scholarly journals Diagnosis, risk stratification, and response evaluation in classical myeloproliferative neoplasms

Blood ◽  
2017 ◽  
Vol 129 (6) ◽  
pp. 680-692 ◽  
Author(s):  
Elisa Rumi ◽  
Mario Cazzola
Keyword(s):  
Bone Marrow ◽  
Diagnostic Criteria ◽  
Myeloproliferative Neoplasms ◽  
Phenotypic Variability ◽  
Vascular Complications ◽  
Prognostic Models ◽  
Lymphoid Tissues ◽  
Driver Genes ◽  
Jak2 Mutation ◽  
Endogenous Erythropoietin

Abstract Philadelphia-negative classical myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The 2016 revision of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues includes new criteria for the diagnosis of these disorders. Somatic mutations in the 3 driver genes, that is, JAK2, CALR, and MPL, represent major diagnostic criteria in combination with hematologic and morphological abnormalities. PV is characterized by erythrocytosis with suppressed endogenous erythropoietin production, bone marrow panmyelosis, and JAK2 mutation. Thrombocytosis, bone marrow megakaryocytic proliferation, and presence of JAK2, CALR, or MPL mutation are the main diagnostic criteria for ET. PMF is characterized by bone marrow megakaryocytic proliferation, reticulin and/or collagen fibrosis, and presence of JAK2, CALR, or MPL mutation. Prefibrotic myelofibrosis represents an early phase of myelofibrosis, and is characterized by granulocytic/megakaryocytic proliferation and lack of reticulin fibrosis in the bone marrow. The genomic landscape of MPNs is more complex than initially thought and involves several mutant genes beyond the 3 drivers. Comutated, myeloid tumor-suppressor genes contribute to phenotypic variability, phenotypic shifts, and progression to more aggressive disorders. Patients with myeloid neoplasms are at variable risk of vascular complications, including arterial or venous thrombosis and bleeding. Current prognostic models are mainly based on clinical and hematologic parameters, but innovative models that include genetic data are being developed for both clinical and trial settings. In perspective, molecular profiling of MPNs might also allow for accurate evaluation and monitoring of response to innovative drugs that target the mutant clone.

Cultivation of Megakaryocytes On a Collagen Matrix: A More Sensitive and Reproducible Test for the Diagnosis of Patients with Essential Thrombocythaemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4970-4970
Author(s):  
Adrian Emanuel Schmidt ◽  
Patricia Darlington ◽  
Lucie Kopfstein ◽  
Elisabeth Ischi ◽  
Elisabeth Oppliger Leibundgut ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Negative Predictive Value ◽  
Predictive Value ◽  
Healthy Subjects ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Essential Thrombocythaemia ◽  
Jak2 Mutation ◽  
Spontaneous Proliferation

