Myeloproliferative Leukemia Virus Research Articles

Signaling properties of murine MPL and MPL mutants after stimulation with thrombopoietin and romiplostim

Thrombopoietin (THPO) and its receptor myeloproliferative leukemia virus oncogene (MPL) regulate hematopoietic stem cell (HSC) quiescence and maintenance, but also megakaryopoiesis. Thrombocytopenias or aplastic anemias can be treated today with THPO peptide mimetics (romiplostim) or small-molecule THPO receptor agonists (e.g., eltrombopag). These THPO mimetics were designed for human application; however, many preclinical studies are performed in murine models. We investigated the activation of wild-type murine MPL (mMPL) by romiplostim. Romiplostim stimulated AKT, ERK1/2, and STAT5 phosphorylation without preference for one of these pathways, however, with a four- to fivefold lower phosphorylation intensity at high concentration. Faster internalization of mMPL after romiplostim binding could be one explanation of reduced signaling. In vitro megakaryocyte differentiation, proliferation, and maturation by romiplostim was less efficient compared with stimulation with mTHPO. We further dissected mMPL signaling by lentiviral overexpression of mMPL mutants with tyrosine (Y)-to-phenylalanine (F) substitutions in the distal cytoplasmic tyrosines 582 (Y582F), 616 (Y616F), and 621 (Y621F) individually and in combination (Y616F_Y621F) and in truncated receptors lacking 53 (Δ53) or 69 (Δ69) C-terminal amino acids. Mutation at tyrosine residue Y582F caused a gain-of-function with baseline activation and increased ERK1/2 phosphorylation upon stimulation. In agreement with this, proliferation in Y582F-32D cells was increased, yet did not rescue in vitro megakaryopoiesis from Mpl-deficient cells. Y616F and Y621F mutated receptors exhibited strongly impaired ERK1/2 and decreased AKT signaling and conferred reduced proliferation to 32D cells upon mTHPO stimulation but a partial correction of immature megakaryopoiesis in vitro.

The effects of mutational profiles on phenotypic presentation of myeloproliferative neoplasm subtypes in Bosnia: 18 year follow-up.

The identification of mutually exclusive somatic mutations shared among myeloproliferative neoplasm (MPN) subtypes has provided a powerful tool for studying disease evolution. Clinical features, gene mutations, and survival over 18 years were analyzed in MPN patients. One hundred thirty-eight MPN patients were subcategorized according to MPN subtypes: essential thrombocythemia (ET, n = 41), polycythemia vera (PV, n = 56), primary myelofibrosis (PMF, n = 10), and MPN unclassified (MPN-U, n = 31). Patient characteristics included clinical parameters, overall survival (OS), and mutational status of the Janus kinase 2 (JAK2), calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) genes. We compared hematologic and clinical features of JAK2V617F-ET vs. CALR-mutated ET vs. JAK2V617F-PV patients. JAK2V617F-patients had higher values of erythrocytes, hemoglobin, and hematocrit compared to CALR-mutated patients (p < 0.05). The mutant allele burden in JAK2V617F-PV and JAK2V617F-ET patients directly correlated with erythrocyte, hemoglobin, and hematocrit values, but it inversely correlated with platelet count. Thus, mutant allele burden was an indicator of the clinical phenotype in JAK2V617F-MPN patients. OS was not affected by the mutational status. In general, mutated JAK2, CALR, and MPL genes left specific hematological signatures.

Hepatic thrombopoietin gene silencing reduces platelet count and breast cancer progression in transgenic MMTV-PyMT mice

