Triple-negative Essential Thrombocythemia Research Articles

Germline MPL Mutations May be a Rare Cause of “Triple-Negative” Thrombocytosis

Background Rare cases of primary thrombocytosis are related to inherited genetic abnormalities and are called hereditary thrombocytosis (HT). HT has clinical features resembling sporadic essential thrombocythemia (ET) but has usually an autosomal dominant (AD) inheritance with variable penetrance. To date, germline mutation involving the MPL, THPO, JAK2 genes have been associated with HT. Methods The study included 933 patients evaluated at our Division because of persistent isolated thrombocytosis for whom secondary reactive causes were excluded. Patients were screened for the three canonical somatic driver mutations associated with myeloproliferative neoplasms: JAK2 V617F mutation, CALR exon 9 mutations, MPL exon 10 mutations. Triple-negative cases with unexplained isolated thrombocytosis which also resulted negative for non-canonical exon 10 MPL mutations were analyzed using next generation sequencing (NGS) to test a panel of 80 myeloid genes. Results Of 933 patients with isolated thrombocytosis screened, 567 were JAK2-mutated, 255 CALR-mutated, 41 MPL-mutated, 2 double-mutated and 68 triple-negative. Two MPL-mutated patients carried a non-canonical mutation located in exon 10: MPL W515* and MPL V501A. Of the 68 triple-negative patients, 32 were evaluated by NGS and one of them showed another non-canonical mutation of MPL located outside exon 10: MPL R102P. These 3 non-canonical MPL mutations resulted to be germline as they were detected also in DNA extracted from T lymphocytes and from hair roots. Due to the germline nature of the mutations, a study of the family was carried out. In 2 out of 3 pedigrees at least a second relative carried the same germline mutation discovered in the proband; these 2 pedigrees are shown in the figure. Patient MPC17_15 was an 80 years-old patient with a history of thrombocytosis. She resulted JAK2- and CALR- wild-type while HRM screening was positive for exon 10 MPL mutations; Sanger sequencing identified the MPL W515* mutation in DNA from granulocytes, T-lymphocytes and hair roots, confirming its germline nature. Her children showed a normal complete blood count (CBC) and were MPL wild-type, thus confirming a segregation between mutation and clinical phenotype. Patient MPC14_674 (panel A) was a 36 years-old women with a history of mild thrombocytosis never investigated before. She was referred to our clinic by Pediatricians after the delivery because her baby (MPC17_167) showed persistent isolated thrombocytosis (826 x10 9/L) during the first months of life despite the exclusion of the most common reactive causes of thrombocytosis. Sanger sequencing identified the MPL V501A mutation in granulocyte and in T-lymphocyte in both cases. To evaluate the segregation of MPL V501A with the disease phenotype, we evaluated the CBC and MPL mutation in the other relatives. Both proband's father (MPC23_597) and sister (MPC23_602) carried germline heterozygous MPL V501A mutation associated with a PLT count above or at the upper limit of normal range (570 x10 9/L and 355 x10 9/L, respectively). Conversely, the husband (MPC17_495), the second son (MPC18_43) and the mother (MPC23_603) of the proband showed a normal CBC and were MPL wild-type, confirming an AD inheritance. Patient MPC07_130 (panel B) was a 44 years-old women with triple-negative isolated thrombocytosis lasting since 2007. NGS analysis showed the presence of a heterozygous germline MPL R102P mutation. As this mutation is located in exon 3, it was not detected through HRM and Sanger sequencing which target only exon 10. Her son (MPC13_672), 18 years-old, was completely asymptomatic but the CBC showed mild thrombocytosis (472 x 10 9/L). He tested negative for the 3 driver mutations but Sanger sequencing of MPL exon 3 identified the heterozygous germline MPL R102P mutation. Conclusions We identified 3 non-canonical germline MPL mutations in 3 different unrelated subjects with isolated thrombocytosis: MPL W515*, MPL R102P, and MPL V501A. Germline MPL mutations may underly HT and should be evaluated by sequencing of the whole exon 10 of MPL gene or by NGS in cases with isolated thrombocytosis negative for the three canonical somatic driver mutations. Namely, patients with HT might present as sporadic cases, and in the initial workup they might be inappropriately diagnosed with triple-negative ET. A correct diagnosis is pivotal to avoid unnecessary treatments.

Real-World Experience of Ropeginterferon-Alfa Treatment of PV and ET - Two Centers Experience

