Hypercellular Bone Marrow Research Articles

Prognostic Significance of Bone Marrow Cellularity in the Outcome of Patients with Myelodysplastic Syndromes Treated with Azacyitidine: A Retrospective Analysis from the Hellenic MDS Study Group

Prognostic Significance of Bone Marrow Cellularity in the Outcome of Patients with Myelodysplastic Syndromes Treated with Azacyitidine: A Retrospective Analysis from the Hellenic MDS Study Group

JAK-2-Positive Latent Myeloproliferative Neoplasms with Splanchnic Vein Thrombosis- from the Hematopathology Perspective

Introduction: Philadephia-negative Myeloproliferative neoplasms (MPNs) include polycythemia Vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The diagnosis of MPNs and further sub classification are based mainly on WHO diagnostic criteria which rely on the presence of an evidence of single or multiple cell lineage proliferation reflected by elevated hemoglobin, and / leukocytosis and /thrombocytosis. In addition, to presence of certain pathologic features in the bone marrow (BM) including hypercellularity , panmyelosis, proliferation of megakaryocytes, the presence of atypical megakaryocytes and or increased marrow fibrosis. This is in addition to the presence of molecular genetic marker specific for MPN including JAK-2, MPL and CALR. Making a diagnosis of MPN in absence of the previously mentioned features is always challenging. Herein, we analyze the clinical, pathologic and molecular genetics features for five cases presented with splanchnic vein thrombosis and found to be positive for JAK-2 V617F. However, from the hematpathologic point of view, apart from JAK-2 positivity, none of these cases had fulfilled the WHO diagnostic criteria for MPNs. Although the BM specimens were reviewed by at least two experienced hematopathologists, it was so difficult to conclude the diagnosis and or do further sub classification into a specific MPN category. Objective: The aim of this report is to highpoint the difficulty in establishing the diagnosis within this group of patients based on the current WHO diagnostic criteria, to focus on BM pathologic features of this group of patients. Patients and Methods: The patients reported here were referred to our center presenting with portal vein, superior mesenteric or splenic vein thrombosis. Five adult female patients were recognized (2015 and 2019). The mean age at diagnosis is 40.8 years (27-56). None of these cases had an increase in peripheral blood counts except for patient (1) which had mild leukocytosis and thrombocytosis. All patients showed normocellular or slightly hypercellular BM. Slight erythroid predominance was reported in patient (2) and (4). Megakaryocytes were increased in all patients except for patient (2). Interestingly, megakaryocytes morphology was similar in all five patients as they showed anisocytosis with some degree of pleomorphism with predominant of large forms, no clustering and no abnormal topography was noted (figure 1 and 2). All patients showed no significant increase in reticulin fibrosis. Sub-classification into a specific MPN category cannot be performed in all patients. Next generation sequence had been used to analyze targeted regions in recurrently mutated genes in myeloid neoplasia. JAK-2V617F mutation was detected in all studied patients. In patient number (2) JAK-2 mutation was detected at a low level (4%), however, on follow-up, the mutation level increased in addition to detection of 3bp insertion/deletion in CALR gene resulting in a mutation other than type I or type II. table (1). Discussion: Splanchnic veins thrombosis (SVT) includes the portal vein thrombosis (PVT), mesenteric (MVT) splenic vein thrombosis, and the Budd Chiari syndrome (BCS). SVTs are rare events, estimated by 0.7 per 100,000 patients per year for PVT [Rajani et al. 2010]. In a laboratory setting, the diagnosis of latent MPN in patients with SVT is challenging due to absence of elevated blood counts, probably caused by portal hypertension and splenomegaly that result from SVT and MPN. However, the reason why this group of patients does not have overt features of myeloproliferation (particularly) the hypercellularity in the BM is not yet clear. It is possible that JAK-2 positivity induced venous thrombosis longer time before development of the full blown picture of MPN. However, follow-up BM studies for this group of patients might help in better characterization of the pathologic features of these patients. These patients are included in the MPN, unclassifiable subcategory in the current WHO classification. However, based on our findings and the previously published data it seems that these patients have distinctive clinical and pathological features that necessitate having them in a separate MPN entity that might have less strict diagnostic criteria that might include clinical symptoms especially venous thrombosis in large vessels. Separating this group of patients will help to study them thoroughly. Disclosures No relevant conflicts of interest to declare.