AbstractIn humans, platelet count within the normal range is required for physiological hemostasis, but, adversely, platelets also support pathological thrombosis. Moreover, by releasing growth factors, they may enhance neoplastic proliferation. We hypothesize that platelet count correlates with platelet-dependent pathologies, even within the range of hemostatic competence. Because platelet production is promoted by thrombopoietin signaling through the myeloproliferative leukemia virus oncogene (cMPL), a receptor expressed on megakaryocytes, we evaluated the feasibility of selective targeting of hepatic thrombopoietin production to test this hypothesis. We synthesized murine- and primate-specific antisense oligonucleotides (THPO-ASO) that silence hepatic thrombopoietin gene (THPO) expression without blocking extrahepatic THPO. Repeated doses of THPO-ASO were administered to mice and a baboon, causing a sustained 50% decline in plasma thrombopoietin levels and platelet count within 4 weeks in both species. To investigate whether reducing platelet count within the translationally relevant hemostatic range could alter a neoplastic process, we administered THPO-ASO to 6-week-old transgenic MMTV-PyMT mice that develop early ductal atypia that progresses into cMPL-negative fatal metastatic breast cancer within 2 to 3 months. THPO-ASO treatment increased the average time to euthanasia (primary humane endpoint) at 2 cm3 combined palpable tumor volume. Our results show that THPO-ASO reduced blood platelet count, plasma platelet factor 4, vascular endothelial growth factor, thrombopoietin levels, bone marrow megakaryocyte density, tumor growth rate, proliferation index, vascularization, platelet and macrophage content, and pulmonary metastases vs untreated controls. These findings confirm that sustained and moderate pharmacological platelet count reduction is feasible with THPO-ASO administration and can delay progression of certain platelet-dependent pathological processes within a safe hemostatic platelet count range.

PF675 JAK2V617F AND MPL MUTATIONS ARE ASSOCIATED WITH WORSENED SYMPTOM BURDEN THAN CALR IN THE MYELOPROLIFERATIVE NEOPLASMS: FINDINGS FROM THE MYMPN PATIENT REGISTRY

Background: The myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) are types of hematologic malignancies accompanied by excessive symptom burden that can contribute to worsened quality of life (Blood 2011). Specific driver mutations (i.e., Jak2V617F, Calreticulin (CalR), myeloproliferative leukemia virus (MPL)) have been associated with disease features, including risk of thrombosis, blood counts and overall survival (Haematologica 2017). However, no studies have yet investigated the role of driver mutations on symptom burden profiles. Aims: In this analysis, we address this gap by utilizing the myMPN Patient Registry to investigate the relationship of symptoms to driver mutational status. Methods: The myMPN patient registry was created by the MPN Research Foundation's myMPN steering committee. Utilizing the Genetic Alliance platform, patients can register and complete questions regarding their MPN as well as authorize disclosures. Patients reported variables including disease features and genetic mutations were collected in the registry.The MPN10 score (JCO 2012) was used to assess patient reported symptom burden. If multiple MPN10 surveys were completed, the mean score was calculated between all completed surveys. ANOVA, Levene's test for equality of variances, and T-tests were used to analyze statistical significance. Results: Participants: Since its initiation in September 2017, the registry comprises a total of 789 unique participants. A total of 583 participants have entered genetic driver mutation data to date (including 474 with JAKV617F, 89 with CALR, and 20 with MPL). The frequencies of driver mutations reported by participants were similar to previously reported studies for ET (73% JAK2V617F, 22% CALR, 4% MPL), PV (99% JAK2V617F, <1% CALR), and MF (67% JAK2V617F, 26% CALR, 7% MPL). Mean age was lower for CALR (56.7 years) than for JAK2V617F (60.6 years) or MPL (59.3 years, ANOVA p = 0.08). Symptom Scores: Participants have completed a mean of 2.1 MPN10 surveys (range 1–50 surveys). As demonstrated in Figure 1, MPN10 scores for CALR patients were significantly less symptomatic than JAK2V617F and MPL patients. When evaluating individual symptom scores, fatigue was significantly different among mutation subtypes (1.1 CALR, 5.3 MPL, 4.5 JAK2V617F, ANOVA p < 0.001). When comparing specific mutations, patients harboring CALR mutations were significantly less symptomatic than those with JAK2V617F mutations for items of fatigue (1.1 vs 4.4, t-test p = 0.001), weight loss (0.3 vs 0.7, t-test p = 0.003), and night sweats (1.1 vs 1.9, t-test p = 0.02). CALR patients had worse early satiety than JAK2V617F patients (2.1 vs 1.5, t-test p = 0.007). CALR patients also reported less symptomatic weight loss (0.3 vs 0.8, t-test p = 0.03) than MPL patients.Summary/Conclusion: This represents the first analysis to identify that genetic mutations may be a contributor to specific symptom phenotypes in MPN populations. This fits with the generally known trend towards a younger age at diagnosis and more indolent clinical course of CALR mutated MPNs, however, the lower age in this cohort may also be a cofounder of less severe symptom burden. The role of symptom etiologies, including thrombotic events, comorbidities, and inflammation, and modifying mutations (i.e., ASXL1), would be ideally investigated in an independently validated database. Although the validity of patient-reported registries may be a caveat to their use, data reported in the myMPN registry mirrors closely with previously published and validated MPN populations.

