaluated. Results The morphology and morphometry of megakaryocytes showed a heterogeneous population in CML. Morphology and morphometric parameters, when compared between the chronic and advanced phases of disease at diagnosis, did not show any statistical difference. Megakaryocytes were variably positive for VEGF, FOXP3, CD150, CD48, osteopontin, N-cadherin, CXCL12, CD44, PDL-1, and IL-7. However, only CD44-positive megakaryocytes were statistically associated with the treatment outcome. The patients with a higher expression of CD44 megakaryocytes progressed to the advanced phase of the disease during therapy compared to those who responded. Conclusion Megakaryocyte morphology and morphometry were heterogeneous in CML; however, they did not show any significant difference with either the phase of the disease or with treatment outcomes. Among the various immunohistochemical markers of the microenvironment, only CD44-positivity on megakaryocytes was associated with poor treatment outcomes.

Dysmorphic megakaryocytes in TAFRO syndrome: A case series from a single institute

TAFRO syndrome is a rare systemic inflammatory disorder of unknown etiology characterized by thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly. The diagnosis of TAFRO syndrome can be challenging; however, prompt diagnosis is vital because TAFRO syndrome is a progressive and life-threatening disease. We have showcased five patients with TAFRO syndrome who had similar bone marrow (BM) findings that could be considered the findings that characterize TAFRO syndrome. All patients were treated with corticosteroids and tocilizumab; three of the five patients (60 %) responded positively to the treatment. The unique BM findings observed in this study were megakaryocytes with distinct multinuclei and three-dimensional and indistinct bizarre nuclei (“dysmorphic megakaryocyte”), similar to the megakaryocyte morphology observed in myeloproliferative neoplasms (MPNs). Notably, dysmorphic megakaryocytes were observed in all five cases, whereas only two of the five patients tested positive for reticulin myelofibrosis, and three of the five patients had megakaryocytic hyperplasia, which are considered typical findings of TAFRO syndrome. Thus, the BM findings of dysmorphic megakaryocytes could help in the correct and immediate diagnosis of TAFRO syndrome.

Diagnostic Value of CD34 and CD117 Immunohistochemistry and Megakaryocyte Morphology in Myelodysplastic Syndromes: A Retrospective Case-control Study.

This study evaluated the diagnostic value of CD34 and CD117 immunohistochemistry(IHC) and megakaryocyte morphology in Myelodysplastic syndromes (MDS). In this study, CD34-positive individual cells (Type I) and small clusters (Type II) were observed in most cases (91.2%). Type II CD34-positive was seen in 24 (49%) MDS cases, and positive percentage was higher than in acute myelogenous leukemia (AML) or aplastic anemia (AA). Type II CD117-positive were observed in 44 (89.8%) MDS cases and Type I were observed in 5 (10.2%) MDS. Type II CD117-positive percentage was higher than in AML or AA. Megakaryocyte counts were normal or increased in most MDS cases except one. Although megakaryocyte counts of AML and AA were predominantly decreased, Most MDS patients (81.6%) had abnormal megakaryocyte, whereas almost none of megakaryocyte abnormality was found in AML and AA. In conclusion, combined detection of CD34 and CD117 and observation of megakaryocyte count and morphology are useful for the diagnosis of MDS.

Three-dimensional spatial mapping of the human hematopoietic microenvironment in healthy and diseased bone marrow.

The bone marrow hematopoietic microenvironment (HME) plays a pivotal role in regulating normal and diseased hematopoiesis. However, the spatial organization of the human HME has not been thoroughly investigated yet. Therefore, we developed a three-dimensional (3D) immunofluorescence model to analyze changes in the cellular architecture in control and diseased bone marrows (BMs). BM biopsies from patients with myeloproliferative neoplasms (MPNs) were stained sequentially for CD31, CD34, CD45, and CD271 with repetitive bleaching steps to realize five color images with DAPI as a nuclear stain. Hematopoietically normal age-matched BM biopsies served as controls. Twelve subsequent slides per sample were stacked to create three-dimensional bone marrow reconstructions with the imaging program Arivis Visions 4D. Iso-surfaces for niche cells and structures were created and exported as mesh objects for spatial distribution analysis in the 3D creation suite Blender. We recapitulated the bone marrow architecture using this approach and produced comprehensive 3D models of endosteal and perivascular BM niches. MPN bone marrows displayed apparent differences compared to the controls, especially concerning CD271 staining density, megakaryocyte (MK) morphology, and distribution. Furthermore, measurements of the spatial relationships of MKs and hematopoietic stem and progenitor cells with vessels and bone structures in their corresponding niche environments revealed the most pronounced differences in the vascular nice in polycythemia vera. Taken together, using a repetitive staining and bleaching approach allowed us to establish a 5-color analysis of human BM biopsies, which is difficult to achieve with conventional staining approaches. Based on this, we generated 3D BM models which recapitulated key pathological features and, importantly, allowed us to define the spatial relationships between different bone marrow cell types. We, therefore, believe that our method can provide new and valuable insights into bone marrow cellular interaction research.