2219 Chronic Active Epstein-Barr Virus Presenting Primarily With Abnormal Liver Enzymes

INTRODUCTION: Epstein-Barr virus (EBV) usually causes asymptomatic infections in adults. In rare cases, infected individuals develop Chronic Active EBV (CAEBV), a systemic disorder characterized by elevated EBV DNA in blood and lymphocytes in tissues. We present a rare case of CAEBV in a 24-year-old woman with serology suggestive of autoimmune hepatitis (AIH). CASE DESCRIPTION/METHODS: A 24-year-old Guatemalan woman presented with RUQ abdominal pain, nausea, vomiting and subjective chills for two weeks. Labs were significant for AST 1,167 U/L, ALT 1,049 U/L, total bilirubin 2.8 mg/dL, INR 1.2, ALP 1,118 U/L, GGT 123 U/L, EBV PCR > 1,000,000 copies, EBV IgM positive, positive ANA, serum IgG 3,690 mg/dL (normal 700 – 1,600), weakly positive smooth muscle antibody (SMA) titers 1:40 (negative < 1:20), and F-actin antibodies 42 units (positive ≥ 31 units). Mild scleral icterus and RUQ abdominal tenderness noted on physical exam. CT abdomen & pelvis showed hepatosplenomegaly. Liver biopsy (LB) showed panlobular hepatitis with focal parenchymal necrosis and a prominent sinusoidal T-lymphocyte dominant infiltrate, many of which were positive for EBV-encoded small RNAs (EBER) on in situ hybridization (Figure 1). Bone marrow biopsy (BMB) showed mildly hypocellular bone marrow without evidence of lymphoproliferative disorder. She left against medical advice (AMA) 6 days later and did not follow up in clinic. Five months later, she returned with similar complaints with pancytopenia and febrile neutropenia. A repeat BMB showed a hypercellular bone marrow with findings suggestive of CAEBV of T-cell type. She left AMA shortly after initiating steroids. A referral to the National Institute of Health for stem cell transplant (SCT) was unsuccessful as she was lost to follow up. DISCUSSION: Hepatic manifestations of CAEBV include acute hepatitis in 47% of patients in the United States with a mixed hepatocellular-cholestatic pattern and hepatomegaly. Her labs were suggestive of AIH such as elevated IgG, positive ANA, weakly positive SMA titers & elevated F-actin antibodies. Several cases report false positive serology suggestive of AIH in patients with active EBV. Inappropriate and possibly dangerous treatment could result if one is unaware of this association. Allogenic hematopoietic SCT is the only known curative therapy to date and without treatment, the average time to death after disease onset is 6.2 years. Given the high mortality associated with delay in treatment, prompt recognition and diagnosis of CAEBV is imperative.

2470 Mysterious Ascites

INTRODUCTION: Hypereosinophilic Syndrome (HES) is a rare disorder most commonly diagnosed in patients 20-50 years of age. It is defined as a persistent absolute eosinophil count greater than 1500 cells/microL and/or pathologic confirmation of tissue hypereosinophilia, as well as evidence of end-organ dysfunction. Prior to making a diagnosis, allergic, rheumatologic, infectious, and neoplastic etiologies should be excluded. CASE DESCRIPTION/METHODS: This is the case of an 87 -year-old male with past medical history of malaria treated with chloroquine who presented with altered mental status, dry cough and ascites de novo. Associated symptoms included unintentional weight loss of 10-15 lbs and intermittent episodes of non- bloody, watery diarrhea. He denied history of alcohol or drug use. Physical exam was remarkable for a distended abdomen with a positive fluid wave and pitting edema +1 in the lower extremities bilaterally. Labs showed a platelet count of 53,000, hypoalbuminemia (1.9 g/dl), increasing creatinine (2.1 mg/dl), alkaline phosphatase of 444 u/l, total bilirubin of 2.8 mg/dl with indirect predominance, AST of 59 u/l, ALT of 55 u/l, and eosinophilia of 72% (absolute eosinophil count of 6,048). Abdominal sonogram with doppler failed to show obstructive pathology and no evidence of hepatic vein thrombosis. Paracentesis showed absence of malignant cells, SAAG >1.1, 0.79 g/dl of protein and abundant eosinophils. Hepatitis, Iron Saturation levels, AMA, ASMA, Schistosoma and Strongyloides workup were negative. Hematology oncology service was consulted for bone marrow biopsy to asses for myeloid and lymphoid disorders. Results showed a hypercellular bone marrow with marked eosinophilia and 50% nucleated cells but C-KIT, PGFR Alpha, PGFRB, BCR/ABL, JAK 2 and TCR were all negative. A diagnosis of Hypereosinophilic Syndrome with suspected eosinophilic liver infiltration was made and the patient was started on prednisone therapy. All prior described multisystemic symptoms resolved. Eosinophilia, thrombocytopenia and all liver enzymes normalized within 24-48 hours after initiation of therapy. Ascites also resolved for which diuretic therapy was discontinued. DISCUSSION: This case is interesting as HES has an incidence of 0.36 per 100,000, and hepatic involvement accounts solely for an estimated 30% of patients. This serves to illustrate that HES should form part of the differential diagnosis when hepatic involvement and eosinophilia are concerned as prompt evaluation and treatment can lead to rapid resolution.