PB2215 HOW TO PREDICT RESPONSE TO RUXOLITINIB IN PATIENTS WITH MYELOFIBROSIS

Background:Myelofibrosis (MF) is a classical myeloproliferative neoplasm characterized both in its primary and secondary form by splenomegaly, constitutional and disease‐related symptoms as well as by cytopenias. Aberrant constitutive activation of the JAK‐STAT pathway is core to its pathogenesis and the inhibitors of this pathway such as ruxolitinib, approved for MF treatment seven years ago, have been proven effective in a majority of patients.Aims:To describe ruxolitinib response and median survival in a group of patients diagnosed with MF in a single center and correlate it with the molecular profile, hematimetric data and previous myeloproliferative neoplasm (polycythemia vera (PV) or essential thrombocytosis (ET)).Methods:Of a total of one‐hundred twenty four patients diagnosed with MF in our center, thirty eight patients were treated with ruxolitinib between April 2012 and September 2018. Diagnosis of myelofibrosis was made based on bone marrow examination and according to the updated WHO criteria. Molecular study was carried out in peripheral blood for the mutation JAK2V617F as well as allele‐specific PCR for mutations type 1 and type 2 of exon 9 of calreticulin (CALR) gene, and exon 10 of myeloproliferative leukemia virus (MPL) gene. The prognostic risk stratification was carried out according to the International Prognostic Index (IPSS). All patients treated with ruxolitinib belonged to the intermediate group‐2 or were classified as high risk. Statistical analyses were performed in SPSS 15.0.Results:Average age of the group was 63 years (range 36‐88). 58% were males and 42% female. Average leukocyte, haemoglobin and platelet counts at diagnosis were 9,8x10e9/l (1.9‐62), 10.3 g/dl (6‐15.7) and 236x10e9/L (52‐789), respectively. 47% of patients had a previous diagnosis of another MPN (50% PV and 50% ET). JAK2V617F mutation was present in 70.3%, and CARL and MPL mutation in 12.5% and 2.6%, respectively. All patients responded to ruxolitinib although 39% finally lost this response, with a 13% evolution to acute leukemia, 40% of this group being subjected to allogeneic SCT. Currently, 73% of patients are alive. Six‐year overall survival of patients with JAK2V617F mutation was 80% compared to 60% of those with wild‐type mutational status, statistical significance not being reached. Patients with previous MPN also had greater six‐year overall survival (70%) than those with idiopathic MF (56%).Summary/Conclusion:Patients with MF with JAK2V617F mutation and previous MPN present a greater duration of response to ruxolitinib than others, probably due to greater inhibition of the preponderant pathogenetic mechanism and the more effective action of the drug on a more differentiated progenitor than in idiopathic MF, although broader studies are required to confirm these results.

Long term follow-up after liver transplantation from a JAK2 mutation positive donor.

Philadelphia-negative myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are characterized by the presence of mutations involving the Janus Kinase 2 ( JAK2 ), calreticulin ( CALR ) or myeloproliferative leukemia virus oncogene ( MPL ) genes (1). Of these, the JAK2 V617F gain-of-function mutation is the most frequently found and is present in >95% of patients with PV and in about 60% of those with ET or PMF whereas JAK2 exon 12 mutations are found in the remaining PV patients (2). JAK2 V617F somatic mutations have been detected in granulocytes and platelets and therefore they could potentially be transmitted to recipients of solid organ transplants (3) and in fact the presence of the JAK2 V617F mutation has been detected in otherwise healthy blood donors (4,5).