BACKGROUND AND AIM: Interferon alpha is an effective treatment for Polycythemia Vera (PV) and Essential Thrombocythemia (ET) and is the only treatment known to induce sustained molecular responses by decreasing JAK2 mutation allelic burden. Long-acting pegylated interferons are better tolerated. Ropeginterferon-alfa is a monopegylated interferon with a longer half-life and is the first interferon to be FDA-approved for the treatment of PV. It is not yet approved for ET, but clinical trials are ongoing. Given the similar disease biology between PV and ET and the efficacy of pegylated interferon-alfa in both PV and ET, this study aims to look at a real-world experience of treating PV and ET with ropeginterferon-alfa since its approval. METHODS: We conducted a retrospective chart review of all our PV and ET patients treated with ropeginterferon-alfa at Huntsman Cancer Institute, University of Utah (SLC cohort), and at the University of North Carolina at Chapel Hill (UNC cohort), since the drug's approval for PV from November 2021 through June 2023. The complete hematologic response (CHR) was defined as the normalization of blood counts within the reference range of the local lab standards. Prior to ropeginterferon-alfa, the participants were managed with therapeutic phlebotomies, or cytoreduced with either hydroxyurea, pegylated interferon-alpha 2a ( Pegasys), JAK2 inhibitors, or were newly diagnosed and previously untreated. RESULTS: A total of 120 patients (76 in SLC cohort and 44 in UNC cohort) were included in this study. Of those, 75 (62.5%) were diagnosed with PV, 37 (31%) with ET, and 1 with congenital polycythemia due to germline JAK2 mutation. Twenty-three (19%) patients had a prior history of thrombosis. JAK2 mutation was present in 100 (83%) patients, while 9 (7.5%) were CALR mutated, 1 with MPL mutation, and 1 with triple-negative ET. Extended myeloid gene panel sequencing was available in 64 (53.3%) patients and 35 of them (54.6%) patients had additional somatic mutations. Most common somatic mutations include TET2 (11 patients) and DNMT3A (8 patients) and CBL (3 patients), among others. Of the cohort, 15 (12%) patients discontinued ropeginterferon-alfa due to various adverse events, which included: fatigue, joint aches, skin rash, and increased anxiety and depression. Of the 105 patients who are on ropeginterferon-alfa, 9 patients were previously untreated patients while 96 (91.4%) were pretreated with other therapies and switched to ropeginterferon-alfa. In many patients, the switch to ropeginterferon-alfa was done because of the increased frequency and aggressivity of squamous cell skin cancers while on hydroxyurea treatment. All of them started ropeginterferon-alfa at a dose of either 50 mcg or 100 mcg every two weeks and were increased at an increment of 50 mcg to achieve a goal of CHR. As most of the patients were switching from prior therapies, 51 (49%) were already in CHR at the time of initiating ropeginterferon-alfa. The median ropeginterferon-alfa dose to maintain CHR was 50mcg in the SLC cohort and 100mcg in the UNC cohort. (See table for more details). We have 6 patients with DNMT3A mutation, 4 (66.6%) have achieved CHR at a median ropeginterferon-alfa dose of 100 mcg (range 50 mcg - 150 mcg), and 2 patients are undergoing dose titration. Of the 11 patients with TET2 mutation, 7 (63.3%) achieved CHR at a median ropeginterferon-alfa dose of 150 mcg (range 50 mcg - 500 mcg). The analysis of JAK2 V617F and CALR allelic burden, transcripts of inflammatory, prothrombotic, and hypoxia-regulated (HIFs) genes, and evaluation of females for reversal of clonal to polyclonal myelopoiesis is ongoing. CONCLUSION: Ropeginterferon-alfa is effective in PV and ET, however, the dose that achieves CHR is highly individualized, necessitating an incremental titration approach. The patients already in CHR from prior therapy needed relatively lower doses of ropeginterferon-alfa to maintain CHR. Clinical trials are ongoing with an alternate accelerated dosing scheme and results are awaited.

A Rare Case of Triple Negative Essential Thrombocythemia Presenting with Splenic Infarction: A Diagnostic Conundrum

Introduction Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by significant thrombocytosis with high propensity for thrombosis formation as well as other vascular complications. Most myeloproliferative neoplasms including ET are associated with Janus kinase ( JAK2) mutation. Other commonly associated mutations include Calreticulin ( CALR) and Myeloproliferative leukemia virus oncogene ( MPL) mutations. About 17% of cases of ET lack mutations in these driver genes. This presents a clinical conundrum for clinicians on initiation of appropriate treatment for these patients. We report a rare presentation of triple-negative ET with splenic infarction. Case Summary A 66-year-old Hispanic female who presented with left sided abdominal pain that gradually worsened over the course of 1 week; she believed it was stomach cramps. Laboratory evaluations were pertinent for platelet counts of 2000 x 10 9/L, hemoglobin level was 10.9 g/dl, and leukocytosis (white blood cell count 13.2k/uL) without left shift. Further work-up with iron studies was unremarkable and molecular studies were negative for BCR-ABL 1, PDGFR alpha/beta, FGFR 1, JAK2 V617F (including exons 12-13), CALR and MPL. A bone marrow biopsy showed an increase in megakaryocytes with some atypical forms with “enlarged size and nuclear hyper lobulation, and mild patchy reticulin fibrosis” suggestive of myeloproliferative neoplasm. Computed Tomography of the abdomen and pelvis with contrast showed evidence of splenic infarct. The patient was started on oral aspirin 81mg and oral hydroxyurea 2000 mg daily. Due to poor response to hydroxyurea and aspirin, she eventually had two cycles of platelet apheresis, and her platelet count improved to 1000 x 10 9/L. A repeat bone marrow biopsy was subsequently done to confirm essential thrombocythemia. At the time of discharge, the patient was asymptomatic and platelet count had decreased to 892 × 10 9/L. Discussion Essential thrombocythemia (ET) is a rare but recognized cause of vascular complications including arterial and venous thrombosis. Triple negative ET is quite uncommon and can pose a great deal of diagnostic challenge which can impact timing of initiation of appropriate therapy. This clinical vignette exemplified the conundrum encountered in making a diagnosis of triple negative ET. Following the World Health Organization (WHO) 2016 criteria for diagnosis of ET, patient has to have all 4 of: sustained platelet counts >450 x 10 9/L, bone marrow biopsy showing increased number of enlarged and mature megakaryocytes with no significant increase or left shift of granulopoiesis or erythropoiesis, presence of driver mutations/clonal markers (including JAK2, CALR and MPL mutations) or absent of evidence of iron deficiency or cause for reactive thrombocytosis and not meeting WHO criteria for Polycythemia vera or other myeloid neoplasms. Our patient met criteria for ET with no driver mutation detected, hence triple negative ET. She was also classified as high-risk ET using the revised International Prognostic Score for Essential Thrombocytopenia (IPSET). Initial work up of this patient was not confirmatory of the essential thrombocythemia due to the bone marrow biopsy not clearly defining features as per WHO stipulations, thus, a more aggressive therapy was not pursued. This can pose a great deal of diagnostic challenge which can impact timing of initiation of appropriate therapy. This is not uncommon especially considering sampling challenge. Studies has shown that early initiation of cytoreductive therapy prophylactically in a high-risk patients can avert vascular complications of ET (including splenic infarction). The index patient responded slowly to platelet apheresis which can be an effective treatment in acute thrombotic complication of ET. Conclusion The diagnosis of triple negative essential thrombocythemia can pose a clinical challenge which impacts effective management of this myeloproliferative neoplasm. There is a dire need for further discussion on this conundrum to provide more applicable recommendations to avert the rare but fatal complications of ET. Platelet apheresis can be an option for emergency treatment in patients presenting with a major vascular complication like splenic infarction.