Abstract Abstract 4970 Background Essential thrombocythaemia (ET) is one of the chronic myeloproliferative neoplasms (MPN), along with polycythaemia vera (PV), primary myelofibrosis (PMF) and chronic myeloid leukaemia (CML). Their common feature is excessive proliferation of a certain stem or progenitor cell in the bone marrow; in the case of ET, the megakaryocytic lineage is affected. Clinical manifestations include thrombotic events and haemorrhage. Diagnosis of ET according to new WHO-criteria requires a sustained high platelet count, bone marrow biopsy showing proliferation of the megakaryocytic lineage with large and mature morphology, demonstration of JAK2 V617F (although only present in about 50% of patients with ET) or another clonal marker and explicit exclusion of other myeloid and myeloproliferative neoplasms as well as signs of reactive thrombocytosis. Additionally, spontaneous proliferation of megakaryocytes obtained from peripheral blood can be detected in in vitro culture assays. Presently, we use agar as a matrix for megakaryocyte cultivation, although this assay has never been validated in connection with ET. The identification of megakaryocytic colonies grown on agar can sometimes be quite difficult. Our aims were therefore to technically evaluate the use of a collagen based matrix and to investigate its suitability to identify patients with ET. Patients and Methods We have examined 63 patients (26 with ET, 21 with PV, 8 with myelofibrosis [MF; including PMF and post-ET/PV-MF], 6 with secondary or idiopathic erythrocytosis and 2 with secondary thrombocytosis; mean age=59.8, male=33, female=30, mean platelet count 457 G/l) and 5 healthy subjects. Following informed consent, both clinical and laboratory data was collected. Medication intake, phlebotomies, smoking habits and regular haemogram results were noted in order to recognise possible confounding factors influencing laboratory results. Results of megakaryocyte cultivation on both agar and collagen matrixes were recorded, considering both spontaneous growth and growth stimulated by megakaryocyte derived growth factor (MDGF). Results Based on our collagen culture results we were able to define 2 or more spontaneously grown megakaryocyte colonies as the most optimal cut-off for the identification of patients with MPN (sensitivity 71%, specificity 100% with positive and negative predictive values of 100% and 45%, respectively). Compared to the agar culture results (where a specificity and a positive predictive value of 100% were demonstrated at a cut-off value of ≥ 10 CFU-Mega) we found a higher accuracy and better reproducibility. In addition, we observed an improved negative predictive value (45% with collagen versus 25% with agar cultures) reducing false negative results. Healthy subjects and patients with secondary thrombocytosis showed no significant spontaneous megakaryocyte proliferation. In patients with MF, we observed strong spontaneous and MDGF-stimulated growth of megakaryocytic colonies. At a cut-off value of ≥ 50 CFU-Mega (after stimulation with MDGF), the collagen assay showed a sensitivity of 100% and a specifity of 70% for this special form of MPN, resulting in a negative predictive value of 100%. We found no confounding clinical or laboratory parameters such as medication intake (particularly cytoreductive treatment with hydroxyurea) or phlebotomies influencing our culture results, and no significant effect of the Jak2-V617F mutation on the growth behaviour of megakaryocytic colonies. Conclusion The results of this ongoing study imply that the collagen based assay is more sensitive, specific, time efficient and user friendly regarding the detection of spontaneous proliferation of megakaryocytes than the currently used agar based culture assay. In addition, the collagen based assay also has the great advantage that it allows isolation of single megakaryocytic colonies for further analyses, for example PCR-based identification of a JAK2 mutation. Furthermore, the collagen based assay facilitates the diagnosis of patients with MPN, especially in cases where conventional diagnostic criteria are lacking, such as in ET without a JAK2 mutation. Ultimately, the new assay may well be able to detect transformation from PV/ET to MF. Disclosures No relevant conflicts of interest to declare.

Clinical Implications for Patients with Low JAK2V617F Allele Burden

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1745-1745
Author(s):  
Marguerite Vignon ◽  
Dorota Jeziorowska ◽  
Pierre Hirsch ◽  
Ollivier Legrand ◽  
Nicole Casadevall ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Phenotype ◽  
Myeloproliferative Neoplasms ◽  
Kinase Domain ◽  
Jak2 Mutation ◽  
Allele Burden ◽  
Vein Thrombosis ◽  
Myeloid Neoplasms ◽  
Exon 10 ◽  
Jak2v617f Allele Burden