Mice with Calr mutations homologous to human CALR mutations only exhibit mild thrombocytosis

Calreticulin (CALR) exon 9 frameshift mutations, commonly detected in essential thrombocythemia (ET) and primary myelofibrosis patients, activate signal transducer and activator of transcription (STAT) proteins in the presence of Myeloproliferative Leukemia Virus (MPL) and induce ET in vivo. Loss of the KDEL motif, an endoplasmic reticulum retention signal, and generation of many positively charged amino acids (AAs) in the mutated C-terminus are thought to be important for disease induction. To test this hypothesis, we generated mice harboring a Calr frameshift mutation using the CRISPR/Cas9 system. Deletion of 19-base pairs in exon 9 (c.1099-1117del), designated the del19 mutation, induced loss of the KDEL motif and generated many positively charged AAs, similar to human mutants. Calr del19 mice exhibited mild thrombocytosis, slightly increased megakaryocytes, and mild splenomegaly. In vitro experiments revealed that the murine CALR del19 mutant had a weaker ability to combine with murine MPL than the human CALR del52 mutant. Consequently, STAT5 activation was also very weak downstream of the murine mutant and murine MPL, and may be the reason for the mild disease severity. In summary, loss of the KDEL motif and positively charged AAs in the C-terminus of CALR is insufficient for MPL binding and ET development.

Endothelial protein C receptor supports hematopoietic stem cell engraftment and expansion in Mpl-deficient mice

AbstractThrombopoietin (Thpo)/myeloproliferative leukemia virus oncogene (Mpl) signaling controls hematopoietic stem cell (HSC) self-renewal and quiescence; however, how these 2 seemingly opposing functions are controlled is not well understood. By transplantation of lentiviral-transduced hematopoietic cells in the Mpl-deficient mouse model, we addressed whether known or predicted Thpo target genes were able to rescue the Mpl-deficient phenotype of the mice. Among the tested genes, we identified endothelial protein C receptor (Epcr) to expand HSCs with the long-term (LT)-HSC surface phenotype in Mpl−/− mice and to enable secondary transplantation of Mpl-deficient bone marrow (BM). Epcr-transduced Mpl−/− HSCs enter quiescence earlier after transplantation than control-transduced Mpl−/− cells, and upregulated expression of the anti-apoptotic gene Bcl-xL. Also, in the wild-type background, Epcr expression marked the engrafting population in the BM. Furthermore, Epcr expression in Mpl−/− hematopoiesis increased the number of megakaryocytes in the BM. In vitro Thpo supported the surface expression of Epcr on primary murine hematopoietic stem and progenitor cells. With these data, we add new insights into Thpo-dependent influence on HSC engraftment after transplantation. This may be of use for the in vitro manipulation of HSCs, also in the context of gene therapy.