Clinical and Molecular Characterization of Triple-Negative Essential Thrombocythemia: Data from the Prospective Spanish Registry of Essential Thrombocythemia

Introduction Triple-negative Essential Thrombocythemia (TN-ET) accounts for up to 10-20% of patients with a histological diagnosis of ET. Despite being considered an indolent disease, it remains a diagnostic and therapeutic challenge as it can be confused with non-clonal thrombocytosis and its prognostic factors and management are not standardized. Objectives The aim of this study was to evaluate the utility of a Next Generation Sequencing (NGS) panel targeting recurrently mutated genes in myeloid pathology for detecting genetic variants in patients diagnosed with TN-ET and to assess the significance of these variants in the risk of thrombosis and hemorrhage, progression to Acute Leukemia (AL) or Myelofibrosis (MF), and overall survival. Materials and Methods Data from 151 TN-ET patients with confirmed biopsy and NGS sequencing from the Spanish Registry of Essential Thrombocythemia of the Spanish Group of Philadelphia-Negative Myeloproliferative Neoplasms was obtained. This registry includes information on diagnosis, molecular biology, clinical characteristics, and molecular biology. In addition, 39 patients whose molecular diagnosis was performed at the Molecular Biology Laboratory of the Hematology Department at Hospital Universitario 12 de Octubre, Madrid, Spain were included. This last panel included the genes: ANKRD26, ASXL1, ATG2B, BCOR, BCORL1, CALR, CBL, CEBPA, CSF3R, CUX1, DDX41, DNMT3A, EGLN1, EPAS1, EPOR, ETNK1, ETV6, EZH2, FLT3, GATA2, GNAS, GNB1, GSKIP, IDH1, IDH2, JAK2, KDM6A, KIT, KMT2A, KRAS, MBD4, MPL, NF1, NFE2, NPM1, NRAS, PHF6, PPM1D, PRPF8, PTPN11, RAD21, RIT1, RRAS, RUNX1, SAMD9, SAMD9L, SETBP1, SF3A1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG2, TERC, TERT, TET2, THPO, TP53, U2AF1, VHL, WT1, ZRSR2. Statistical analyses were performed using SPSS v.29.0. Results A canonical mutation was detected in 20 out of the 190 studied patients, ( JAK2 V617F n=13, CALR n=3 and MPL n=4) being discarded from subsequent studies. Among the other 170 samples, 206 genetic variants were detected in a total of 86 patients (median 2, range 1-9). A total of 39 pathogenic (P) or probably pathogenic (PP) variants were found in 28 patients, whereas 59 uncertain significance (US) and 108 benign (B) variants were found in 44 and 54 patients, respectively. The distribution of genes in which P/PP/US variants were detected is shown in Figure 1. Patients with P /PP mutations had a statistically higher age compared to the remaining (50 vs. 64.8 years; p<0.001). The prevalence of males was higher in the group where P/PP mutations were detected (57.14% vs. 28.87%; p=0.004). No differences were found regarding cardiovascular comorbidity or symptomatology at diagnosis, but the P/PP group presented a higher frequency of thrombosis prior to diagnosis (28.57% vs. 5.64%; p=0.001). There were no differences in hemoglobin, leukocyte, or platelet counts at diagnosis according to the presence of P/PP variants. The presence of P/PP mutations was associated with a higher risk of progression to AL and a lower overall survival. A higher risk of thrombosis or progression to MF was not found (Figure 2). No differences were found in the risk of thrombosis, transformation, or overall survival between patients with or without the presence of US, PB or B variants. Conclusions The use of NGS technology is useful for the diagnosis and prognostication of TN-ET. The detection of P/PP mutations in patients diagnosed with TN-ET is associated with a higher risk of transformation to AL and lower overall survival, but not with higher risk of thrombosis or progression to MF. It is necessary to evaluate the individual risk of each of these mutations and the potential prognostic role of variants of US.

Individual with concurrent chest wall tuberculosis and triple-negative essential thrombocythemia: A case report

In recent years, because of the growing desire to improve the noninvasiveness and safety of tumor treatments, sonodynamic therapy has gradually become a popular research topic. However, due to the complexity of the therapeutic process, the relevant mechanisms have not yet been fully elucidated. One of the widely accepted possibilities involves the effect of reactive oxygen species. In this review, the mechanism of reactive oxygen species production by sonodynamic therapy (SDT) and ways to enhance the sonodynamic production of reactive oxygen species are reviewed. Then, the clinical application and limitations of SDT are discussed. In conclusion, current research on sonodynamic therapy should focus on the development of sonosensitizers that efficiently produce active oxygen, exhibit biological safety, and promote the clinical transformation of sonodynamic therapy.

Triple-negative Thrombocythemia and Subsequent Acute Lymphoblastic Leukemia with Additional Somatic Mutations.

