Single Centre Analysis of JAK2 V617F and Exon 12 Negative Idiopathic Erythrocytosis

Introduction The molecular categorisation of myeloproliferative neoplasms (MPNs) has changed the landscape of diagnosis and treatment. Polycythaemia vera (PV) is characterised by red blood cell proliferation and JAK2 V617F or Exon 12 mutations in up to 98% of patients 1. However, some patients without such mutations have an arduous diagnostic course with varying management and prognostic outcomes 2. We present our experience in managing this challenging cohort and aim to illuminate a potential diagnostic pathway for patients. Method We searched electronic records of patients attending haematology clinics over the last 20 years at University College Hospital with a prior diagnosis of PV / erythrocytosis (presenting with raised Haemoglobin (Hb) &/ Haematocrit (Hct)) with no evidence of JAK2 exon 12 or 14 mutations on bone marrow or peripheral blood molecular analysis (multiplex PCR sensitivity 0.1%). We reviewed their diagnostic workup, which included full blood count & where available, bone marrow myeloid (Illumina TruSight Myeloid) & erythroid next generation sequencing (NGS) panels. Results 37 patients with JAK2 V617F & Exon 12 mutation negative erythrocytosis were identified. Patients were categorised in to 3 groups 2; idiopathic erythrocytosis (IE), secondary polycythaemia (SP) & high affinity haemoglobinopathies (HAH); patient characteristics are summarised in Table 1. The median age of IE & HAH was younger, their presenting Hb/Hct levels was higher compared to SP, with a male predominance. Constitutional symptoms were only reported in the IE cohort. Erythropoietin (EPO) was elevated in HAH & IE patients but within normal range in SP. Thrombotic events occurred in all cohorts, most frequently in IE. Splenomegaly was reported in 4/21 IE, 1/13 SP, but was not a feature in HAH patients. When performed, IE red cell mass (RCM) studies were raised but within normal range in SP patients. Table 2 details IE cohort erythroid mutations. Myeloid NGS only identified MPL and BCOR mutations of pathogenic significance and multiple single nucleotide polymorphisms of no known significance. No abnormalities were demonstrated in 15% of SP patients that underwent bone marrow myeloid mutational analysis. Venesections (VS) were instigated in 95% of the IE cohort, antiplatelets (AP) in 52%, anticoagulation (AC) in 14% and cytoreductive therapy (CT) in 19% due to intolerance/failure of VS. VS programme was instigated in 46% of SP patients, AP in 7% and AC in 47%. Discussion The median age of our IE cohort was 48 yrs with a 19% incidence of thrombosis. Where performed, bone marrow histology demonstrated hypercellularity but was not consistent with MPN diagnostic criteria. Myeloid NGS panel mutations such as BCOR may represent clonal haematopoieis of indeterminate potential. Heterozygous VHL C598T & C376A mutations, in keeping with Chuvash polycythaemia, were demonstrated. Mutations in EGLN1 & BPGM,were detected in our patients, however there was an absence of correlating haematological parameters or family history to support a diagnosis of congenital erythrocytosis (2). Variants of unknown significance were also detected in SH2B3, BMP6 & EGLN3 gene duplication. SP patients were older (median age 68 years) and where performed had normal RCM and no myeloid mutations identified. The initial approach adopted at our centre for diagnosing and managing JAK2 V617F & Exon 12 negative erythocytosis begins with clinical evaluation for secondary causes. This is followed by assessment of EPO level, RCM study, extended molecular mutational analysis involving screening for high affinity haemoglobins, congenital erythrocytosis and bone marrow histology. Where patients are symptomatic or considered at high risk for thrombosis, we venesect to a personalised target and patients are offered AP and/or AC. CT is instigated where VS is not tolerated or ineffective, however this approach is not corroborated in the literature. Our experience highlights the clinical heterogeneity of JAK2 negative erythrocytosis and the need to develop a robust and systematic diagnostic and treatment algorithm with further clarification of the role of molecular profiling. 1. William W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017 Feb; 129(6):667-679 2. McMullin MF. Idiopathic erythrocytosis: a disappearing entity. Hematology Am Soc Educ Program. 2009; 2009(1):629-635 Figure 1 Figure 1. Disclosures Sekhar: Novartis: Consultancy, Research Funding.

Immunophenotype of Erythroid Precursors in Patient with Pure Red Cell Aplasia (PRCA): Utility of Analysis of Erythroid Maturation

Pure Red Cell Aplasia (PRCA) is an infrequent disease [1,2], which usually presents as hypogenerative normochromic anemia, and is characterized by a significant decrease (including absence) of erythroid precursors [3]. Its etiology can be congenital or acquired, and its correct diagnosis requires exclusion of alternative cases of refractory anemia, so the bone marrow histology plays a crucial role. Myelodisplastic Syndromes (MDS) should always be considered in its differential diagnosis. The use of laboratory tools, specifically Flow Cytometry (FCM) is gained importance in the study of malignant and benign hematology pathologies. In MDS, FCM is not yet considered a standard of care, however it provides valuable information [4,5] and there are numerous publications and scores for its usual clinical use (for example Ogata score and RED-score [6,7]). In relation to the rise of FCM in MDS, enormous progress has been made in the description of the erythroid precursors immunophenotype [8-10]. An example of normal erythroid maturation is presented in Figure 1, showing proerythroblasts with immunophenotype CD71+ CD105+ CD117+, basophilic erythroblasts CD71+ CD105+ CD117-, polychromatophilic and orthochromatophilic erythroblasts CD71+ CD105- CD117- distinguishing by size in Forward Scatter (FSC) versus CD36 respectively. Characteristic maturation curve in CD117 versus CD105 analysis evidenced a predominance towards more mature erythroblasts.