MPL-Mutated Essential Thrombocythemia: A Morphologic Reappraisal

Background:Myeloproliferative leukemia virus oncogene (MPL)-mutated essential thrombocythemia (ET) is uncommon, with a cited incidence of less than 5%, while that of MPL-mutated primary myelofibrosis (PMF) is at least twice as frequent (Blood. 2006;108:3472;Blood.2008;112:141). MPL-mutated ET cohorts also have higher reported rates of fibrotic progression than their MPL wild type counterparts (Blood. 2014;124:2507). These observations suggest the possibility that some instances of MPL-mutated ET might actually represent prefibrotic PMF.Methods:Patients were recruited from institutional databases of the Mayo Clinic, Rochester, MN, USA. Diagnoses were according to the 2016 World Health Organization (WHO) criteria (Blood. 2016;127:2391). Study inclusion criteria required the availability of bone marrow biopsy slides for central review by one of the authors (C.A.H.). Central pathology review included assessment of bone marrow cellularity and extent of tri-lineage proliferation, megakaryocyte morphology and grading of reticulin fibrosis. Data was additionally collected from MPL-mutated patients with PMF, for comparison. Standard statistical methods were used for statistical analysis, including calculation of survival data (JMP® Pro 13.0.0, SAS Institute, Cary, NC, USA).Results:A total of 665 patients with ET were annotated for their driver mutational status; 18 (2.7%) were reported out as being MPL-mutated; by comparison, among 867 patients with PMF, 47 (5.4%) were signed out as MPL-mutated. Among the 18 cases with MPL-mutated ET, bone marrow slides were available for central pathology review in 14 patients (Table 1). The latter were subsequently reassigned the diagnosis of either prefibrotic PMF (n=8; 57%) or were felt to be morphologically consistent with true WHO-defined ET (n=6; 43%). Comparison of these two distinct histopathological patterns, i.e. true ET vs reassigned prefibrotic PMF, was respectively characterized by lower (median 35%, range 30-50) vs higher (median 65%, range 40-80) bone marrow cellularity (P<0.001), ET (n=6) vs PMF (n=8) consistent megakaryocyte morphology (P<0.001) and presence of tri-lineage proliferation (0% vs 100%; P<0.001); in contrast the degree of reticulin fibrosis was similar between the two (P=0.1) (Table 2).The reassigned prefibrotic PMF (n=8), vs confirmed ET (n=6), cases also displayed a higher frequency of increased serum levels of lactate dehydrogenase (60% vs 0%; P=0.02), higher likelihood of displaying hemoglobin levels below the sex-adjusted reference range values (29% vs 0%; P=0.1), leukoerythroblastosis (14% vs 0%; P=0.2), constitutional symptoms (13% vs 0%; P=0.2) and a higher incidence of thrombosis history at presentation (38% vs 0%; P=0.04) (Table 2). Interestingly, reassigned prefibrotic PMF also displayed a narrower MPL mutational spectrum compared to those confirmed as ET (MPLW515L/K incidence 100% vs 60%; P=0.04). The incidences of abnormal karyotype and high risk molecular mutations (ASXL1, SRSF2 and U2AF1) were similar between the two.We documented a higher incidence of post-diagnosis thrombosis in prefibrotic PMF (25% vs 0%; P=0.1) but similar rates of leukemic transformation (0% for both) and fibrotic progression (38% vs 33%); when all 665 ET patients were assessed for myelofibrosis-free survival, MPL-mutated cases (prior to central review) displayed significantly worse outcome, compared to patients with other driver mutations (Figure 1a). Median overall survival in confirmed MPL-mutated ET, ET re-classified as PMF, and MPL-mutated PMF was not reached, 11.6 and 5.3 years, respectively (confirmed ET vs PMF, P=0.01; ET re-classified vs PMF, P=0.04; confirmed ET vs ET re-classified as PMF, P=0.54) (Figure 1b).Conclusions:The current study suggests that the majority of routinely-assigned MPL-mutated ET probably represents prefibrotic PMF, when morphologically scrutinized. Prior to central pathology review, we show a higher rate of fibrotic progression in MPL-mutated ET, compared to patients with other driver mutations; accordingly, after central pathology review, the similar rate of fibrotic progression between morphologically confirmed MPL-mutated ET and those reassigned as prefibrotic PMF further suggests the latter to be biologically more akin to PMF, despite its ET-consistent morphologic features. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Icaritin Provokes Serum Thrombopoietin and Downregulates Thrombopoietin/MPL of the Bone Marrow in a Mouse Model of Immune Thrombocytopenia.

Immune thrombocytopenia (ITP) is a common acquired autoimmune disease, and thrombopoietin (TPO) is an important cytokine that regulates the production of megakaryocytes and platelets. We have identified a biologically active component, icaritin, from a Chinese herba epimedii extract. Icaritin promotes platelet production and regulates T cell polarization, but its mechanism is not clear. In this study, the BALB/c mouse model of ITP was established by injection of an antiplatelet antibody every other day for seven total times. The antiplatelet sera were derived from guinea pigs immunized with the platelets of BALB/c mice. Mice with ITP were treated with icaritin at low, moderate, or high doses of 4.73, 9.45, and 18.90 mg/kg, respectively, for fourteen consecutive days. The present study shows that icaritin can significantly increase peripheral blood platelet counts and thrombocytocrit, increase the TPO level in serum, attenuate splenomegaly, and reduce the abnormal proliferation of megakaryocytes in the spleen and bone marrow. Icaritin can also downregulate the expression of bone marrow TPO, myeloproliferative leukemia virus oncogene (MPL), and p-Stat3. Our results suggest that icaritin can significantly improve the health of mice with ITP via possible downregulation of p-Stat3 expression in the JAK2/Stat3 phosphorylation signaling pathway and regulation of bone marrow TPO/MPL metabolism.