The Role of Bone Marrow Biopsy Morphology and Clinical Characteristics in Facing the Challenges of Diagnosing Primary Myelofibrosis, Polycythemia Vera, and Essential Thrombocythemia at Cipto Mangunkusumo Hospital

BackgroundMyeloproliferative neoplasm (MPN) is a neoplasm characterized by the proliferation of one or more myeloid cells and their derivatives. The limitations of molecular examination in Indonesia make the diagnosis of MPN based on clinical and histopathological examination very crucial. The aim evaluate the clinicopathological profile of primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocythemia (ET) and identify the typical morphological characteristics of bone marrow biopsy.MethodsA retrospective study of cases diagnosed with MPN was conducted at the Department of Anatomic Pathology FMUI/RSCM in 2015-2019. Clinical data and evaluation of bone marrow morphology consisting of cellularity, erythroid myeloid ratio, cluster and megakaryocyte morphology, blast cells, fibrosis, osteosclerosis, and sinus dilatation were analyzed.ResultsA total of 172 cases were diagnosed as MPN BCR-ABL1-negative (PMF: 74; ET: 56; PV: 42). On routine blood examination, there was an increase in hemoglobin (Hb) and hematocrit (Ht) in PV, a decrease in Hb in PMF and an increase in platelets in ET (p<0.001). Splenomegaly is mostly found in PMF. Myeloid erythroid ratio was decreased in PV, normal in ET and increased in PMF (p<0.001). Megakaryocytes were arranged in loose clusters in 88.1% of PV cases and 96.4% of ET, dense clusters were found in 91.9% of PMF cases. Staghorn-like megakaryocytes were found in all ET cases and bulbous/cloud-like megakaryocytes were found in 97.3% of PMF cases. Most cases of PV (90.5%) and all cases of ET showed pre-fibrotic bone marrow (grade 0-1), while 77% of PMF cases showed fibrosis grade 2-3.ConclusionIn diagnosing PV, ET, and PMF, correlation between clinical data, laboratory, and bone marrow histomorphological evaluations, especially cellularity, myeloid:erythroid ratio, cluster and megakaryocyte morphology, degree of fibrosis, osteosclerosis, and sinus dilatation is required.

Atypical Megakaryocyte Morphology: an Important Diagnostic Clue for JAK2 Mutation–Associated Disorders

Atypical Megakaryocyte Morphology: an Important Diagnostic Clue for JAK2 Mutation–Associated Disorders

A Novel Approach to 3D Spatial Mapping of the Human Hematopoietic Microenvironment in Normal and Diseased Bone Marrow

The bone marrow hematopoietic microenvironment (HME) plays a pivotal role in regulating normal and diseased hematopoiesis. Important cellular components of the human (HME) include the bone marrow (BM) stromal cells (Li, BioRxive 2022) and other cell types, such as vascular endothelial cells (ECs), and megakaryocytes (MKs). Perturbations of the HME can affect normal and diseased hematopoiesis. In Philadelphia-negative myeloproliferative neoplasms (MPNs), which include Essential Thrombocythemia (ET), Polycythemia Vera (PV), Primary Myelofibrosis (PMF), and its precursor, pre-fibrotic MF (pre-PMF), the HME is of critical importance for disease development and progression (Mead, Blood 2017). However, despite this important role, little is known about the spatial organization of the different HME elements in MPNs. Therefore, we aimed to develop a novel method of immunofluorescence (IF) multiplexing with markers for HME and hematopoietic cells based on sequential staining and bleaching in combination with scanning IF microscopy and standard imaging analysis (Fig.1). Herein we show that this approach allowed us to generate 3D reconstitutions of standard biobank-stored BM samples from MPN patients and age-matched hematopoietically normal controls. Furthermore, we demonstrate how the free and open-source 3D computer graphics software toolset Blender (Stichting Blender Foundation, Amsterdam) can be utilized to analyze cell distributions and spatial relationships in 3D. BM biopsies from patients with MPN and controls were stained sequentially for CD31, CD34, CD45, and CD271 with repetitive bleaching steps to realize five color images with DAPI as a nuclear stain. Twelve subsequent slides per sample were stacked to create three-dimensional BM reconstructions with the imaging program Arivis Vision 4D. Iso-surfaces for niche cells and structures were created and exported as mesh objects for spatial distribution analysis in Blender. We recapitulated the BM architecture using this approach and produced comprehensive 3D models of endosteal and perivascular BM niches. MPN bone marrows displayed considerable differences between the different MPN types, especially concerning megakaryocyte (MK) morphology and distribution, as well as CD271 staining density and CD271+ cell numbers. Whereas ET and pre-PMF were comparable to their paired normal control, we found that PMF and especially PV samples showed considerably increased numbers and densities of CD271+ cells. In PMF the generated MSC objects formed a thick fibrillary structure reminiscent of classic fibrosis staining that was particularly dense close to vessels, bone surfaces, and MKs. Measurements of the spatial relationships of MKs and hematopoietic stem and progenitor cells (HSPC) to vessels and bone structures, respectively, revealed the most pronounced differences in PV, with median distances of PV-MKs to vessels being significantly lower than in controls (PV 7.62 µm vs 8.20 µm control, p<0.05). Also, the median distance in PV of HSPCs to vessels (2,88 µm) and bones (52.23 µm) was less than in the controls (46.31 µm and 236.7 µm, respectively; p<0.01). Taken together, we report a novel sequential staining and bleaching technique for multi-color analysis of normal and diseased BMs. The three-dimensional reconstruction technique developed herein revealed important aspects of the spatial HME composition in MPNs and normal BMs and provides a platform for a deeper investigation of BM pathophysiology, for example by increasing numbers of antibodies and including visualization of the expression of relevant genes and proteins. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clonal Megakaryocyte Dysplasia with Isolated Thrombocytosis Is a Distinct Myeloproliferative Neoplasm Phenotype