Study of haematological disorders detected by bone marrow examination at tertiary care hospital

Bone marrow aspiration & biopsy examination is often required for diagnosis & management of haematological disorders which have diverse modes of presentation. Mostly of the haematological disorders present as anaemia, which are diagnosed by bone marrow aspiration (BMA) and bone marrow biopsy (BMB). I n the department of pathology, the study was conducted to compare the role of bone marrow aspirate and trephine biopsy for diagnosing the haematological diseases and indications. Materials and Methods: To check the role of bone marrow aspiration and biopsy in the diagnosis of haematological disorders this is the aim of study. In the department of pathology at tertiary care teaching hospital this study was conducted over a period of 2 years from November 2016 to October 2018. A total of 572 cases were included in this study. Results: In the present study, both aspiration and biopsy cases were available are 572 were included. The age ranged from 6 months to 84 years. The 326(56.99%) were males and 246(43%) were females with M: F=1.32 :1. Clinical indications for bone-marrow examination, The most common indication was pancytopenia of 201 cases (35.13%) followed by anaemia 167 cases (29.19%), malignancy 79 cases (13.81%), Hepatosplenomegaly 48 cases (8.39%), thrombocytopenia 47 cases (8.21%) and fever 30 cases (5.24 %). Most of the cases had hypercellular bone marrow (53%) followed by normocellular marrow (32%) and hypocellular marrow (15%). Keywords: Bone marrow, Biopsy, Examination, Haematological disorders.

CLINICO HAEMATOLOGICAL PROFILE IN PATIENTS OF PANCYTOPENIA AND ROLE OF NUTRITIONAL DEFICIENCIES AS IMPORTANT AETIOLOGICAL AND PREVENTABLE FACTOR IN CAUSING PANCYTOPENIA IN INDIA

Observational and descriptive cross-sectional study-150 patients, >18 yrs age,evaluated clinically with haematological parameters and bone marrow aspiration.RESULTS-Majority patients presented with fatigue, breathlessness(87.3%) and fever(52.7%). Commonest sign was pallor, followed by splenomegaly, hepatomegaly.Commonest BM picture was hypercellular.Most common aetiology was megaloblastic anaemia(34%),followed by infections(21.3%),hypersplenism(11.3%) and iron deficiency anaemia (8%).Nutritional deficiencies constituted 42% of cases.36% patients had normocellular bone marrow,54.6% patients had hypercellular bone marrow and 9.3% patients had hypocellular bone marrow.Hypocellular BM patients-severe anaemia(42.8%),moderate leucopenia (64.28%)& severe thrombocytopenia.Hypercellular BM patients-moderate anaemia(53.65%),moderate leucopenia(53.65%).CONCLUSION-clinical history & physical examination along with baseline haematological investigations and BMA(biopsy if needed) provide invaluable information, helping in systematic planning of further investigations to diagnose and acertain the cause,avoiding unnecessary tests which not only add to expense of treatment but sometimes may also delay diagnosis and treatment in pancytopenia patients. Pancytopenia is a triad of findings resulting from disease processes primarily or secondarily involving bone marrow ranging from megaloblastic anaemia, aplastic anaemia, disseminated kochs, enteric fever, leishmaniasis to lethal haematological malignancies so all these causes should be kept in mind while planning investigations for the workup of pancytopenic patients.Megaloblastic anaemia and iron deficiency anaemia were the most common causes of pancytopenia in our study which is easily treatable and reversible cause of pancytopenia and thus has a good prognosis.Its early diagnosis is important to prevent further complications.

PB2226 PHILADELPHIA‐NEGATIVE MYELOPROLIFERATIVE NEOPLASMS, CLINICAL MANIFESTATION AND OUTCOME. EXPERIENCE AT HOSPITAL ITALIANO DE BUENOS AIRES

Background:BCR‐ABL negative myeloproliferative neoplasms(MPNs) are a heterogeneous group of clonal hematopoietic disorders including polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (MF) and prefibrotic primary myelofibrosis (prePMF). Clinically they share the characteristics of hypercellular bone marrow, increased incidence of thrombosis or hemorrhage and a higher rate of transformation to acute leukemia. There are scare data about latin american population, due to the scarce data available we conducted a study to evaluate patient characteristics and outcomes in the region.Aims:Primary endpoint was to evaluate the association between molecular and laboratory characteristics and the presence of thrombosis. Secondary endpoint was to evaluate time to leukemia transformation and global survival.Methods:Observational retrospective cohort study conducted at a Universal hospital in Buenos Aires. All patients evaluated between January 2004 and December 2017 with a diagnosis of MNPs confirmed by biopsy were included. Clinical and molecular data were extracted from the electronic medical history. Survival analysis was performed with Kaplan Meier curves and cox regression model using IBM SPPS software.Results:Two hundred and sixty four patients were included in the analysis. Patients characteristics are shown in table 1. A mutational state was performed in 194 patients(73.5%),132 were JAK2 V617F mutated, 6 cases had CALR mutation type 1 (3MF and 3ET), in 2 cases the CALR mutation type 2 was detected (1MF and 1ET), 2 patients presented the MPL mutation (1MF and TE) and 17 patients were triple negative. In PV, the JAK2 V617F mutation was detected in 88% of the patients analyzed, in ET and in MF 60% were positive for this mutation. Fifty two patients (19,6%) presented thrombotic events: thirty seven patients (13.9%) presented venous thrombosis, eleven patients (4.1%) arterial events, four patients (1.5%) presented both arterial and venous event. We found no significant association between thrombosis and JAK2 mutation, LDH, leukocytosis or platelet count.The risk of venous thrombosis was higher in PV (HR 1.92 CI 95% 0.90‐4.07) and FM HR 1.18 (CI 95% 0.54 a 2.61) than ET. There were not difference in arterial event between the three groups (OR 1.5, p0.47). Fourteen patients (5,3%) suffer transformation: seven patients (2.6%) evolved to AML, of them 2 had initial diagnosis of PV, 4 of MF and 1 patients of ET. Seven patients progressed to MF, two with previous diagnosis of PV and five with ET. The median time of transformation in the total population was 196 months, in ET it was also of 196 months. There were not relationship between age, hemoglobin value, leukocyte count and the risk of disease transformation. At the end of the follow‐up, 94% of the PV and the 96.3% of the MF, have not reach the median of progression‐free survival. The median follow‐up of the entire cohort was 53 months, 47 months for PV, 48 months for ET and 31,5 month for MF. Forty nine patients (18,6%) died. The median overall survival for the entire cohort was 288 months.Summary/Conclusion:The analysis showed that the characteristics of our population are similar to those reported in the literature, with rates of global survival and transformation similar to the global population, in PV and MF and higher in ET.image