Triple-negative essential thrombocythemia (ET) is a condition in which mutations in JAK2, CALR and MPL are all negative. Transformation to acute myeloid leukemia may occur during the course of ET, while B-acute lymphoblastic leukemia (B-ALL) is rare. We experienced a case diagnosed as B-ALL during the course of triple-negative ET. Notably, cytoreduction was required for the excessive increase in blood cells during the bone marrow recovery period after chemotherapies. Whole-exome sequencing identified 17 somatic mutations: 9 were identified in both ET and B-ALL samples, while 8 were specific to B-ALL, suggesting that these 8 might have caused the transformation.

Identification of putative noncanonical driver mutations in patients with essential thrombocythemia.

Essential thrombocythemia (ET) cases without canonical JAK2, CALR, or MPL mutations, that is, triple-negative (TN) ET, have been found in 10%-20% of ET cases. Owing to the limited number of TN ET cases, its clinical significance remains unclear. This study evaluated TN ET's clinical characteristics and identified novel driver mutations. Among 119 patients with ET, 20 (16.8%) had no canonical JAK2/CALR/MPL mutations. Patients with TN ET tended to be younger and had lower white blood cell counts and lactate dehydrogenase values. We identified putative driver mutations in 7 (35%): MPL S204P, MPL L265F, JAK2 R683G, and JAK2 T875N were previously reported as candidate driver mutations in ET. Moreover, we identified a THPO splicing site mutation, MPL*636Wext*12, and MPL E237K. Four of the seven identified driver mutations were germline. Functional studies on MPL*636Wext*12 and MPL E237K revealed that they are gain-of-function mutants that increase MPL signaling and confer thrombopoietin hypersensitivity with very low efficiency. Patients with TN ET tended to be younger, although this was thought to be due to the inclusion of germline mutations, hereditary thrombocytosis. Accumulating the genetic and clinical characteristics of noncanonical mutations may help future clinical interventions in TN ET and hereditary thrombocytosis.

Comparison of Clinical and Hematological Parameters of Janus Kinase 2, Calreticulin or Myeloproliferative Leukemia Virus Oncogene Mutant Essential Thrombocythemia and Triple-Negative Essential Thrombocythemia.

IntroductionEssential thrombocythemia (ET) is one of the chronic myeloproliferative neoplasms. While Janus kinase 2 (JAK2) V617F mutation is defined in more than half of the patients with ET, calreticulin (CALR) or myeloproliferative leukemia virus oncogene (MPL) mutations are encountered more rarely. The discovery of the JAK2 V617F mutation in 2005, followed by the recognition of MPL and CALR mutations, brought up the idea of subdividing ET according to the mutation status. Our aim in this study is to investigate whether genetic mutations detected in patients diagnosed with ET cause a different clinical phenotype compared to triple-negative ET.MethodsThis retrospective study was conducted by evaluating the patients who were followed up with the diagnosis of ET in the hematology clinic of two tertiary centers in Turkey between 2009 and 2021. Patients with negative JAK2, CALR, and MPL mutations and meeting the diagnostic criteria for ET were defined as triple-negative ET. The patients were divided into two groups as triple-negative ET and mutation-positive ET according to the presence of a mutation. It was investigated whether there was a difference between these two groups in terms of demographic, laboratory, and clinical characteristics.ResultsA total of 109 patients were included in the study. The mean age of these patients was 54 (18-91) years and 85 (78%) patients were females. A total of 48 patients (44.0%) had JAK2 mutation, six (5.5%) had CALR mutation, and one (0.9%) had MPL mutation. It was observed that there was a significant difference between the two groups in terms of gender, mean age, and hemoglobin value. While 87% of patients with triple-negative ET were females, this rate was 69% in patients with mutation-positive ET (p = 0.036). The mean age was 41.8 years in triple-negative ET and 67.1 years in patients with mutation-positive ET (p = 0.0001). While the mean hemoglobin value was 12.9 g/dl in patients with triple-negative ET, it was 14.4 g/dl in patients with mutation-positive ET (p = 0.0001).ConclusionIt has been observed that ET with JAK2, CALR, or MPL mutations may have different phenotypic features compared to triple-negative ET, resulting in a clinical condition consisting of older patients with a higher erythrocyte count.

Clinical and molecular biological features of triple-negative essential thrombocythemia

Clinical and molecular biological features of triple-negative essential thrombocythemia

Cell-autonomous megakaryopoiesis associated with polyclonal hematopoiesis in triple-negative essential thrombocythemia

A subset of essential thrombocythemia (ET) cases are negative for disease-defining mutations on JAK2, MPL, and CALR and defined as triple negative (TN). The lack of recurrent mutations in TN-ET patients makes its pathogenesis ambiguous. Here, we screened 483 patients with suspected ET in a single institution, centrally reviewed bone marrow specimens, and identified 23 TN-ET patients. Analysis of clinical records revealed that TN-ET patients were mostly young female, without a history of thrombosis or progression to secondary myelofibrosis and leukemia. Sequencing analysis and human androgen receptor assays revealed that the majority of TN-ET patients exhibited polyclonal hematopoiesis, suggesting a possibility of reactive thrombocytosis in TN-ET. However, the serum levels of thrombopoietin (TPO) and interleukin-6 in TN-ET patients were not significantly different from those in ET patients with canonical mutations and healthy individuals. Rather, CD34-positive cells from TN-ET patients showed a capacity to form megakaryocytic colonies, even in the absence of TPO. No signs of thrombocytosis were observed before TN-ET development, denying the possibility of hereditary thrombocytosis in TN-ET. Overall, these findings indicate that TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and is paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis.