Introduction: About 15% of people with a myeloproliferative neoplasm (MPN) are identified as MPN, unclassifiable using the 2016 WHO classification. Methods: We tested whether persons with platelet concentration ≥450 × 10E+9/L, bone marrow megakaryocyte morphology typical of prefibrotic/early myelofibrosis (pre-MF), and no minor criteria of pre-MF should be classified as a distinct MPN subtype, clonal megakaryocyte dysplasia with isolated thrombocytosis (CMD-IT). Results: 139 subjects meet these criteria who we compared with primary myelofibrosis (PMF) including 402 with pre-MF and 521 with overt myelofibrosis. CMD-IT subjects were more likely female and younger. They had lower frequencies of JAK2^V617F compared with persons with PMF (55% vs. 70%; p < 0.001) and higher frequencies of CALR mutations (37% vs. 17%; p < 0.001). They also had lower frequency of variations associated with JAK2^V617F susceptibility, JAK2 46/1 (35% vs. 47%; p = 0.021), and VEGFA rs3025039 (12% vs. 17%; p = 0.030). Subjects with CMD-IT had lower incidences of thrombotic events compared with those with pre-MF (9.7% vs. 26%; p < 0.001) and longer survival (median, not reached vs. 23 years; HR = 0.34 (0.10, 0.30); p < 0.001). Conclusion: Our data indicate CMD-IT is a distinct MPN subtype and should be included in the classification of myeloid neoplasms.

Hirsutine, a novel megakaryopoiesis inducer, promotes thrombopoiesis via MEK/ERK/FOG1/TAL1 signaling

BackgroundThrombocytopenia (TP) remains a challenge in clinical hematology. TP may have serious consequences, such as recurrent skin and mucosal bleeding and increased risk of intracranial and internal organ hemorrhage. However, effective and safe therapeutic drugs for the long-term management of TP are still lacking. PurposeThis study aimed to identify more effective active compounds for TP therapy. MethodsLiquid chromatography-mass spectrometry-nuclear magnetic resonance analysis was used to confirm the medicinal species and chemical structure of Hirsutine (HS). The proliferation of HS was examined by Cell Counting Kit (CCK-8) assay on cells lines. The effect of HS on megakaryocyte differentiation was analyzed by evaluating the expression of CD41, CD42b, and DNA ploidy via flow cytometry (FCM). The morphology of megakaryocytes and intermediate cells was observed using an optical microscope. K562 cells were then stained with Giemsa and benzidine. qRT-PCR was used to examine the mRNA expression of GATA-1, GATA-2, FOG-1, TAL-1, RUNX-1, NF-E2, and KLF-1 in K562 cells. Protein levels of the transcription factors were analyzed by western blotting. An MEK inhibitor was used to verify the relationship between the MEK/ERK signaling pathway and CD41/CD42b (FCM), FOG-1, and TAL-1. The Kunming thrombocytopenia mouse model was established by X-ray irradiation (4 Gy) and used to test HS activity and related hematopoietic organ index in vivo. Finally, computer simulations of molecular docking were used to predict the binding energies between HS-MEK and HS-ERK. ResultsWe preliminarily identified HS by screening a plant-sourced compound library for natural compounds with megakaryocytic differentiation and maturation (MKD/MKM)-promoting activity. We found that HS not only enhanced MKD/MKM of K562 and Meg01 cells, but also suppressed the decline of peripheral platelet levels in X-ray-induced myelosuppressive mice. In addition, HS promoted MKD via activation of MEK-ERK-FOG1/TAL1 signaling, which may be the key molecular mechanism of HS action in TP treatment. Molecular docking simulations further verified that HS targets the signaling protein MEK with high-affinity. ConclusionIn this study, we report for the first time that hirsutine boosts MKD/MKM through the MEK/ERK/FOG1/TAL1 signaling pathway and thus represents a promising treatment option for TP.

Pedigree Analysis of ACTN1-Related Thrombocytopenia Attributed to A Novel Mutation