Protective Effect of the c-mpl Agonist Romiplostim on Megakaryocytopoiesis of Human CD34+Hematopoietic Progenitor Cells Exposed to Ionizing Radiation

A thrombopoiesis-stimulating protein, the myeloproliferative leukemia virus protooncogene (Mpl) ligand romiplostim (RP), is currently approved as a therapeutic agent for idiopathic thrombocytopenic purpura in many countries. Although the action of the initial MPL ligand thrombopoietin (TPO) on human megakaryocytic regeneration from irradiated human hematopoietic stem cells has been examined, there are few reports on the action of RP. In the present study, freshly prepared nonirradiated and 2-Gy X-irradiated human CD34 positive (CD34+) cells from placental umbilical cord blood were cultured with a combination of RP and various cytokines. As a result, the effect of RP on cell proliferation of nonirradiated CD34+ cells was found to be comparable to that of TPO. However, the stimulating activity of RP on megakaryocytic progenitor-derived colony formation was markedly lower compared with TPO. Regarding the action of RP with various cytokines, the present results showed that a combination of RP with interleukin-3 (IL-3) or IL-3 plus stem cell factor (SCF) showed a high regenerative effect on cell proliferation, megakaryopoiesis, thrombopoiesis, and megakaryocyte colony formation from X-irradiated CD34+ cells. The present study showed that human recombinant RP has potential effects on human megakaryocytic regeneration from X-irradiated human CD34+ cells and synergistically acts with IL-3 and IL-3 plus SCF, just as observed with TPO.

Driver mutations in primary myelofibrosis and their implications.

Primary myelofibrosis (PMF) is one of the classic BCR-ABL1 negative myeloproliferative neoplasms (MPN). Oncogenic driver mutations in PMF include Janus kinase 2, calreticulin (CALR), and myeloproliferative leukemia virus oncogene. These mutations are not only pathogenetically relevant but might also influence disease outcome. Our objective for the current communication is to comprehensively review the distinct phenotypic, therapeutic, and prognostic implications of driver mutations in PMF. The discovery of driver mutations has revolutionized our understanding of pathogenic mechanisms and clinical heterogeneity in MPN, including PMF. Recently, there have been further advances in our knowledge of the molecular pathogenesis of MPN, particularly pertaining to CALR and its mutation. Moreover, the type and number of additional mutations, their order of acquisition, and their myriad combinatorial interactions with driver mutations may have dynamic pathogenic and clinical consequences. There are also additional data supporting the role of these genetic lesions and their associated allele burdens in modulating clinical features, including outcomes following treatment. Literature exists to support both phenotypic and prognostic correlates of conventional driver mutations in PMF. As the genetic landscape becomes increasingly complex, establishing the functional impact of these mutations and defining their interactions with other molecular, cytogenetic, and extrinsic factors will further our insight and potentially alter our clinical approach.

Selected Parameters of Angiogenesis and the JAK2, CALR, and MPL Mutations in Patients With Essential Thrombocythemia.

The aim of the study was to evaluate selected angiogenic factors in patients with essential thrombocythemia (ET) depending on JAK2V617F, calreticulin gene (CALR) and myeloproliferative leukemia virus oncogene (MPL) mutations. Sixty ET patients and 20 healthy volunteers were enrolled in the study. The following tests were performed: vascular endothelial growth factor- A (VEGF-A), soluble vascular endothelial growth factor receptor-1 (sVEGFR-1),soluble vascular endothelial growth factor receptor-2 (sVEGFR-2), platelet-derived growth factor( PDGF-BB), and stromal-derived factor-1α (SDF-1α). We observed an increased PDGF-BB level in patients with ET compared to the controls. Patients with CALR mutation had significantly higher concentration of PDGF-BB and lower concentration of SDF-1α than patients with JAK2V617F mutation. High concentration of PDGF-BB and low concentration of SDF-1α in patients with CALR(+) ET may indicate a contribution of these chemokines in disturbed Ca2+ metabolism in platelets.

Effect of CALR and JAK2 mutations on the clinical and hematological phenotypes of the disease in patients with myelofibrosis - long-term experience from a single center.

Primary myelofibrosis (PMF) is a chronic clonal myeloid disorder. Together with essential thrombocythemia (ET) and polycythemia vera (PV), it belongs to a group of Philadelphia chromosome-negative myeloproliferative neoplasms. An integral part of laboratory tests carried out in this disease group is detecting the presence of mutations in the Janus kinase 2 gene at position 617 (JAK2 V617F) and in the gene encoding for the receptor for thrombopoietin (myeloproliferative leukemia virus oncogene, MPL) found in approximately 60% of PMF patients. The discovery of mutations affecting exon 9 of the calreticulin (CALR) gene was of great benefit to the diagnosis of the diseases in JAK2 V617F and MPL unmutated cases. This is a study of the effect of a mutation in the CALR gene on the clinical course in patients with primary, post-ET and post-PV myelofibrosis. Analysis of 66 patients (54.5% JAK2 V617F; 34.8% CALR; 6.1% MPL; 3.0% triple negative; 1.5% coincidence of CALR and JAK2 V617F) confirmed a different phenotype of the disease in CALR-mutated patients as compared with CALR-unmutated individuals. Those with CALR mutation were significantly younger and had borderline higher platelet counts, less pronounced splenomegaly and less frequent B symptoms at diagnosis. The study suggests that the driver mutation types define variations in the biological basis, clinical manifestations and course of the disease. The presence of CALR mutation has been shown to be an independent prognostic favorable factor. Careful risk stratification of these patients is of great importance to adequate therapeutic decision-making and aids in selecting high-risk patients eligible for allogeneic hematopoietic stem cell transplantation which continues to be the only treatment modality for myelofibrosis having curative potential.