Non-Driver Mutations and Gene Sequence Variants Identified By Targeted Deep Sequencing in Polycythemia Vera and Essential Thrombocythemia

Background:Driver mutations in myeloproliferative neoplasms involve mutations in the JAK2, MPL, and CALR genes. However, incorporation of next generation sequencing (NGS) testing into routine clinical practice may identify for mutations or variants in additional hematologic malignancy-associated genes, the roles of which have not been extensively elucidated in patients with polycythemia vera (PV) or essential thrombocythemia (ET). The purpose of this study is to characterize the clinical characteristics of ET/PV patients harboring additional non-driver mutations or gene variants.Methods:Eligible cases included patients with PV/ET diagnosed and treated at MD Anderson Cancer Center from January 2012 to January 2017. NGS sequencing of 28 genes known to be mutated in hematologic malignancies was performed on samples from 240 ET/PV patients. Non-driver mutations detected by the NGS platform were classified based on the allele frequency recorded in our database in conjunction with currently available and periodically updated public reference databases of somatic mutations or variants of unclear significance.Results:A total of 110 ET and 130 PV cases had NGS performed on samples collected at the time of initial referral. Among them, 21 (19%) ET cases and 28 (21.5%) PV cases had ≥1 non-driver somatic mutation detected. The distribution of non-driver somatic mutations in ET and PV are shown in Figure 1A. On grouping non-driver genes into functional pathways, the most frequent mutational targets were in genes associated with epigenetic regulation (PV, 22; ET, 25), mainly DNA methylation (PV, 18; ET, 20) followed by epigenetic modifier (PV, 4; ET, 5). In subjects with PV, TET2 (n = 12) was the most frequently mutated gene, followed by DNMT3A (n = 7), IDH1/2 (n = 5), and ASXL1 (n = 4). A similar pattern was noted in ET, with TET2 mutations (n = 8) being most frequent followed by DNMT3A (n = 7), and ASXL1 (n = 5). Older patients were more likely to display non-driver mutations in both ET (p < 0.001) and PV (p = 0.02). The combined prevalence of non-driver somatic mutations was 27% and 32% in PV and ET, respectively. Over a short median follow up of 3.9 (range, 1.0-37.1) months among PV patients with non-driver somatic mutations, no leukemic transformations occurred. Similarly, no leukemic transformations were observed in ET patients with additional non-driver somatic mutations over a median follow up of 3.8 (range, 1.1-36) months. Overall survival (OS) was significantly longer in patients without additional mutations in epigenetic modifiers (Figure 1C). The prognostic relevance of non-driver somatic mutations on OS was not determined due the small sample size and limited follow up duration. Of 13 (12%) triple-negative ET patients without non-driver somatic mutations, nine had non-synonymous gene sequence variants; the most common being in DNA methylation genes namely TET2 &IDH1/2 genes (Figure 1B).Conclusion:The spectrum of non-driver somatic mutations in our ET and PV study cohorts was dominated by mutations in the TET2, DNMT3A, ASXL1 genes, and were correlated with age (Buscarlet Blood, 2017). A larger sample size and longer follow-up is needed to determine the impact of non-driver somatic mutations on transformation-free and overall survival. Germline TET2 variants or their combinations with other variants/non-driver mutations may constitute low penetrance predisposition factors for myeloid neoplasms and their functional relevance warrants further exploration. [Display omitted] DisclosuresBose:Incyte Corporation: Honoraria. Kantarjian:Amgen: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Delta-Fly Pharma: Research Funding. Verstovsek:Astrazeneca: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Roche: Research Funding; Blueprint Medicines Corp: Research Funding; Galena BioPharma: Research Funding; Genentech: Research Funding; Galena BioPharma: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Blueprint Medicines Corp: Research Funding; Lilly Oncology: Research Funding; Incyte: Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Research Funding; Lilly Oncology: Research Funding; CTI BioPharma Corp: Research Funding; Pfizer: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding; CTI BioPharma Corp: Research Funding; Bristol Myers Squibb: Research Funding; Astrazeneca: Research Funding; Gilead: Research Funding; Celgene: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding.

Functional Consequences of Mutations in Myeloproliferative Neoplasms.

Driver mutations occur in Janus kinase 2 (JAK2), thrombopoietin receptor (MPL), and calreticulin (CALR) in BCR-ABL1 negative myeloproliferative neoplasms (MPNs). From mutations leading to one amino acid substitution in JAK2 or MPL, to frameshift mutations in CALR resulting in a protein with a different C-terminus, all the mutated proteins lead to pathologic and persistent JAK2-STAT5 activation. The most prevalent mutation, JAK2 V617F, is associated with the 3 entities polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF), while CALR and MPL mutations are associated only with ET and MF. Triple negative ET and MF patients may harbor noncanonical mutations in JAK2 or MPL. One major fundamental question is whether the conformations of JAK2 V617F, MPL W515K/L/A, or CALR mutants differ from those of their wild type counterparts so that a specific treatment could target the clone carrying the mutated driver and spare physiological hematopoiesis. Of great interest, a set of epigenetic mutations can co-exist with the phenotypic driver mutations in 35%–40% of MPNs. These epigenetic mutations, such as TET2, EZH2, ASXL1, or DNMT3A mutations, promote clonal hematopoiesis and increased fitness of aged hematopoietic stem cells in both clonal hematopoiesis of indeterminate potential (CHIP) and MPNs. Importantly, the main MPN driver mutation JAK2 V617F is also associated with CHIP. Accumulation of several epigenetic and splicing mutations favors progression of MPNs to secondary acute myeloid leukemia. Another major fundamental question is how epigenetic rewiring due to these mutations interacts with persistent JAK2-STAT5 signaling. Answers to these questions are required for better therapeutic interventions aimed at preventing progression of ET and PV to MF, and transformation of these MPNs in secondary acute myeloid leukemia.

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.