All the family members' peripheral blood was collected for routine blood tests, blood smear, coagulation function, and platelet aggregation test. Flow cytometry was used to detect the expression of platelet CD41 and CD61. The proband and her father were tested bone marrow cytomorphology. Whole-exome sequencing techniques were performed to detect and uncover mutant loci of suspected pathogenic genes. Bioinformatics was used to assess the conserved nature of the mutated loci and to analyze the effect of the mutated genes leading to the function of the corresponding amino acid sequences. The platelet count of the proband was 88×109/L, and the blood smear showed dumbbell-shaped platelets, snake-shaped platelets and platelets of various sizes. Her bone marrow cytomorphology revealed normal megakaryocyte morphology with a count of 270. The platelet count of the proband's father was 74×109/L, with large platelets and platelets of various sizes observed in the blood smear, and the morphology of megakaryocytes was normal in bone marrow with a megakaryocyte count of 239. Her grandfather had a platelet count of 83×109/L, with snake-shaped platelets and platelets of various sizes on blood smears. Other family members were normal in all tests. The missense mutation c.2396G > A in exon 20 of the ACTN1 gene in the proband resulted in the mutation of 799 amino acids of the encoded protein, i.e., Arg, to His. The sequencing results of her father and grandfather at this locus were found to be consistent with her. Furthermore, bioinformatics analysis indicated that the locus was highly conserved across species and that variation in this locus might lead to functional impairment of the protein. The protein model analysis demonstrated that α-actin-1 at position 799 Arg and Glu at position 811 could form a critical salt bridge which stabilizes the conformation of the Ca2+ binding loop within the calmodulin-like motif. the mutation of R799H lost this critical salt bridge and destabilized this structural domain. In the present study, the newly uncovered missense mutation c.2396G＞A in exon 20 of the ACTN1 gene is potentially the molecular mechanism for the thrombocytopenia.

CHANGES IN MEGAKARYOCYTES IN VARIOUS HEMATOLOGICAL CONDITIONS : A STUDY OF BONE MARROW ASPIRATION IN A TERTIARY CARE CENTRE

INTRODUCTION Dysmegakaryopoiesis is characterized by various Megakaryocytic alterations in bone marrow and includes both dysplastic and non dysplastic features.Dysplastic changes are mostly seen in myelodysplastic syndrome however megakaryocyte alteration have also been noted in some bone marrow aspiration in non myelodysplastic conditions. AIMS OBJECTIVE To evaluate changes in megakaryocytes in various hematological conditions in bone marrow aspiration examination. MATERIAL AND METHODS Study was done at Central Pathology lab in the Department of Pathology GAJRA RAJA MEDICAL COLLEGE GWALIOR over a period of 1.5 year FROM NOV 2019 TO MAY 2021.Total 84 bone marrow were received out of 14 were dilute so excluded from study and results were prepared from 70 cases.Age range was 9 months to 75 years RESULTS AND OBSERVATIONS In this study out of 70 patients, 43 were male and 27 were female.Patients were categorised into neoplastic and non neoplastic category.Out of 70 cases 46 were non neoplastic forming majority while 24 were neoplastic. Out of non neoplastic, megaloblastic anemia was most common while acute leukemia was most frequent in neoplastic category.Bone marrow was evaluated for cellularity. 47 were hypercellular marrow forming majority with 15 cases showing normal cellularity and 08 as hypocellular. Acute leukemia and megaloblastic anemia were two conditions which had hypercellular marrow. The number of the megakaryocytes was considered normal when one megakaryocyte per 3 low power fields was encountered,increased if more than two megakaryocytes per low power field were seen and decreased when one megakaryocyte per five to ten low power fields was documented and absent when no megakaryocytes were seen per ten low power field. Our study showed 27 cases of megakaryocytic hypocellularity, 22 cases normal number of megakaryocytes and 18 as increased number of megakaryocytes. Normally megakaryocytes have four to sixteen nuclear lobes. Immature megakaryocytes were defined as young forms of megakaryocytes with scant bluish cytoplasm and lacking lobulation of the nucleus. Dysplastic megakaryocytes are those with single, multiple/separated nuclei. Micro megakaryocytes were defined as megakaryocytes whose size was that of large lymphocyte or monocyte and which had a single / bi-lobed nucleus. Our study – showed normal megakaryocyte morphology in 32/70 (45%) cases and 35/70 (50%) were with altered morphology. CONCLUSION Dysplastic Morphologic changes in megakaryocyte were not only seen in MDS but also in various non MDS conditions which should be considered during diagnosis.Understanding and detailed knowledge of changes in megakaryocytes,including both cellularity and morphology,can improve the diagnostic accuracy for a wide range of hematological disorders

Characterization of the Role of Integrin α5β1 in Platelet Function, Hemostasis, and Experimental Thrombosis.

Objective Integrins are key regulators of various platelet functions. The pathophysiological importance of most platelet integrins has been investigated, with the exception of α5β1, a receptor for fibronectin. The aim of this study was to characterize the role of α5β1 in megakaryopoiesis, platelet function, and to determine its importance in hemostasis and arterial thrombosis. Approach and Results We generated a mouse strain deficient for integrin α5β1 on megakaryocytes and platelets (PF4Cre-α5 −/− ). PF4Cre-α5 −/− mice were viable, fertile, and presented no apparent signs of abnormality. Megakaryopoiesis appears unaltered as evidence by a normal megakaryocyte morphology and development, which is in agreement with a normal platelet count. Expression of the main platelet receptors and the response of PF4Cre-α5 −/− platelets to a series of agonists were all completely normal. Adhesion and aggregation of PF4Cre-α5 −/− platelets under shear flow on fibrinogen, laminin, or von Willebrand factor were unimpaired. In contrast, PF4Cre-α5 −/− platelets displayed a marked decrease in adhesion, activation, and aggregation on fibrillar cellular fibronectin and collagen. PF4Cre-α5 −/− mice presented no defect in a tail-bleeding time assay and no increase in inflammatory bleeding in a reverse passive Arthus model and a lipopolysaccharide pulmonary inflammation model. Finally, no defects were observed in three distinct experimental models of arterial thrombosis based on ferric chloride-induced injury of the carotid artery, mechanical injury of the abdominal aorta, or laser-induced injury of mesenteric vessels. Conclusion In summary, this study shows that platelet integrin α5β1 is a key receptor for fibrillar cellular fibronectin but is dispensable in hemostasis and arterial thrombosis.