The orphan nuclear receptor EAR-2 (NR2F6) inhibits hematopoietic cell differentiation and induces myeloid dysplasia in vivo

BackgroundIn patients with myelodysplastic syndrome (MDS), bone marrow cells have an increased predisposition to apoptosis, yet MDS cells outcompete normal bone marrow (BM)-- suggesting that factors regulating growth potential may be important in MDS. We previously identified v-Erb A related-2 (EAR-2, NR2F6) as a gene involved in control of growth ability.MethodsBone marrow obtained from C57BL/6 mice was transfected with a retrovirus containing EAR-2-IRES-GFP. Ex vivo transduced cells were flow sorted. In some experiments cells were cultured in vitro, in other experiments cells were injected into lethally irradiated recipients, along with non-transduced bone marrow cells. Short-hairpin RNA silencing EAR-2 was also introduced into bone marrow cells cultured ex vivo.ResultsHere, we show that EAR-2 inhibits maturation of normal BM in vitro and in vivo and that EAR-2 transplant chimeras demonstrate key features of MDS. Competitive repopulation of lethally irradiated murine hosts with EAR-2-transduced BM cells resulted in increased engraftment and increased colony formation in serial replating experiments. Recipients of EAR-2-transduced grafts had hypercellular BM, erythroid dysplasia, abnormal localization of immature precursors and increased blasts; secondary transplantation resulted in acute leukemia. Animals were cytopenic, having reduced numbers of erythrocytes, monocytes and granulocytes. Suspension culture confirmed that EAR-2 inhibits granulocytic and monocytic differentiation, while knockdown induced granulocytic differentiation. We observed a reduction in the number of BFU-E and CFU-GM colonies and the size of erythroid and myeloid colonies. Serial replating of transduced hematopoietic colonies revealed extended replating potential in EAR-2-overexpressing BM, while knockdown reduced re-plating ability. EAR-2 functions by recruitment of histone deacetylases, and inhibition of differentiation in 32D cells is dependent on the DNA binding domain.ConclusionsThis data suggest that NR2F6 inhibits maturation of normal BM in vitro and in vivo and that the NR2F6 transplant chimera system demonstrates key features of MDS, and could provide a mouse model for MDS.

Successful Salvage with Inotuzumab Ozogamicin in Relapsed/Refractory Lymphoid Blast Crisis of Chronic Myeloid Leukemia after Failure of Multiple Lines of Therapy Including Blinatumomab

Background: Lymphoid blast crisis, one of the two major forms of chronic myeloid leukemia (CML) blast crisis (BC), is comprised of lymphoblasts and occurs in about 30% of BC patients. These individuals often respond to the same treatment strategies used in Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). Blinatumomab, a bispecific T engager, or BiTE, is an antibody with dual specificity against both CD3 and CD19, and is approved for use in relapsed/refractory ALL. We present the first known case of relapsed/refractory lymphoid blast phase CML, including treatment failure with blinatumomab, who achieved a complete response to inotuzumab ozogamicin.Case report: In 2004, the patient was initially diagnosed with CML and received treatment with hydroxyurea followed by imatinib. He was intermittently compliant, later changed to nilotinib, and was ultimately lost to follow-up. Approximately 11 years after diagnosis, he returned with pancytopenia. Bone marrow aspiration and biopsy showed a hypercellular bone marrow with 10-15% lymphoid blasts that expressed CD34, CD19, cCD79a, HLA-Dr, CD10 and nTdt. Testing for the BCR-ABL translocation (at breakpoint P210) was positive confirming lymphoid blast phase CML. He received hyperCVAD + dasatinib, responded, and was referred for allogeneic hematopoietic cell transplantation (HCT) but deferred for social reasons. He was ultimately lost to follow-up.The patient returned with relapsed disease in February 2017 and bone marrow aspiration and biopsy was consistent with CD19+ lymphoid blast phase CML with BCR-ABL major breakpoint p210 and new F317L mutation of ABL kinase domain. He received salvage therapy with vincristine/prednisone/bosutinib, however, repeat bone marrow testing confirmed persistent CD19+ lymphoid blasts (20%). He was treated with blinatumomab, entered a minimal residual disease (MRD)-positive morphologic remission with 1.9% abnormal B lymphoblasts by flow cytometry. A second cycle of blinatumomab was complicated by bone pain and repeat bone marrow testing showed progressive disease with 50-60% CD19+, CD22+ lymphoblasts. Salvage therapy with fludarabine, cytarabine, and idarubicin (FLAG-Ida) in September 2017 was unsuccessful and palliative treatment with vincristine, prednisone, and methotrexate was initiated. He developed new onset neurologic symptoms in March 2018 and lumbar puncture showed evidence of disease in his central nervous system (CNS) The patient received concurrent intrathecal methotrexate and inotuzumab ozogamicin which led to clearance of CNS disease and a morphologic remission in his marrow. He continues with MRD-positivity with 0.15% B lymphoblasts and is on ponatinib maintenance while allogeneic HCT evaluation is underway.Discussion: This is the first reported patient with lymphoid blast phase CML, refractory to BiTE therapy, that was successfully salvaged with inotuzumab ozogamycin. Recent data from acute lymphoblastic leukemia (ALL) reported that the majority of BiTE failures remained CD19+ and the mechanisms of CD19 immunotherapy failure are poorly understood (Jabbour E, et al, AJH 2017). These patients may be successfully salvaged by CD19-directed chimeric antigen receptor (CAR) T cells or other directed therapies such as inotuzumab ozogamycin.Conclusion: Patients with relapsed/refractory lymphoid blast phase CML should receive directed salvage therapies. This case report demonstrates that failure of one line of directed therapy does not predict for future failures. DisclosuresNo relevant conflicts of interest to declare.