Recurrent thrombosis onset in triple-negative essential thrombocythemia – more than a difficult diagnosis. Case report

Recurrent thrombosis onset in triple-negative essential thrombocythemia – more than a difficult diagnosis. Case report

CREB3L1 overexpression as a potential diagnostic marker of Philadelphia chromosome-negative myeloproliferative neoplasms.

Discrimination of Philadelphia‐negative myeloproliferative neoplasms (Ph‐MPNs) from reactive hypercytosis and myelofibrosis requires a constellation of testing including driver mutation analysis and bone marrow biopsies. We searched for a biomarker that can more easily distinguish Ph‐MPNs from reactive hypercytosis and myelofibrosis by using RNA‐seq analysis utilizing platelet‐rich plasma (PRP)‐derived RNAs from patients with essential thrombocythemia (ET) and reactive thrombocytosis, and CREB3L1 was found to have an extremely high impact in discriminating the two disorders. To validate and further explore the result, expression levels of CREB3L1 in PRP were quantified by reverse‐transcription quantitative PCR and compared among patients with ET, other Ph‐MPNs, chronic myeloid leukemia (CML), and reactive hypercytosis and myelofibrosis. A CREB3L1 expression cutoff value determined based on PRP of 18 healthy volunteers accurately discriminated 150 driver mutation–positive Ph‐MPNs from other entities (71 reactive hypercytosis and myelofibrosis, 6 CML, and 18 healthy volunteers) and showed both sensitivity and specificity of 1.0000. Importantly, CREB3L1 expression levels were significantly higher in ET compared with reactive thrombocytosis (P < .0001), and polycythemia vera compared with reactive erythrocytosis (P < .0001). Pathology‐affirmed triple‐negative ET (TN‐ET) patients were divided into a high– and low–CREB3L1‐expression group, and some patients in the low‐expression group achieved a spontaneous remission during the clinical course. In conclusion, CREB3L1 analysis has the potential to single‐handedly discriminate driver mutation–positive Ph‐MPNs from reactive hypercytosis and myelofibrosis, and also may identify a subgroup within TN‐ET showing distinct clinical features including spontaneous remission.

PB2200 PREVALENCE OF JAK2V617F, CALR AND MPL MUTATIONS IN PATIENTS OF MYELOPROLIFERATIVE NEOPLASMS AND SPLANCHNIC VEIN THROMBOSIS IN INDIA

Background:Recurrent somatic mutations in the JAK2, MPL, and CALR genes have been described in patients diagnosed with Philadelphia‐negative Myeloproliferative neoplasms (MPNs), including Polycythemia vera (PV), Essential thrombocythemia (ET) and Primary myelofibrosis (PMF). MPN is also the most common etiology in splanchnic vein thrombosis (SVT). MPNs account for approximately 40% of Budd Chiari syndrome (BCS) and 31% of portal vein thrombosis (PVT).The present study was aimed at studying the prevalence of JAK‐2V617F, MPLW515L/K and CALR exon 9 mutations in BCR‐ABL1 negative myeloproliferative neoplasms (MPNs) and splanchnic vein thrombosis (SVT) patients. There is paucity of such data in the Indian patients.Aims:The study had two aims: Identification of incidence of JAK2V617F, CALR exon 9 mutations and MPLW515L/K mutations in BCR‐ABL1 negative MPNs in India Identification of incidence of JAK2V617F, CALR exon 9 mutations and MPLW515L/K mutations in Indian patients of SVT MethodsA total number of 60 patients of SVT and 59 MPN patients (10 PV, 19 ET, and 30 PMF) were included in the study. The mutation analysis for JAK2V617F, CALR exon 9 and MPLW515L/K mutations were performed using the Allele specific oligonucleotide‐polymerase chain reaction (ASO‐PCR) was used to detect JAK‐2, CALR and MPL mutations.Results:Among 59 patients of MPNs and 60 SVT; JAK‐2 mutation was found in 61.0% (36/59) and 23.3% (14/60) respectively. On sub‐classification, JAK‐2 mutation was detected in 80.0% (8/10) of PV; 42.1% (8/19) of ET and 66.7% (20/30) of PMF patients.All the patients of MPNs and SVT were subjected to CALR mutation analysis. The overall rate of CALR mutations in PMF and ET was 10% (3/30) and 21.0% (4/19) respectively. Limiting the analysis to JAK2V617F negative patients of PMF and ET, it was seen that 30% (3/10) PMF and ∼36.36% (4/11) ET cases were CALR mutated. Of these 7 CALR mutations; L367fs∗46 (type‐1) and K385fs∗47 (type‐2) mutations were found in 5 and 2 patients respectively. MPL mutation was identified in 5.26% (1/19) ET cases. Triple negative ET and PMF were ∼31.5% (∼6/19) and ∼23.3% (7/30) respectively. None of the SVT patients were observed to be CALR mutated and none showed the MPLW515L/K mutations.Summary/Conclusion:Indian patients exhibit a lower incidence of CALR mutations in Primary myelofibrosis than that seen in the western literature. However, the incidence of CALR mutations is the same as that reported earlier in Indian patients of Essential thrombocythemia. Triple negative cases constitute 31.5% of all cases of Essential thrombocythemia and 23.3% of Primary myelofibrosis Indian patients show a Testing for CALR mutations and MPLW515L/K mutations in splanchnic vein thrombosis is not justified.