Enhanced Autophagy Suppresses Proplatelet Formation in Pediatric Immune Thrombocytopenia

To investigate the effect of enhanced autophagy in megakaryocyte to proplatelet formation in children with immune thrombocytopenia(ITP). Giemsa staining and immunofluorescence staining were used to observe megakaryocyte morphology and proplatelet formation, Western blot was used to determine the expression of cytoskeleton protein and autophagy related protein. Autophagr regulation drugs Rap or 3-MA was used to regulate autophagy of megakaryocytes. Some vacuole-like structures was found in ITP megakaryocytes of the children, the expression of LC3II/I (ITP 1.32±0.18； Ctrl 0.49±0.16，P<0.05) and Atg5-Atg12 (ITP 0.69±0.17； Ctrl 0.12±0.08，P<0.05) was significantly higher in ITP children as compared with those in control group. The immu- nofluorescence staining showed that the cytoskeleton arrangement in megakaryocytes of ITP children was abnormal, and the phosphorylation of myosin light chain was also increased(ITP 0.74±0.09, Ctrl 0.05±0.02，P<0.05). In vitro, inducer or inhibitor of autophagy could regulate the production of proplatelet and the expression of cell cycle related protein, including CyclinD1（Veh 1.08±0.12; Rap 0.46±0.04; Rap+3-MA 0.70±0.03）, CyclinD2（Veh 0.47±0.04; Rap 0.27±0.04; Rap+3-MA 0.41±0.03）, P21（Veh 0.15±0.01; Rap 0.04±0.01; Rap+3-MA 0.05±0.01）. Enhanced autophagy is the key factor of poor proplatelet formation in megakaryocytes of ITP children.

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 Multicenter, Open-Label, Pilot Study of Alisertib (MLN8237), a Novel Inhibitor of Aurora Kinase a, in Myelofibrosis

Background: Primary/Post-Essential/Post-Polycythemic Myelofibrosis (MF) is characterized by impaired maturation of GATA1 deficient megakaryocytes, which we hypothesize directly contribute to disease. (J Clin Invest 2017). The selective AURKA inhibitor alisertib (MLN8237) was shown to promote megakaryocytic maturation, amelioration of bone marrow fibrosis and lessen disease in animal models (Nat Med 2015).Methods: In an investigator sponsored multi-center pilot study (clinical trials.gov Identifier NCT 02530619), alisertib was administered to MF patients with at least DIPSS intermediate 1 risk who were in need of treatment, and were intolerant/refractory to or unlikely to benefit from JAK inhibitor therapy. Eligibility criteria also included an absolute neutrophil count ≥ 1 x109/L and platelet count ≥ 50 x109/L. Alisertib was provided by Takeda and administered at a dose of 50 mg by mouth twice daily for 7 days every three weeks. Adverse events (AEs) were monitored by common terminology criteria (Version 4.0). Laboratory correlative studies included assessment of the effects of alisertib and ruxolitinib on diagnostic samples and evaluation of changes in megakaryocytes and GATA1 expression in the bone marrow with therapy.Results:i) Patient characteristicsAs of June 2017, 19 patients (63% males; median age 72 years) have been enrolled, including 68% primary MF and 32% post PV/ET MF; 26% were DIPSS high and 74% intermediate-risk.11 (89%) patients had abnormal karyotype, including 7(64%) with unfavorable risk. Driver mutational frequencies were JAK2 58%, CALR 32%, and MPL 10%. Prior treatments included JAK inhibitors in 68% of patients.13 (68%) patients remain on treatment (median number of cycles=4; range 1-16). Median follow up of patients that were alive was 5 months (range; 1-10 months).ii) Safety dataTreatment related grade 4 hematological AEs included neutropenia, lymphopenia and thrombocytopenia seen in one patient each (5%). Grade 3 related hematological AEs included lymphopenia (n=4; 21%), anemia (n=3, 16%), febrile neutropenia (n=2, 11%), thrombocytopenia (n=1; 5%). The incidence of grade 3 related non- hematological AEs was as follows- vertigo (n=1; 5%), diarrhea (n=1; 5%); elevated lipase, alanine aminotransferase, and creatinine in one patient (5%) each. There was one unrelated grade 5 event -pneumonia with renal failure.iii)Preliminary efficacy dataa) Spleen response : 11(58%) patients had palpable splenomegaly ≥ 5 cm at time of study entry; 3 (27%) achieved ≥ 50% reduction in palpable spleen size.b) Erythroid response: 8 (62%) patients were transfusion dependent at baseline, of which none achieved transfusion independence. One patient displayed a durable rise in hemoglobin of ≥ 1g/dl.c) Leukocytosis/Thrombocytosis/Leukoerythroblastosis/LDH responses: 6 (32%) patients presented with leukocytosis and all (100%) had resolution of leukocytosis with treatment. Two patients (11%) had baseline thrombocytosis and both displayed ≥ 50% reduction in platelet count. Ten patients (53%) demonstrated leukoerythroblastosis and 2 (20%) displayed ≥ 50% improvement. All 19 patients had increased serum lactate dehydrogenase levels at time of study entry and 6 (32%) displayed ≥ 50% reduction with treatment.d) Symptom response: Of the 9 patients with constitutional symptoms at presentation, 5 (56%) reported improvement/resolution of symptoms. Moreover, 4 of 16 (25%) patients with at least moderate symptom burden achieved ≥ 50% reduction in the MF-SAF score.iv) Correlative studies: Alisertib, but not ruxolitinib, robustly promoted polyploidization and apoptosis of megakaryocytes derived from patient CD34+ cells at diagnosis. Morphological analysis of bone marrow revealed that alisertib led to improved megakaryocyte morphology and restoration of GATA1 staining (Figure 1).Conclusions: In this study we have reproduced, in the setting of a clinical trial, our previous preclinical observations that alisertib appears to normalize megakaryopoiesis in the MPNs. The reduction in peripheral counts is consistent with studies that suggest atypical MF megakaryocytes promote expansion of leukocytes. Although Alisertib had limited activity beyond demonstrated myelosuppressive activity in patients treated an average of four cycles, it was relatively safe. Updated results including overall response assessment and bone marrow review will be presented. [Display omitted] DisclosuresWatts:Jazz Pharmaceuticals: Consultancy, Speakers Bureau. Altman:Syros: Consultancy; NCCN: Other: Educational speaker; BMS: Consultancy; Celgene: Consultancy; Astellas: Consultancy; Ceplene: Consultancy; Janssen Pharmaceuticals: Consultancy; Novartis: Consultancy; ASH: Other: Educational speaker. Giles:Novartis: Consultancy, Research Funding.