Abstract 37: An unusual case of myelofibrosis with a JAK2 H538QK539L mutation associated with nephrotic syndrome

Abstract Introduction: Activating Janus kinase 2 (JAK2) mutations have been described in myeloproliferative neoplasms (MPNs) since 2005. Since its initial discovery, the JAK2 V617F mutation has been described in more than 95% of patients with polycythemia vera (PV) and in 50-60% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Subsequent efforts were made to identify molecular mutations in MPN cases lacking the V617F mutation and further mutations in the exon 12 domain of the JAK2 were identified in PV patients. However, there have been only a few studies evaluating the presence of these JAK2 exon 12 mutations in PMF patients. Case Report: A 60-year-old Malay gentleman with no known prior medical history of note presented at our Emergency Department with a 1-week history of bipedal edema and exertional dyspnea. Physical examination revealed bibasal coarse crepitations with moderate hepatosplenomegaly. Laboratory investigations revealed normochromic, normocytic anemia, thrombocytopenia, hypoalbuminemia, and elevated creatinine levels. A spot urine sample showed a protein to creatinine ratio of 93 mg/mmol with an estimated total urinary protein (TUP) of 0.8 g/day. Further workup of his renal impairment, which included an autoimmune screen, hepatitis, and humman immunodeficiency tests, were all unremarkable. A myeloma screen revealed elevated immunoglobulin G levels of 29.18 g/L but with no monoclonal bands detected. Transthoracic echocardiography showed mild pulmonary hypertension with a pulmonary artery systolic pressure (PASP) of 45 mmHg, but the ejection fraction was normal at 65%. He was also noted incidentally to have a prolonged prothrombin time (PT), activated partial thromboplastic time (APTT), and thrombin clotting time. Fibrinogen levels were normal and a lupus anticoagulant was not detected. Bone marrow aspiration and biopsy results were consistent with myelofibrosis and diffuse coarse reticulin fibers of grade 4 were reported. Quantitative BCR-ABL messenger ribonucleic acid (mRNA), JAK2 V617F, and calreticulin (CALR) mutations were negative. However, a JAK2 H538QK539L (mutation leading to a substitution of glutamine for histidine at position 538 and leucine for lysine at position 539) in exon 12 was detected. Incidentally, iron stores were absent on the bone marrow aspirate sample. Ultrasound (US) and computed tomography (CT) scans showed the presence of hepatosplenomegaly with portal hypertension with no evidence of any venous thrombosis. A nonobstructive left renal calculi was seen but no other renal abnormality was detected. He was started on low-salt diet, fluid restriction, diuretic, and iron supplements. Within 1 month, his hemoglobin increased from 7.7g/dL to 14 g/dL with a rise in hematocrit to 52.1%. His iron supplements were stopped and he required 3 sessions of venesection in total to control his hematocrit. Subsequently, he was commenced on hydroxyurea for cytoreduction. His spleen reduced in size and his proteinuria normalized after 10 months. However, he developed worsening anemia and hydroxyurea had to be stopped. On follow-up, his hemoglobin increased to 12 g/dL with a baseline platelet count of &amp;gt; 100 X 109/L. Throughout this period, his bipedal edema remained chronic but he was not consistent with his renal follow-ups. Within 12 months, his proteinuria started increasing and is now within the nephrotic range. Discussion: We describe here a patient who was diagnosed with a JAK2 V617F negative PMF. Subsequent analysis revealed that he was harboring a JAK2 exon12 mutation instead. This was associated with a deranged coagulation profile and proteinuria which responded to hydroxyurea. This patient was never diagnosed with PV previously and thus did not meet the criteria for post-PV myelofibrosis. Also, we believe that this is the first case of a JAK2 exon12 mutation occurring in a myelofibrotic patient. Given the recent description of MPN-related glomerulopathy and the response of this patient to hydroxyurea, we believe further investigations into the associations of triple-negative (JAK2 V617F, myeloproliferative leukemia virus oncogene (MPL) and CALR negative) PMF with JAK2 exon12 mutations and the associated organ involvements may increase our knowledge of the pathogenesis of this rare condition. Citation Format: Moon Ley Tung, Hersharan Kaur Sran. An unusual case of myelofibrosis with a JAK2 H538QK539L mutation associated with nephrotic syndrome [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 37.