Distinct Microrna Profiles in Patients with Essential Thrombocythemia: Clinical and Prognostic Correlation

Background: Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm characterized by increased number of mature megakaryocytes in the bone marrow and sustained thrombocytosis in the peripheral blood. ET is associated with an increased risk of hemorrhagic and thrombotic complications and leukemic transformation. In addition to somatic mutations, microRNA (miRNA) deregulation has been proposed to play roles in the molecular pathogenesis and disease phenotype in ET patients. The aims of this study were to investigate the plasma miRNA profiles in ET patients with those obtained from healthy adults (HA) and its correlation with clinical and prognostic features.Methods: ET patients seen at the MacKay Memorial Hospital were enrolled into this study. The clinical and laboratory characteristics at the time of diagnosis or referral were determined retrospectively by chart review. Total plasma miRNA was derived from bone marrow or peripheral blood in patients or HA. Mutational status of JAK2V617F,CALR and MPL were detected by Sanger sequencing and/or allele-specific PCR. Plasma miRNA profiling was carried out on PanelChip™ Analysis System (Quark Biosciences, Taiwan) using a customized miRNA panel including 165 miRNAs called mirSCAN™ PanCancer Chips 1 & 2. KEGG pathways that miRNAs were associated with were analyzed using DIANA TOOLS - mirPath v.3. Differentially expressed miRNAs were identified by student t-test (P < 0.05) and significantly affected miRNAs in ET were identified by a change in expression of two-fold or more compared to the expression in HA. Statistical analysis was performed using SPSS.Results: A total of 53 ET patients (median age at diagnosis 59 years; 60.4% females) were enrolled. Frequency of the 3 driver mutations was 64% for JAK2V617F, 15% CALR, 4% JAK2V617F and CALR co-mutations, and none for MPL. 17% of patients were classified as triple-negative. A total of 40 differentially expressed miRNAs were identified (Figure left axis) including 4 miRNAs (hsa-miR-940, hsa-miR-411-5p, hsa-miR-596 and hsa-miR-376c-3p) with higher expression levels. The putative target genes of these 40 differentially expressed miRNAs were enriched in signaling pathway including TGF-beta signaling pathway (p-value = 1.86E-12), Hippo signaling pathway (p-value = 1.84E-07), MAPK signaling pathway (p-value = 0.03), and FoxO signaling pathway (p-value < 0.001). According to the expression levels of these 40 miRNAs, samples were grouped into two clusters, i.e. low expression group and high expression group. ET patients with JAK2V617F were significantly associated with high expression of 40 miRNAs (n = 31 of 34, 91%) when compared with triple-negative ET patients (n = 5 of 9, 56%) (p = 0.03). ET patients with low expression of 40 miRNAs were significantly associated with acute leukemia transformation (n = 2 of 11, 18%) when compared with high expression group (n = 0 of 42, 0%) (p = 0.04). However, the expression levels of 40 miRNAs were not statistically correlated with other clinical features including hemogram, secondary solid cancer, myelofibrosis transformation, or thrombotic/hemorrhagic events.Conclusions: In this cohort of ET patients, distinct plasma miRNA profiles correlated with JAK2V617F mutation and leukemic transformation. Larger cohort is warranted to validate our findings. [Display omitted] DisclosuresChen:Quark Biosciences, Inc.: Employment. Kang:Quark Biosciences, Inc.: Employment. Huang:Quark Biosciences, Inc.: Employment. Lin:Quark Biosciences, Inc.: Employment. Wang:Quark Biosciences, Inc.: Employment.

Single-Cell RNA Sequencing Discloses Distinct Transcriptomic Profiling in Essential Thrombocythemia

BackgroundMyeloproliferative neoplasm (MPN) is a heterogeneous group of clonal disorders. The underlying mechanisms of pathogenesis, especially in subtype specification and stochastic malignant transformation, are still largely unknown. Single-cell RNA sequencing (scRNA-seq) is a novel tool that can be used to identify the transcriptomic signature of individual cells. In the current study, we aimed to employ scRNA-seq to analyze genetic profiling of individual cells at different hematopoietic hierarchy in essential thrombocythemia (ET) patients.MethodsWe enrolled 7 ET patients and one healthy adult. Individual CD34+ progenitor cells were enriched from peripheral blood (PB). Harvested viable cells were barcoded and sequenced with the Illumina HiSeq 4000. Data was visualized with 10x Genomics Loupe software. We performed t-distributed stochastic neighbor embedding (t-SNE) plotting to dissect the scRNA-seq data and cluster cells with transcriptional similarity. Cellular sub-populations were stratified by the surface markers, including hematopoietic stem cells (HSC, CD34+CD38-Lin-), common myeloid progenitors (CMP, CD34+KIT+FLT3+IL3RAlowLin-), megakaryocyte-erythroid progenitor (MEP, CD34+CD38+PTPRC-) and granulocyte-macrophage progenitor (GMP, CD34+CD38+PTPRC+). Differentially expressed genes were subjected to gene ontology analysis. Gene Set Enrichment Analysis (GSEA) and the Reactome analysis were also employed. To clarify the distinct genetic background of these patients, targeted deep sequencing of PB granulocytes was also performed.ResultsAmong the 7 ET patients, two carry JAK2 mutation [one heterozygous (h-JAK2)and one homozygous (H-JAK2)] and three carry CALR mutation (1 type I, 2 type II). The remaining two cases are triple-negative (TN). Integrative analysis showed significant activation of JAK-STAT signaling in ET patients. Compared with control, the t-SNE analysis revealed disparate expression profiling in ET patients across various hematopoietic lineages. The discrepancy grew wider as the hematopoiesis became more lineage-restricted. Significant heterogeneity existed even among different ET patients, suggesting the high diversity of the disease. In the two JAK2-mutated patients, the t-SNE analysis demonstrated divergent transcriptomic profiling which scarcely overlapped. In the H-JAK2 sample, the HSCs exhibited a distinct profile different from the rest of hematopoietic progenitors. The CMPs were more closely related to MEP, which possibly suggested skewed differentiation and resultant ET phenotype. The phenomenon, however, was not similarly seen in the h-JAK2 sample. Using GSEA, we identified a subset of miR-21-targeted genes that were down-regulated in the h-JAK2 sample. Furthermore, there was apparent aberrant signaling activity of TGF-β, widely considered a regulator of miR-21, in this ET sample as compared with the H-JAK2 sample. Therefore, it was probably not a coincidence that, two months after blood sampling, the h-JAK2 patient suffered from disease transformation to secondary myelofibrosis. Among the three CALR-mutated patients, the expression patterns and the mutational profiles were also significantly discrepant. In the patient with type I CALR mutation, the CD34+ cells exhibited aberrant activity in epigenetic regulators. Coupled with the identified somatic mutations in some epigenetic modifiers from the targeted sequencing results, it is speculated that these mutations occur in a very early hematopoietic stage and contribute to ET pathogenesis through abnormal epigenetic regulation in this patient. Lastly, principal component analysis showed that the pathognomonic molecular events initiated at different hierarchical level of hematopoiesis in the 2 TN ET patients. Reactome analysis also disclosed one patient had altered DNA repair activity, and targeted sequencing confirmed the presence of TP53 mutation. Clinically, this patient exhibited highly aggressive, treatment-refractory disease.ConclusionWe demonstrate that scRNA-seq extends our knowledge of clonal diversity and inter-individual heterogeneity in patients with ET. Combined with the results from targeted sequencing, we were able to uncover unique transcriptomic pattern in samples carrying specific somatic mutations. The obtained information could potentially leapfrog our effort in the elucidation of the pathogenesis of ET. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Characteristic of "triple-negative" Essential Thrombocythaemia Patients and Mutation Analysis by Targeted Sequencing