Acute megakaryoblastic leukaemia with erythrophagocytosis

A 42-year-old man with Down syndrome presented with fever and facial ecchymosis. A full blood count showed: white blood cells 21.0 × 109/l, haemoglobin concentration 89 g/l and platelets 14 × 109/l. The blood film showed 30% small to medium sized blast cells with scant cytoplasm and anisopoikilocytosis with dacrocytes. A bone marrow aspiration was performed. The bone marrow was hypercellular, with the presence of 40% of heterogeneous blast cells. Most blast cells were similar to those in the peripheral blood, being small with round to slightly irregular nuclei and scant agranular cytoplasm, but medium to large blast cells with irregular nuclei and cytoplasmic blebs were also present. Moreover, a small proportion of blasts were very large, either multinucleated or with polylobulated nuclei with a moderate amount of cytoplasm with blebs and, sometimes, coarse myeloperoxidase (MPO)-negative granules (all images, ×100 objective). Dysplasia was present in 12% of erythroid cells. Abundant examples of erythrophagocytosis by leukaemic blast cells were present (top images). Multiple mitosis were observed with some mitotic figures indicating that the cells were hyperdiploid (bottom left image). The blasts were positive for cluster of differentiation (CD)117, CD33, CD34, CD41 and CD61, and negative for MPO, CD71 and CD14 by flow cytometry. There was a complex karyotype with monosomy of 7 and 16, a marker chromosome and the known germline trisomy of 21. GATA-binding protein 1 (GATA1) Sanger sequencing showed no mutations. A diagnosis of myeloid leukaemia associated with Down syndrome (acute megakaryoblastic leukaemia) was made. However, the age of the patient and the absence of a GATA1 mutation suggest that this case differs from the cases usually reported in the first 5 years of life. The patient received induction treatment with ‘3+7’ (idarubicin and cytarabine) and FLAG-IDA (fludarabine, cytarabine and idarubicin) regimens; however, the disease was refractory to treatment and the patient died at 4 months after diagnosis. Erythrophagocytosis by leukaemic blasts is a rare finding in acute myeloid leukaemia. It has been reported in leukaemias with monocytic or megakaryocytic morphology and the presence of t(8;16)(p11.21;13.3), inv(8)(p11.21q13.3) or tetraploidy.

Platelet enhancement by Carica papaya L. leaf fractions in cyclophosphamide induced thrombocytopenic rats is due to elevated expression of CD110 receptor on megakaryocytes