Abstract Abstract 1745 In classical Philadelphia-negative myeloproliferative neoplasms (MPN), JAK2V617F is considered as a driver mutation when the threshold of 1% JAK2V617F/JAK2total allele burden is reached. However a lower ratio is sometimes detected with highly sensitive assays. We investigated the clinical significance of such minor clones by describing the characteristics of 27 patients with a low JAK2V617F allele burden ranging from 0.1% to 0.99%. Material and Methods A commercially available quantitative ASO-PCR assay of 0.1% sensitivity (MutaQuant® kit, Ipsogen) was used. Two thousand five hundred consecutive blood samples were sent to our lab for JAK2V617F mutation between 2009 and 2012. Total blood DNA was extracted by an automated standardized procedure (Qiasymphony®, Qiagen). All samples were tested in duplicate. The 27 samples of our cohort were controlled using a second assay of 0.01% sensitivity (Larsen et al, BJH 2007). Thirty samples from healthy donors were also tested. High resolution melting curve (HRM) analysis of JAK2 exon 14 ruled out the possibility of an additional mutation hampering the annealing of a primer. Patients with a known classical MPN clinical phenotype were also tested for JAK2 exons 12–17 (entire pseudo-kinase domain) or for MPL exon 10 depending on the context. Results Laboratory Findings Among the 2500 samples, 735 (29.4%) were positive above 1%, 27 (1.1%) had low JAK2V617F allele burden ranging from 0.12 to 0.99%. The patient with the lowest ratio (0.12%) was not confirmed by the second assay and therefore was excluded from the study. This allowed the median to settle at 0.40%. No associated mutations were found in the JAK2 pseudo-kinase domain in patients with polycythemia vera (PV) and in MPL exon 10 in patients with essential thrombocytosis (ET) and primary myelofibrosis (PMF). Healthy patients were all tested JAK2V617F negative. Clinical Aspects The cohort included 19 men and 7 women ranging from 28 to 95 years of age (median 63 years old). Two patients had secondary acute myeloid leukaemia following JAK2V617F positive MPN indicating the presence of residual JAK2V617F cells and the negativity of the myeloblastic population. Thirteen patients (50%) had a classical MPN with a median ratio of 0.36%: 7 ET, 5 PV and 1 PMF according to WHO 2008 criteria. However a bone marrow biopsy was available for only two patients (1 ET, 1 PMF). None of them had received pegylated interferon alpha-2a. Four patients had a prior history of thrombosis: two strokes, one pulmonary embolism, two portal vein thrombosis (PVT). For one PV patient, a 6 months follow-up blood and bone marrow sample confirmed a low allele burden in the same range (0.4%) and in vitro Epo-independant erythroid colonies were observed. Five patients had other chronic myeloid neoplasms (two myelodysplastic/myeloproliferative neoplasms, one chronic eosinophilic leukaemia, one chronic myeloid leukaemia, one refractory anaemia with ring sideroblasts). Among these five, four had an abnormal karyotype. We did not observe any thrombotic event in these patients. We cannot conclude on hematological diagnosis for the last six patients: four patients were screened for JAK2 mutation because of PVT. One patient had chronic polycythemia in a context of alcohol and tobacco abuse. One patient had homozygous hemochromatosis with a normal haemoglobin level in spite of repeated phlebotomies. Discussion In this single centre study low JAK2V617F allele burden represented 1% of all samples sent for JAK2V617F study and 3.5% of JAK2V617F positive patients. Seventeen patients (65%) had classical MPN or splanchnic vein thrombosis. To our knowledge PV patients with such low JAK2V617F allele burden have not been reported in the absence of associated JAK2 pseudo-kinase domain mutation. A larger screen for cooperating mutations responsible for the PV phenotype is under process. In the context of other chronic myeloid neoplasms, the JAK2V617F mutation is thought to belong to a more complex clonal architecture mostly implicating chromatin remodeling genes. Here, the presence of a JAK2 mutation could argue in favour of clonal haematopoiesis. In conclusion the clinical phenotype of low JAK2V617F patients overlaps with classical JAK2V617F MPN. The technical implications might be challenging for molecular diagnostic platforms. More data are needed to further characterize these patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Two Cases of Severe Hypertension in JAK2 Mutation-Positive Myeloproliferative Neoplasms

2020 ◽  
Vol 2020 ◽  
pp. 1-6
Author(s):  
Raunak Rao ◽  
Spoorthy Kulkarni ◽  
Ian B. Wilkinson
Keyword(s):  
Bone Marrow ◽  
Renal Artery ◽  
Renal Artery Stenosis ◽  
Bone Marrow Biopsy ◽  
Blood Count ◽  
Severe Hypertension ◽  
Myeloproliferative Neoplasms ◽  
Artery Stenosis ◽  
Full Blood Count ◽  
Jak2 Mutation