Essential thrombocythemia is a subgroup of myeloproliferative neoplasms. Previous studies identified mutations of JAK2, CALR, and MPL that are closely related with the pathogenesis of myeloproliferative neoplasms. All these mutations contribute to the hyperactivation of JAK2/STAT pathway. However, a small proportion of essential thrombocythemia patients does not display such mutations. The pathogenesis of "triple-negative" form of essential thrombocythemia remains unknown. To investigate the clinical characteristics of triple-negative essential thrombocythemia and related mutation genes. To identify the mutations associated with triple-negative essential thrombocythemia, next-generation sequencing was used to conduct targeted sequencing of 360 genes in samples from 68 patients. At least one missense mutation was detected in all the patients and all the detected genes. After screening the data, it was observed that 10 genes with the 10 highest mutation were follows: FLT3, SH2B3, ASXL1, ADAMTS1, TET2, TP53, EGFR, CUX1, GATA2, and MPL.When only rare genes (i.e., with a frequency in Asian populations lower than 5%, as estimated by the 1000 Genomes Project) were analyzed, the most frequently mutated genes in the patients were TET2 (33.82%), SH2B3(29.41%), and ASXL1 (23.53%). Our study identified some mutations that did not previously reported. Although all these mutations need further validation, high incidence rates may indicate relevance of the respective mutations to essential thrombocythemia pathogenesis. Some of the detected mutations have been previously reported; these mutations were also found in a large proportion of our subjects. whole-exon sequencing can provide a higher level of accuracy for gene mutation analysis and assist in identifying mutations that contribute to illustrate the pathogenesis of essential thrombocythemia.

Quantification of IGF-1 Receptor May be Useful in the Differential Diagnosis of Essential Thrombocytosis from Reactive Thrombocytosis- Suggested to Add IGF-1Receptor Quantification As the Minor Criteria for the Diagnosis of ET

Introduction. The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia lists the criteria for the diagnosis of essential thrombocythemia (ET ) as 1) Platelet count more than 450 x 109/L 2) BM biopsy 3) Not meeting WHO criteria for BCR-ABL CML, PV, PMF, myelodysplastic syndromes, or other myeloid neoplasms, 4) Presence of JAK2, CALR, or MPL mutation The minor criteria included Presence of a clonal marker or absence of evidence for reactive thrombocytosis .Among these criteria , the most reliable criteria for diagnosis of ET is the presence of JAK2, CALR, or MPL mutation, these mutually exclusive mutations with respective incidences of 55%, 25%, and 3% , so approximately 17% are so called ET . These 17 % cases will depend on other criterias to make a definite diagnosis as ET. Bone marrow criteria is helpful in some cases but in many cases, the differential from re-active thrombocytosis (RT) could be misleading , So a substantial portion of triple-negative patients with ET have evidence of polyclonal: the use of cytoreductive drugs in these patients is questionable We have recently published using IGF-1R quantification by flow cytometry could be helpful in differentiating polycythemia from secondary polycythemia Now we also found quantification of IGF-1R by flow cytometry to be helpful in the diagnosis of ET from reactive thrombocytosis. Materials and Methods: Nineteen patients with ET (treatment naive), 20 with reactive thrombocytosis (RT) (5 sickle cell disease, 5 arthritis, 5 iron deficiency, 3 metastatic Cancer, 2 with infection, one with bleeding) and 23 normal volunteer controls were studied. Flow cytometry: MNC were isolated from peripheral blood then incubated with fluorophore-conjugated primary antibody or isotype controls for 30 min at room temperature and analyzed by FACS Calibur System (BD Biosciences, San Jose, CA). PE-conjugated antibody against human IGF-1R was purchased from R&D Systems. Results: Untreated ET patients had significantly higher IGF-1R (290.8, 86.83-475.2) than RT patients (127.0, 47.75-177.2),(P =220 means that a patient belongs to ET group, with sensitivity of 47.4% and specificity equal to 94.7%. Conclusion: Quantification of IGF-1 receptor may be useful in the differential diagnosis of Essential thrombocythemia from reactive thrombocytosis, especially in those cases where no driver mutations were detected (triple negative). Disclosures Wang: Celgen: Research Funding.