Ethnopharmacological relevanceCarica papaya leaf juice/decoction has been in use in folk medicine in Srilanka, Malaysia and in few parts of India for enhancing the platelet counts in dengue. In Siddha medicine, a traditional form of medicine in India, papaya leaf juice has been used for increasing the platelet counts. Papaya leaf has been reported to enhance blood volume in ancient Ayurveda books in India. Carica papaya leaf is well known for its platelet enhancement activity. Although many preclinical and clinical studies have demonstrated the ability of papaya leaf juice for platelet enhancement, but the underlying mechanisms are still unclear. Aim of the studyThe study is aimed at identifying the key ingredients of papaya leaf extract and elucidate the mechanism (s) of action of the identified potent component in mitigating thrombocytopenia (Thp). Materials and methodsC. papaya leaf juice was subjected for sequential fractionation to identify the anti-thrombocytopenic phytochemicals. In vivo, stable thrombocytopenia was induced by subcutaneous injection of 70 mg/kg cyclophosphamide (Cyp). After induction, rats were treated with 200 and 400 mg/kg body weight papaya leaf juice and with identified fractions for 14 days. Serum thrombopoietin level was estimated using ELISA. CD110/cMpl, a receptor for thrombopoietin on platelets was measured by western blotting. ResultsAdministration of cyclophosphamide for 6 days induced thrombocytopenia (210.4 ± 14.2 × 103 cells/μL) in rats. Treating thrombocytopenic rats with papaya leaf juice and butanol fraction for 14 days significantly increased the platelet count to 1073.50 ± 29.6 and 1189.80 ± 36.5 × 103 cells/μL, respectively. C.papaya extracts normalized the elevated bleeding and clotting time and decreased oxidative markers by increasing endogenous antioxidants. A marginal increase in the serum thrombopoietin (TPO) level was observed in Cyp treated group compared to normal and treatment groups. Low expression of CD110/cMpl receptor found in Cyp treated group was enhanced by C. papaya extracts (CPJ) and CPJ-BT. Furthermore, examination of the morphology of bone marrow megakaryocytes, histopathology of liver and kidneys revealed the ability of CPJ and fractions in mitigating Cyp-induced thrombocytopenia in rats. ConclusionC. papaya leaf juice enhances the platelet count in chemotherapy-induced thrombocytopenia by increasing the expression of CD110 receptor on the megakaryocytes. Hence, activating CD110 receptor might be a viable strategy to increase the platelet production in individuals suffering from thrombocytopenia.

Morphologic and Immunophenotypic Differences in Juvenile Myelomonocytic Leukemias With CBL and Other Canonical RAS-pathway Gene Mutations: A Single Institutional Experience.

The diagnostic criteria for juvenile myelomonocytic leukemia have recently been revised to include clinical findings and RAS-pathway gene mutations per the 2016 World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues. Differing clinical behaviors have been observed in cases with CBL versus other RAS-pathway gene (RAS-p) mutations, notably the patients with CBL mutations can be self-limiting with spontaneous resolution. Additional clinical characteristics and histopathologic findings between these subsets are less well-described. We performed a retrospective search and identified cases with either CBL or RAS-p mutations, as per targeted and/or massively parallel sequencing. Eight patients had sufficient material for review, including cytogenetic studies and peripheral blood, bone marrow aspirate, and/or biopsy with flow cytometry analyses. Three patients showed CBL mutations and lower percentages of hemoglobin F and peripheral blood absolute monocyte counts, lesser degrees of leukocytosis compared with the RAS-p cohort, and normal megakaryocyte morphology and myeloblast immunophenotypes. Two of these patients were managed with observation only and experienced resolution of their disease. The patients with RAS-p mutations had severe thrombocytopenia, moderate to severe anemia, and experienced variable clinical outcomes. Abnormal megakaryocyte morphology and decreased numbers of megakaryocytes were seen in cases with RAS-p mutations. In addition, 3 of 4 cases with flow cytometry data demonstrated aberrant CD7 expression in myeloblasts. Our study is the first to identify morphologic and immunophenotypic differences between juvenile myelomonocytic leukemia cases with CBL or RAS-p mutations, and further supports previous reports of significantly different clinical behaviors between these subsets of patients.

Triple-Negative Essential Thrombocythemia: Clinical-Pathological and Molecular Features. A Single-Center Cohort Study.

Lack of demonstrable mutations affecting JAK2, CALR, or MPL driver genes within the spectrum of BCR-ABL1-negative myeloproliferative neoplasms (MPNs) is currently referred to as a triple-negative genotype, which is found in about 10% of patients with essential thrombocythemia (ET) and 5–10% of those with primary myelofibrosis (PMF). Very few papers are presently available on triple-negative ET, which is basically described as an indolent disease, differently from triple-negative PMF, which is an aggressive myeloid neoplasm, with a significantly higher risk of leukemic evolution. The aim of the present study was to evaluate the bone marrow morphology and the clinical-laboratory parameters of triple-negative ET patients, as well as to determine their molecular profile using next-generation sequencing (NGS) to identify any potential clonal biomarkers. We evaluated a single-center series of 40 triple-negative ET patients, diagnosed according to the 2017 WHO classification criteria and regularly followed up at the Hematology Unit of our Institution, between January 1983 and January 2019. In all patients, NGS was performed using the Illumina Ampliseq Myeloid Panel; morphological and immunohistochemical features of the bone marrow trephine biopsies were also thoroughly reviewed. Nucleotide variants were detected in 35 out of 40 patients. In detail, 29 subjects harbored one or two variants and six cases showed three or more concomitant nucleotide changes. The most frequent sequence variants involved the TET2 gene (55.0%), followed by KIT (27.5%). Histologically, most of the cases displayed a classical ET morphology. Interestingly, prevalent megakaryocytes morphology was more frequently polymorphic with a mixture of giant megakaryocytes with hyperlobulated nuclei, normal and small sized maturing elements, and naked nuclei. Finally, in five cases a mild degree of reticulin fibrosis (MF-1) was evident together with an increase in the micro-vessel density. By means of NGS we were able to identify nucleotide variants in most cases, thus we suggest that a sizeable proportion of triple-negative ET patients do have a clonal disease. In analogy with driver genes-mutated MPNs, these observations may prevent issues arising concerning triple-negative ET treatment, especially when a cytoreductive therapy may be warranted.