Background. Myeloproliferative neoplasms are a heterogeneous group of disorders resulting from the abnormal proliferation of one or more terminal myeloid cells—established complications include thrombosis and haemorrhagic events; however, there is limited evidence to suggest an association with arterial hypertension. Herein, we report two independent cases of severe hypertension in JAK2 mutation-positive myeloproliferative neoplasms. Case Presentations. Case 1: a 39-year-old male was referred to our specialist hypertension unit with high blood pressure (BP) (200/120 mmHg), erythromelalgia, and headaches. We recorded elevated serum creatinine levels (146 μM) and panmyelosis. Bone marrow biopsy confirmed JAK2-mutation-positive polycythaemia vera. Renal imaging revealed renal artery stenosis. Aspirin, long-acting nifedipine, interferon-alpha 2A, and renal artery angioplasty were employed in management. BP reached below target levels to an average of 119/88 mmHg. Renal parameters normalised gradually alongside BP. Case 2: a 45-year-old male presented with high BP (208/131 mmHg), acrocyanosis, (vasculitic) skin rashes, and nonhealing ulcers. Fundoscopy showed optic disc blurring in the left eye and full blood count revealed thrombocytosis. Bone marrow biopsy confirmed JAK2-mutation-positive essential thrombocytosis. No renal artery stenosis was found. Cardiac output was measured at 5 L/min using an inert gas rebreathing method, providing an estimated peripheral vascular resistance of 1840 dynes/s/cm5. BP was well-controlled (reaching 130/70 mmHg) with CCBs. Conclusions. These presentations highlight the utility of full blood count analysis in patients with severe hypertension. Hyperviscosity and constitutive JAK-STAT activation are amongst the proposed pathophysiology linking myeloproliferative neoplasms and hypertension. Further experimental and clinical research is necessary to identify and understand possible interactions between BP and myeloproliferative neoplasms.

scholarly journals Nonproliferative and Proliferative Lesions of the Rat and Mouse Hematolymphoid System

2019 ◽  
Vol 47 (6) ◽  
pp. 665-783 ◽  
Author(s):  
Cynthia L. Willard-Mack ◽  
Susan A. Elmore ◽  
William C. Hall ◽  
Johannes Harleman ◽  
C. Frieke Kuper ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Great Britain ◽  
North America ◽  
Lymph Nodes ◽  
Diagnostic Criteria ◽  
Lymphoid Tissues ◽  
The World ◽  
Tertiary Lymphoid Structures ◽  
Lymphoid Structures ◽  
Rats And Mice

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative changes in rats and mice. The purpose of this publication is to provide a standardized nomenclature for classifying changes observed in the hematolymphoid organs, including the bone marrow, thymus, spleen, lymph nodes, mucosa-associated lymphoid tissues, and other lymphoid tissues (serosa-associated lymphoid clusters and tertiary lymphoid structures) with color photomicrographs illustrating examples of the lesions. Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. The nomenclature for these organs is divided into 3 terminologies: descriptive, conventional, and enhanced. Three terms are listed for each diagnosis. The rationale for this approach and guidance for its application to toxicologic pathology are described in detail below.

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

2021 ◽  
Vol 11 ◽  
Author(s):  
Daniele Cattaneo ◽  
Giorgio Alberto Croci ◽  
Cristina Bucelli ◽  
Silvia Tabano ◽  
Marta Giulia Cannone ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Essential Thrombocythemia ◽  
Triple Negative ◽  
Clinical Laboratory ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Molecular Profile ◽  
Single Center ◽  
Driver Genes ◽  
Molecular Features

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.

JAK2 V617F Mutation in Essential Thrombocythemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4925-4925
Author(s):  
Jeong Yeal Ahn ◽  
Pil Whan Park ◽  
Yiel Hea Seo ◽  
Dong-Bok Shin ◽  
Jae-Hoon Lee ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Essential Thrombocythemia ◽  
Statistical Significance ◽  
Vascular Complications ◽  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Janus Kinase ◽  
Jak2 Mutation ◽  
Hematopoietic Stem ◽  
Specific Association