JAK2 or CALR Mutations Status and Bone Marrow Histology in Patients with Essential Thrombocythemia in Chinese

Objectives: To characterize the clinical, and bone marrow histopathological features of essential thrombocythemia (ET) with different gene mutations status of CALR and JAK2.Methods: 159 patients of ET were centrally analyzed from January 2016 to December 2019, including 59 cases with CALR mutation, 96 JAK2 mutation, 2 MPL mutation, and 2 cases were triple-negative (TN). Bone marrow pathology observation and determination were performed by 2 immobilized experienced morphological specialists.Results: Compared to ET with JAK2 mutation, patients with CALR mutation were younger (p=0.000), showed lower count of white blood cell (WBC) and level of hemoglobin (p=0.001, p=0.001), and higher count of platelet (p=0.001). In the bone marrow (BM) biopsy, the median number of megakaryocyte and clusters of megakaryocytes in each high power field (HPF) of vision in patients with CALR mutations were lower than JAK2 mutations patients (p=0.001, p=0.001), thrombotic events in two group was different (5% vs 11.5%) (p=0.03).Conclusion: In Chinese ET patients, patients with CALR mutations were younger, and had lower levels of Hb, and count of WBC, the lower thrombotic evens although with higher platelet counts than those with JAK2 mutation. Patients with JAK2 mutations had a higher median number of megakaryocytes and median number of clusters of megakaryocytes, the clinical significance is worth exploring.

Essential thrombocythaemia

Essential thrombocythaemia is a classic myeloproliferative neoplasm characterized by thrombocytosis, increased risk of thrombotic and/or haemorrhagic complications, and a trend to transformation to myelofibrosis and acute leukaemia. Mutations in JAK2, CALR, and MPL genes besides bone marrow histology are crucial elements of diagnosis. Treatment is aimed to prevent the appearance of thrombotic complications that are the main cause of morbidity and mortality. Accordingly, thrombosis risk stratification is of the utmost importance to select the appropriate treatment. Antiplatelet therapy as primary antithrombotic prophylaxis in low-risk patients should be tailored according to the existence of extreme thrombocytosis and presence of JAK2V617F mutation and/or cardiovascular risk factors. Cytoreductive treatment options are discussed with reference to results of randomized clinical trials. Practical approach to unusual and risk situations as surgery, pregnancy, and paediatric essential thrombocythaemia are also reviewed.

Naxitamab-based chemoimmunotherapy for resistant high-risk neuroblastoma: Preliminary results of HITS pilot/phase II study.

10025 Background: Chemoresistant and relapsed disease are major obstacles to curing high-risk neuroblastoma (HR-NB). Anti-GD2 monoclonal antibody (MoAb) is effective in preventing relapse after remission but responses in relapsed or progressive disease (PD) are rare. We investigated the combination of humanized anti-GD2 MoAb naxitamab, (previously termed Hu3F8), irinotecan, temozolomide and sargramostim (GM-CSF): a pilot HITS protocol against resistant HR-NB now expanded to a phase II study (NCT03189706). Methods: Salient eligibility criteria included evaluable or measurable chemoresistant disease. Prior anti-GD2 MoAb and/or irinotecan/temozolomide (I/T) therapy was permitted. Each cycle comprised of irinotecan 50 mg/m2/day intravenously (IV) plus temozolomide 150 mg/m2/day IV or orally (days 1-5); naxitamab 2.25 mg/kg/day IV over 30 minutes, days 2, 4, 8 and 10 (total 9 mg/kg or 270 mg/m2 per cycle), and GM-CSF 250 mg/m2/day subcutaneously, days 6-10. Toxicity was measured by CTCAE v4.0 and responses by modified International Neuroblastoma Response Criteria. Results: Forty-six (23 enrolled on protocol and 23 on compassionate-use basis) heavily prior-treated patients (median age at enrollment: 6.6 years; median number of prior relapses: 2) have received 175 (median 2; range 1-12) cycles to date. At enrollment, 7 patients had HR-NB refractory to induction chemotherapy while 39 had prior relapse. Toxicities included myelosuppression and diarrhea expected with I/T, and pain and hypertension expected with naxitamab. No other > grade 2 related toxicities occurred; treatment was outpatient. Early responses, assessed after 2 cycles, were documented in 18 (39%) patients and were complete (n = 9), partial (n = 8), and mixed (n = 1); 13 patients had stable disease. Responses were achieved in refractory (3/7;43%) and PD (15/39;38%) subgroups, in patients who had previously received I/T (12/34;35%) and/or anti-GD2 MoAb (14/36;39%), and in soft tissue (6/22; 27%) MIBG-avid skeletal sites (20/36;56%) and on bone marrow histology (9/12; 75%). Conclusions: High-dose naxitamab-based chemoimmunotherapy is safe and effective against chemoresistant HR-NB. This ongoing phase II study may define a broader role for naxitamab which was recently granted breakthrough designation by the FDA. Clinical trial information: NCT03189706.