Clinicopathological Description from the Largest Series of Patients with the Novel Pro106Leu MPL gene Mutation Associated with Hereditary Thrombocythemia

Background: Hereditary thrombocythemia (HT) has been reported in Japanese and African populations in association with S505N, and N35K c-Mpl mutations, respectively. A novel Pro106Leu germ-line mutation in the c-Mpl gene has recently been shown to be associated with HT in Arabic population. Clinical and bone marrow (BM) features of Pro106Leu mutation are largely unknown. Methods: The molecular genetic databases at two tertiary hospitals in Riyadh, King Abdulaziz medical city (KAMC) and King Saud University medical city (KSUMC), were searched to identify all patients (pts) with MPL Pro106Leu mutation. Clinical and pathological data were retrospectively collected. BM aspiration and biopsy were independently reviewed retrospectively by two consultant hematopathologists and agreement was reached by a consensus review. Simple descriptive statistics were used to summarize the results. A univariate subgroup analysis, comparing the hematologic parameters between the homozygous and heterozygous genotypes was conducted using Pearson Chi-Square and t-tests. Results: A total of 115 pts with Pro106Leu MPL mutation were included, 86 (75%) from KAMC and 29 (25%) from KSUMC. All pts were ethnically Arabs. Median age was 33 years (yrs) (range: 0.4-68), 65 (56.5%) were female, and 31 (27%) were pediatric pts (age &amp;lt;18 yrs). MPL Pro106Leu mutation was homozygous in 87 (75.7%) pts, and heterozygous in 28 (24.3%). Spleen was enlarged in 3 (3%) pts, not documented in 15 (13%), and normal in 97 (84%). History of bleeding was documented among 11 (10%) pts. Thrombosis history was positive in 5 (4%) pts only, unavailable in 6 (5%), and negative in 104 (90%). Family history of thrombocytosis was reported in 46 (40%), but family history was not documented in 20 (17%). Common comorbidities include: autoimmune disease in 33 (29%), diabetes 21 (18%), and hypertension 20 (17%). Reasons for MPL testing was: abnormal routine blood work 79 (69%), family history of thrombocytosis 23 (20%), or others 13 (11%). Thrombocytosis [platelet (plt) counts &amp;gt; 450 x109/L] was documented in 107 (93%) pts, normal in 4 (3.5%), and low in 4 (3.5%) at the time of diagnosis of Pro106Leu mutation. See figure 1. The median plt count at the time of diagnosis of MPL Pro106Leu mutation was 667 x 109/L (range: 13-1473). The median mean plt volume was 8 fL (range 6.1-10.2), white blood cell count 8.4 x 109/L (2.46-68.35), absolute neutrophil count 5 x 109/L (1.01- 21.19), hemoglobin 132 gm/L (85-148), mean corpuscular volume 84.1 fL (57-117.3), mean corpuscular hemoglobin 27.7 pg (18-37.5), mean corpuscular hemoglobin concentration 327 g/dL (300-351), and red cell distribution width 13.6 (10.9-22.8). Ferritin less than 30 was seen in 40 (35%) pts, 27 of whom were women. No ferritin done in 14 pts. Iron stores (based on bone marrow, ferritin and iron saturation) were adequate in 56 (49%), inadequate in 49 (43%), and not documented in 10 (9%). BCR-ABL, JAK2 and CALR were only detected in 1 pt each. One pt was not tested for CALR mutation. All other pts were negative for the three mutations. Out of all 115 pts, 33 (29%) had an evaluable BM. BM cellularity ranged from 20-100 %, 12/33 (36%) were hypocellular, 17/33 (52%) normocellular, and 4/33 (12%) hypercellular. Megakaryocyte (meg) morphology revealed dysplastic changes in 20 (60%) (hypolobated megs or with separated lobes), only 7 (21%) of cases had cloudlike megs but none had staghorn or giant megs as described in essential thrombocythemia. BM megs were increased in 29 (87%). Small size megs were seen in 15/33 (45%). Clustering of megs was seen in the majority of the cases 30/33 (90%) of whom, 29(87%) had loose and 20 (60%) dense clusters. On univariate analysis (see tables 1-2), homozygous genotype was associated with higher plt count. Total of 65 (57%) pts were prescribed aspirin, and 16 (14%) hydroxyurea. At the time of last follow-up 114 (99%) of pts were alive. The median follow-up was 7.8 yrs from the time of thrombocytosis (ranged from 0-24.8). No case developed disease progression to myelofibrosis. One pt was diagnosed with T-lymphoblastic lymphoma and later died from treatment complications and another pt was diagnosed with CML. Conclusion: Pro106Leu mutation is associated with marked thrombocytosis at a younger age with a low risk of thrombosis. Homozygous genotype is associated with a significantly higher plt count. BM usually shows either normo- or hypocellular marrow with increased megs proliferation, and clustering. Disclosures No relevant conflicts of interest to declare.