From Eosinophilic Gastroenteritis to Chronic Eosinophilic Leukemia: A Case of Hypereosinophilia

Introduction Hypereosinophilia is generally defined as a peripheral blood eosinophil count greater than 1500/mm3 and can be associated with reactive (secondary) or hematologic (primary) disorders, which can lead to end-organ damage. We present a patient with gastrointestinal symptoms in the setting of absolute eosinophilia. Work-up was initially suggestive of eosinophilic gastroenteritis, a rare inflammatory disorder characterized by eosinophilic tissue infiltration with no known natural progression to date. However, additional studies revealed a clonal genetic abnormality on chromosomal analysis in the diagnosis of chronic eosinophilic leukemia, not otherwise specified (CEL, NOS). Case Description An 82-year-old female with history of psoriatic arthritis presented with a 2-week history of watery, nonbloody bowel movements (5-20 per day) along with abdominal cramping, nausea and worsening oral intolerance. Vital signs and physical exam were unremarkable. Laboratory studies were significant for a WBC of 25,000 (40% eosinophils) and elevated IgE level at 138IU/mL. CT of the abdomen was also unremarkable.2578_A Figure 1. Duodenal biopsy, increased eosinophils infiltrating epithelium and lamina propria.2578_B Figure 2. Bone marrow biopsy, hypercellular bone marrow with eosinophilia.An esophagogastroduodenoscopy was performed, demonstrating diffuse duodenal erythema, edema and villous blunting as well as erythematous and hypertrophied gastric folds. Random duodenal, gastric, esophageal, and colonic biopsies revealed increased eosinophils (60 to >100 eos/hpf). Bone marrow biopsy was notable for hypercellularity and marked eosinophilia. The chromosomal analysis detected a clonal cytogenetic abnormality, del(11q), satisfying the 2017 WHO diagnostic criteria for CEL, NOS. The patient was treated with corticosteroids with resolution of absolute eosinophilia and gastrointestinal symptoms. Discussion This case elucidates the importance of undergoing a complete evaluation for hypereosinophilia, beginning with assessing for secondary causes, followed by thorough hematologic diagnostic studies. The localization of the patient's symptoms in the gastrointestinal tract, coupled with biopsy-proven eosinophilic infiltration, pointed initially towards eosinophilic gastroenteritis. However, the abnormal chromosomal analysis proved the diagnosis of CEL, NOS. As more cases suggestive of eosinophilic gastroenteritis emerge, the hope is to better understand the pathogenesis and natural progression of this rare disease, as well as to compare the effectiveness of therapeutic strategies with that of the hematologydriven processes.

Novel Clinical, Laboratory, Molecular and Pathological (2018 CLMP) Criteria for the Differential Diagnosis of three Distinct JAK2, CALR and MPL Mutated Myeloproliferative Neoplasms: The Role of Driver Mutation Analysis and Bone Marrow Histology