Abstract Background: Essential thrombocythemia (ET) is thought to reflect transformation of a multipotent hematopoietic stem cell, but its molecular pathogenesis has remained obscure. But tyrosine kinase, especially Janus kinase 2 (JAK2) has been implicated in myeloproliferative disorders other than chronic myeloid leukemia. We investigated the incidence and its correlation with other clinicopathologic variables of JAK2 mutation in patients with ET and reactive thrombocytosis (RT). Method: JAK2 mutation analysis, using allele-specific polymerase chain reaction, was undertaken on genomic DNA from bone marrow aspirates of 24 patients with ET and peripheral blood in 36 patients with RT. Results: JAK2 mutation was detected in 11 patients (46%) among the 24 patients with ET and was not found in 36 patients with RT. In patients with ET, older age and leukocytosis were related with JAK2 mutation without statistical significance (p=0.172 and 0.094, respectively). But this mutation was not correlated with sex, hemoglobin, platelet count, splenomegaly, increased cellularity of bone marrow, bone marrow fibrosis and vascular complications. Conclusions: The current observation strengthens the specific association between JAK2 mutation and ET. At the diagnosis of ET, identification of JAK2 mutation should be incorporated in foundation for new approaches.

JAK2 Mutation Analysis and Function of Bone Marrow Mesenchymal Stem Cells in Myeloproliferative Neoplasms

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5179-5179
Author(s):  
Hong Tian ◽  
Yang Xu ◽  
Guanghua Chen ◽  
Man Qiao ◽  
Wu Depei
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Jak2v617f Mutation ◽  
Primary Study ◽  
Jak2 Mutation ◽  
Healthy Donors ◽  
Marrow Mesenchymal Stem Cells

Abstract Abstract 5179 Background: JAK2V617F and JAK2 exon12 mutations in haematopoietic cells were partially responsible for the pathogenesis of myeloproliferative neoplasms (MPN).But it was still unclear whether bone marrow mesenchymal stem cells (BMSCs), the significant component of hemopoiesis microenvironment, were participated in the pathogenesis of MPN. Objective: To study the physiopathology characteristics and analyze JAK2 mutation in BMSCs from MPN patients. Methods: By searched for the JAK2V617F mutation and exon 12 mutation in 135 MPN patients' blood /bone marrow samples, 20 patients with JAK2V617F mutation, 10 patients with JAK2 exon 12 mutation, 5 JAK2-mutation-negetive patients and 10 healthy donors were recruited. The phenotype, mesenchymal differentiation capacity, expression of hematopoietic and immune molecules and JAK2 mutation of isolated bone marrow BMSCs were detected. Results: BMSCs derived from the four groups were found to be similar in morphology, differentiation ability and expression of hematopoietic and immune molecules. Primary study indicated that the isolated BMSCs from patients groups were not able to harbor JAK2 mutation in spite of positive or negative JAK2 mutation in blood /bone marrow samples. Conclusion: BMSCs from MPN patients had similar biological characteristics to healthy donors, and BMSCs were not likely involved in pathogenesis of MPN. Disclosures: No relevant conflicts of interest to declare.

scholarly journals JAK2S523L, a novel gain-of-function mutation in a critical autoregulatory residue in JAK2V617F− MPNs

2020 ◽  
Vol 4 (18) ◽  
pp. 4554-4559
Author(s):  
Friederike Pastore ◽  
Aishwarya Krishnan ◽  
Henrik M. Hammarén ◽  
Olli Silvennoinen ◽  
Benedict Yan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Data ◽  
Myeloproliferative Neoplasms ◽  
Negative Regulation ◽  
Erythropoietin Receptor ◽  
The Novel ◽  
Jak2 Mutation ◽  
Linker Region ◽  
Somatic Alterations ◽  
Linker Domain

Abstract The SH2-JH2 linker domain of JAK2 has been implicated in the negative regulation of JAK2 activity. In 2 patients with myeloproliferative neoplasms (MPNs), we identified and characterized the novel JAK2 mutation S523L, which occurs in a key residue in the linker region. In 1 case, acquisition of JAK2S523L was associated with thrombocytosis and bone marrow megakaryocytic hyperplasia, and there were no other somatic alterations in this patient. The second patient with JAK2S523Lmutation presented with increased hematocrit and had concurrent mutations in RUNX1 and BCORL1. Consistent with the genetic and clinical data, expression of JAK2S523L causes interleukin-3–independent growth in Ba/F3 cells transduced with the erythropoietin receptor by constitutively active Jak2/Stat5 signaling.