The broad spectrum of JAK2V617F mutated trilinear phenotypes varies from essential thrombocythemia (ET), prodromal polycythemia vera (PV), masked PV, erythrocythemic PV, classical PV, and PV complicated by splenomegaly and myelofibrosis (MF). ET heterozygous for the JAK2V617F mutation is associated with normal life expecancy. JAK2V617F mutation load increases from low to 40% in ET, from below to above 50% in early stage PV and above 50% up to 100% in overt and advanced PV and MF. Pretreatment bone marrow morphology and cellularity distinguish JAK2V617F mutated trilinear MPN from calreticulin (CALR) and MPL mutated MPN. The morphology of clustered mature enlarged pleomorphic megakaryocytes with hyperlobulated nuclei are similar in JAK2V67F ET and PV patients. MPL515 mutated thrombocythemia is featured by monolinear proliferation of large to giant mature megakaryocytes with hyperlobulated nuclei in a normocellular or hypocellular bone marrow. CALR mutated thrombocythemia shows characteristic bone marrow features of primary dual megakaryocytic granulocytic myeloproliferation (PMGM) in a normocellular or hypercellular bone marrow without features of PV. JAK2V617F, CALR and MPL515 allele burden slowly increases to values around 50% together with the degree of splenomegaly, myelofibrosis and constitutional symptoms during life long follow-up. Natural history and life expectancy relate to the degree of splenomegaly, bone marrow fibrosis, anemia and the acquisition of epigenetic mutations at increasing age predict unfavorable outcome in JAK2V617F, CALR and MPL515 mutated MPN. Low dose aspirin in JAK2V617F mutated ET and PV and phlebotomy on top of aspirin in PV is mandatory to prevent platelet-mediated microvascular circulation disturbances. Pegylated interferon is the first line myeloreductive treatment option in prodromal and early stage JAK2V617F mutated PV and in CALR and MPL mutated thrombocythemia to postpone the use of hydroxyurea and ruxolitinib as long as possible.

Abnormal folate metabolism causes age-, sex- and parent-of-origin-specific haematological defects in mice.

Key points Folate (folic acid) deficiency and mutations in folate‐related genes in humans result in megaloblastic anaemia.Folate metabolism, which requires the enzyme methionine synthase reductase (MTRR), is necessary for DNA synthesis and the transmission of one‐carbon methyl groups for cellular methylation.In this study, we show that the hypomorphic Mtrrgt/gt mutation in mice results in late‐onset and sex‐specific blood defects, including macrocytic anaemia, extramedullary haematopoiesis and lymphopenia.Notably, when either parent carries an Mtrrgt allele, blood phenotypes result in their genetically wildtype adult daughters, the effects of which are parent specific.Our data establish a new model for studying the mechanism of folate metabolism in macrocytic anaemia aetiology and suggest that assessing parental folate status might be important when diagnosing adult patients with unexplained anaemia. The importance of the vitamin folate (also known as folic acid) in erythrocyte formation, maturation and/or longevity is apparent since folate deficiency in humans causes megaloblastic anaemia. Megaloblastic anaemia is a type of macrocytic anaemia whereby erythrocytes are enlarged and fewer in number. Folate metabolism is required for thymidine synthesis and one‐carbon metabolism, though its specific role in erythropoiesis is not well understood. Methionine synthase reductase (MTRR) is a key enzyme necessary for the progression of folate metabolism since knocking down the Mtrr gene in mice results in hyperhomocysteinaemia and global DNA hypomethylation. We demonstrate here that abnormal folate metabolism in mice caused by Mtrrgt/gt homozygosity leads to haematopoietic phenotypes that are sex and age dependent. Specifically, Mtrrgt/gt female mice displayed macrocytic anaemia, which might be due to defective erythroid differentiation at the exclusion of haemolysis. This was associated with increased renal Epo mRNA expression, hypercellular bone marrow, and splenic extramedullary haematopoiesis. In contrast, the male response differed since Mtrrgt/gt male mice were not anaemic but did display erythrocytic macrocytosis and lymphopenia. Regardless of sex, these phenotypes were late onset. Remarkably, we also show that when either parent carries an Mtrrgt allele, a haematological defect results in their adult wildtype daughters. However, the specific phenotype was dependent upon the sex of the parent. For instance, wildtype daughters of Mtrr+/gt females displayed normocytic anaemia. In contrast, wildtype daughters of Mtrr+/gt males exhibited erythrocytic microcytosis not associated with anaemia. Therefore, abnormal folate metabolism affects adult haematopoiesis in an age‐, sex‐ and parent‐specific manner.

59-Year-Old Man With Fatigue, Weight Loss, and Hepatomegaly

59-Year-Old Man With Fatigue, Weight Loss, and Hepatomegaly

Role of bone marrow aspiration and biopsy in elaborating the diagnosis of pancytopenia

Background and Aims: Pancytopenia may be a manifestation of a wide variety of disorders which primarily or secondarily affects the bone marrow. Bone marrow failure syndromes and malignancies are serious and lifethreatening causes, but certain non-malignant conditions such as infection, and nutritional anemia are equally important. The severity of pancytopenia and the underlying pathology determine the implementation of correct management and prognosis. Materials and Methods: This was a prospective study conducted for a period of two years on 134 patients of pancytopenia. Detailed history, thorough clinical examination, complete hemogram, peripheral smear examination, reticulocyte count evaluation and bone marrow aspiration was performed in all cases. In addition, trephine biopsy was done in the same setting wherever indicated. Results: The age ranged from 15 to 75 years with a mean age of 30.9 years. The most common cause of pancytopenia was Megaloblastic anemia (37%) followed by Nutritional anemia (31%), aplastic anemia (9%) and Leukemia (1.75%). Majority (79%) of the patients had hyper cellular bone marrow followed by hypocellular (13%) and normal cellular marrow (8%). Conclusion: Elaborate hematological investigations and bone marrow examination, both, in correlation with clinical findings are vital for the diagnosis of pancytopenia. Bone marrow Aspiration and Trephine biopsy, complement each other in diagnosis of challenging cases.