Identifying Myeloid Mutations By NGS in Patients with Unexplained Erythrocytosis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 47-47
Author(s):  
Eri Kawata ◽  
Anargyros Xenocostas ◽  
Cyrus C. Hsia ◽  
Alejandro Lazo-Langner ◽  
Michael Levy ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Care Center ◽  
Tertiary Care ◽  
Clinical Impact ◽  
Jak2v617f Mutation ◽  
Lymphoid Tissues ◽  
Driver Genes ◽  
Jak2 Mutation ◽  
Clone Size ◽  
Assay Sensitivity

Introduction: Unexplained erythrocytosis is a common reason for consultation in hematology. Identification of JAK2V617F mutation has facilitated the diagnosis of Polycythemia Vera (PV), but a proportion of patients without clear secondary causes for erythrocytosis remain undiagnosed or presumptively diagnosed with an either Exon 12 mutation or JAK2 negative PV. Since 2005, our institution has been performing JAK2V617F testing by PCR. In 2018 we switched to an NGS panel which includes JAK2/exon 12 and 40 other genes implicated in myeloid malignancies. We reviewed all previously diagnosed PV who had NGS myeloid panel performed to determine whether patients with a clinical diagnosis of JAK2 negative PV had other myeloid mutations that might explain their erythrocytosis and alter their management. Methods: We identified all cases with clinically suspected PV or confirmed JAK2 mutated PV who went on to have had NGS testing performed between January 2018 and February 2019 at London Health Sciences Centre, a tertiary care center servicing a population of approximately 2.5 million in Ontario, Canada. The Oncomine Myeloid NGS panel (Thermo-Fisher, MA, USA) examines DNA sequence variants in 40 genes (17 full genes and 23 hotspot genes) along with an RNA-based panel of 29 fusion driver genes and their over 600 fusion partners. Diagnosis was based on the WHO 2016 Classification of Tumours of Haematopoietic and Lymphoid Tissues. The clinical impact was assessed from retrospective review of electronic medical record to determine whether there was a diagnostic or management impact. Results: A total of 143 patients followed for PV or unexplained erythrocytosis had NGS testing during the study period. Of those, 137/143 (95.8%) patients had previous JAK2V617F PCR tested and 48/137 (35%) were identified with JAK2V617F mutation. Of the 48 patients with previous JAK2V617F PCR detected, NGS confirmed JAK2 mutation in 40/48 (83.3%) with additional non-JAK2 mutations in 17/40 (42.5%) patients. Of note 8/48 (16.7%) patients previously detected JAK2V617F by PCR had undetectable JAK2 mutation when repeat testing was performed by NGS. Of those 89/137 (65%) patients with previous JAK2V617F PCR negative result, NGS revealed JAK2 exon 12 mutation in 3/89 (3.4%) patients and JAK2V617F/JAK2L611V mutations in 1/89 (1.1%) patient resulting in diagnosis as PV, whereas non-JAK2 mutations in 6/89 (6.7%) patients. No MPL or CALR positive cases were identified in this cohort. Remaining 79/89 (88.8%) had no mutations identified (Figure1) and in this group, 13/79 (16.5%) patients were discharged from hematology clinic, 7/79 (8.9%) had therapies such as phlebotomy, aspirin or hydroxyurea stopped or reduced, whereas 2/79 (2.5%) patients had further evaluation or testing for unexplained erythrocytosis. (Table 1) Conclusions: In the unexplained erythrocytosis JAK2V617F PCR negative group, JAK2exon 12 mutation was identified in 3.4% in keeping with known incidence of this mutation. Some previously positive PCR JAK2V617F mutation were not identified by NGS panel (16%) which may reflect changes in clone size either with time or therapy or inherent differences in assay sensitivity (2.5% mutational alleles for NGS versus 0.1% for PCR). Lack of identifiable myeloid mutation and clonal hematopoiesis by NGS testing influenced clinical management. Specifically, mutation negative patients were more likely assigned to non-MPN group and called secondary erythrocytosis which resulted in reducing interventions. Non-JAK2 mutations occurred in more than 1/3 of previously identified JAK2 positive PCR tested PV. The clinical impact of most these mutations is uncertain and requires longer follow up. Disclosures No relevant conflicts of interest to declare.

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