Myelodysplastic syndrome with myelofibrosis in a 12-year-old patient – A case report

Abstract Myelodysplastic syndromes are a heterogeneous group of clonal disorders characterized by peripheral blood cytopenia and normal or hypercellular bone marrow with dysplasia in more than one blood cell lineage, unfavorable prognosis, and lack of response to treatment. We present the case of a 12-year-old male patient who was referred to the Hematology and Oncology Department of Pediatric Clinic I Târgu- Mures in May 2016, with splenomegaly and pancytopenia. The osteomedullary biopsy revealed myelofibrosis, discrete dysplasia of the myeloid series and megakaryocytes, blasts CD34+ approximately 10%, which led to the diagnosis of myelodysplastic syndrome with myelofibrosis. The myeloid precursors indicated a high risk of transformation into acute myeloid leukemia, so chemotherapy associated with corticosteroids was started, leading to slight improvements. Although myelodysplastic syndrome associated with myelofibrosis is rare at this age, despite the treatment and favorable progression in the case presented, in the absence of hematopoietic stem cell transplantation the prognosis remains unfavorable.

Acute myeloid leukemia with t(4;12)(q12;p13): an aggressive disease with frequent involvement of PDGFRA and ETV6.

We describe the clinical, morphologic, immunophenotypic and molecular genetic features of 15 cases of acute myeloid leukemia (AML) with t(4;12)(q12;p13). There were 9 men and 6 women, with a median age of 50 years (range, 17–76). Most patients had hypercellular bone marrow with a median blast count of 58% and multilineage dysplasia. Flow cytometry analysis showed myeloid lineage with blasts positive for CD13, CD33, CD34, CD38, CD117 and HLA-DR. Interestingly, aberrant CD7 expression was detected in 12/14 cases, and myeloperoxidase was either negative (3/15) or positive in only a small subset of the blasts (12/15). t(4;12)(q12;p13) was detected at time of initial diagnosis in 4 and at relapse or progression in 9 patients. The initial karyotype was unknown in 2 cases. FISH analysis showed PDGFRA-ETV6 rearrangement in all 7 cases assessed. FLT3 ITD was detected in 2/11 cases and IDH2 and JAK2 mutation were each detected in 1/2 cases assessed. There were no mutations of KRAS (0/8), NRAS (0/8), CEBPA (0/3), KIT (0/3), NPM1 (0/3) or IDH1 (0/2). All patients received aggressive multiagent chemotherapy; 7 patients additionally received stem cell transplantation. With a median follow-up of 10 months (range, 6–51), 13 patients died of AML, 1 patient had persistent disease, and 1 patient was lost to follow-up. In summary, AML with t(4;12)(q12;p13) is usually associated with myelodysplasia, aberrant CD7 expression, weak of absent myeloperoxidase expression, frequent PDGFRA-ETV6 fusion, and an aggressive clinical course. The molecular findings suggest that there may be a role for tyrosine kinase inhibitors in patient management.

Atypical Chronic Myeloid Leukemia: A Rare Entity with Management Challenges

The aim of our study was to review the clinicopathologic features and management of atypical chronic myeloid leukemia (aCML). Relevant manuscripts published in English were searched using PubMed. aCML is diagnosed as per WHO 2016 classification in the presence of leukocytosis≥13×109/l with circulating neutrophil precursors≥10%, monocytes less than 10%, minimal basophils, hypercellular bone marrow with granulocytic proliferation and dysplasia, bone marrow blast less than20% and absence of BCR/ABL fusion gene. Common cytogenetic features and mutations include trisomy 8, and mutations in SETBP1 and ETNK1. Median survival is 1-2years. Hematopoietic stem cell transplant may be the only curative option. Ruxolitinib and dasatinib are emerging therapeutic options. Thus,aCML is a rare entity with poor survival. Novel therapies are needed.

Evaluation of the Carcinogenic Potential of Roxadustat (FG-4592), a Small Molecule Inhibitor of Hypoxia-Inducible Factor Prolyl Hydroxylase in CD-1 Mice and Sprague Dawley Rats.

The carcinogenic potential of roxadustat (FG-4592), a novel orally active, heterocyclic small molecule inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PH) enzymes in clinical development for treatment of anemia, was evaluated in CD-1 mice and Sprague Dawley rats. Inhibition of HIF-PH by roxadustat leads to a rapid increase in cytoplasmic HIF-α concentrations, followed by translocation of HIF-α to the nucleus and upregulation of HIF-responsive genes, including erythropoietin. Roxadustat was dosed by oral gavage 3 times weekly (TIW) for up to 104 weeks in mice at 0, 15, 30, and 60 mg/kg and in rats at 0, 2.5, 5, and 10 mg/kg. Treatment-associated changes in hematology parameters were consistent with the pharmacologic activity of roxadustat and included elevations in hematocrit in mice at 30 and 60 mg/kg TIW and elevations in erythrocyte count, hemoglobin, hematocrit, and red cell distribution width in rats at 10 mg/kg TIW. No increase in mortality or neoplastic effects compared with vehicle controls was observed after roxadustat treatment in either species. No treatment-related nonneoplastic findings were observed in mice, whereas nonneoplastic microscopic findings in rats were limited to atrial/aortic thromboses at 10 mg/kg TIW males and bone marrow hypercellularity in all treated male and female groups, consistent with the pharmacology of roxadustat. In conclusion, roxadustat administered by oral gavage to mice and rats TIW for up to 104 weeks resulted in dose-dependent exposure and hematologic effects with no effect on survival or development of neoplastic lesions at up to 60 mg/kg in mice and up to 10 mg/kg in rats.

Bone marrow pathologic abnormalities in familial platelet disorder with propensity for myeloid malignancy and germline RUNX1 mutation

A subset of patients with familial platelet disorder with propensity to myeloid malignancy and germline RUNX1 mutation develops hematological malignancies, often myelodysplastic syndrome/acute myeloid leukemia, currently recognized in the 2016 WHO classification. Patients who develop hematologic malignancies are typically young, respond poorly to conventional therapy, and need allogeneic stem cell transplant from non-familial donors. Understanding the spectrum of bone marrow morphologic and genetic findings in these patients is critical to ensure diagnostic accuracy and develop criteria to recognize the onset of hematologic malignancies, particularly myelodysplastic syndrome. However, bone marrow features remain poorly characterized. To address this knowledge gap, we analyzed the clinicopathologic and genetic findings of 11 patients from 7 pedigrees. Of these, 6 patients did not develop hematologic malignancies over a 22-month follow-up period; 5 patients developed hematologic malignancies (3 acute myeloid leukemia; 2 myelodysplastic syndrome). All patients had thrombocytopenia at initial presentation. All 6 patients who did not develop hematologic malignancies showed baseline bone marrow abnormalities: low-for-age cellularity (n=4), dysmegakaryopoiesis (n=5), megakaryocytic hypoplasia/hyperplasia (n=5), and eosinophilia (n=4). Two patients had multiple immunophenotypic alterations in CD34-positive myeloblasts; 1 patient had clonal hematopoiesis. In contrast, patients who developed hematologic malignancies had additional cytopenia(s) (n=4), abnormal platelet granulation (n=5), bone marrow hypercellularity (n=4), dysplasia in ≥2 lineages including megakaryocytes (n=3) and acquired clonal genetic aberrations (n=5). In conclusion, our study demonstrated that specific bone marrow abnormalities and acquired genetic alterations may be harbingers of progression to hematological malignancies in patients with familial platelet disorder with germline RUNX1 mutation.

New evidence suggesting a dissociated etiology for cribra orbitalia and porotic hyperostosis.

Porotic hyperostosis (PH), characterized by porotic lesions on the cranial vault, and cribra orbitalia (CO), a localized appearance of porotic lesions on the roof of the orbits, are relatively common osteological conditions. Their etiology has been the focus of several studies, and an association with anemia has long been suggested. Anemia often causes bone marrow hypertrophy or hyperplasia, leading to the expansion in trabecular or cranial diploic bone as a result of increased hematopoiesis. Hypertrophy and/or hyperplasia is often coupled with a disruption of the remodeling process of outer cortical bone, cranially and/or postcranially, leading to the externally visible porotic lesions reported in osteological remains. In this article, we investigate whether individuals with CO have increased thickness of the diploë, the common morphological direct effect of increased hematopoiesis, and thus test the relationship between the two conditions, as well as explore the type of anemia that underlie it. An analysis of medical CT scans of a worldwide sample of 98 complete, young to middle-aged adult dry skulls from the Duckworth Collection was conducted on male and female cribrotic individuals (n = 23) and noncribrotic individuals (n = 75), all of whom lacked any evidence of porotic lesions on the vault. Measurements of total and partial cranial thickness were obtained by virtual landmark placement, using the Amira 5.4 software; all analyses were conducted in IBM SPSS 21. Cribriotic individuals have significantly thinner diploic bone and thicker outer and inner tables than noncribriotic individuals, contrary to the expected diploic expansion that would result from anemic conditions associated to bone marrow hypertrophy or hyperplasia. Additionally, individuals without CO and those with the condition have distinctive cranial thickness at particular locations across the skull and the severity to which CO is expressed also differentiates between those with mild and those with a moderate to severe form of the condition. Our results suggest a complex pattern of causality in relation to the pathologies that may lead to the formation of porotic lesions on the vault and the roof of the orbits. A form of anemia may be behind the osteological changes observed in PH and CO, but it is unlikely to be the same type of anemic condition that underlies both types of osteological lesions. We suggest that CO may be associated to anemias that lead to diploic bone hypocellularity and hypoplasia, such as those caused by anemia of chronic disease and, to a lesser extent, of renal failure, aplastic anemia, protein deficiency, and anemia of endocrine disorders, and not those that lead to bone marrow hypercellularity and hyperplasia and potential PH. This leads us to the conclusion that the terms PH and CO should be used to reflect different underlying conditions.