Enhanced Risk for Specific Somatic Myeloproliferative Neoplastic Mutations in Patients with Stroke.

Somatic mutations of Janus kinase 2 (JAK2V617F), calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) are the major clonal molecules that drive the pathogenesis of myeloproliferative neoplasms (MPN). It is well recognized that MPN patients carry an excessive risk of thrombohemorrhagic complications. However, little is known about the prevalence of these clonal markers in patients with cerebral vascular disease. To address this issue, 153 consecutive stroke patients in Taiwan were enrolled in the study. Allele-specific PCR (AS-PCR), real-time AS-PCR, and Illumina paired-end sequencing were employed to detect the presence of MPL, JAK2V617F, and CALR exon 9 mutations, respectively. JAK2V617F mutation was detectable in 13 samples (8.5%), but the allele burdens (AB) were greater than 1% in only six (3.9%) of them. Compared to JAK2-unmutated patients, those with JAK2V617F AB &gt; 1% had significantly higher white blood count (p = 0.01), although four of the six did not exhibit MPN phenotypes. Two patients had a heterozygous CALR exon9 mutation locating outside the coding region and did not alter the amino acid sequence of this protein. On the other hand, there were no patients carrying the MPL mutations. Using patient age, baseline hemogram, and stroke-relevant risk factors, we developed a predictive model that could successfully identify stroke patients at risk of carrying clonal JAK2V617F mutation. The prevalence of JAK2V617F mutation in stroke patients was higher than that seen in general population. Based on our newly developed probability stratification model, genotyping of JAK2V617F mutation in selected patients with stroke might be warranted.

Molecular determinants of pathogenesis and clinical phenotype in myeloproliferative neoplasms.

The myeloproliferative neoplasms are a heterogeneous group of clonal disorders characterized by the overproduction of mature cells in the peripheral blood, together with an increased risk of thrombosis and progression to acute myeloid leukemia. The majority of patients with Philadelphia-chromosome negative myeloproliferative neoplasms harbor somatic mutations in Janus kinase 2, leading to constitutive activation. Acquired mutations in calreticulin or myeloproliferative leukemia virus oncogene are found in a significant number of patients with essential thrombocythemia or myelofibrosis, and mutations in numerous epigenetic regulators and spliceosome components are also seen. Although the cellular and molecular consequences of many of these mutations remain unclear, it seems likely that they interact with germline and microenvironmental factors to influence disease pathogenesis. This review will focus on the determinants of specific myeloproliferative neoplasm phenotypes as well as on how an improved understanding of molecular mechanisms can inform our understanding of the disease entities themselves.

Targeted deep sequencing in polycythemia vera and essential thrombocythemia

AbstractPolycythemia vera (PV) is characterized by JAK2 and essential thrombocythemia (ET) by JAK2, calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) mutations; we describe the occurrence and prognostic relevance of DNA sequence variants/mutations other than JAK2/CALR/MPL. A myeloid neoplasm–relevant 27-gene panel was used for next-generation sequencing of bone marrow or whole blood DNA and conventional tools were used for analysis. “Adverse variants/mutations” were identified by age-adjusted multivariable analysis of impact on overall, leukemia-free, or myelofibrosis-free survival. Fifty-three percent of 133 Mayo Clinic patients with PV and 53% of 183 with ET harbored 1 or more sequence variants/mutations other than JAK2/CALR/MPL; the most frequent were TET2 and ASXL1. “Adverse variants/mutations” in PV included ASXL1, SRSF2, and IDH2 and in ET SH2B3, SF3B1, U2AF1, TP53, IDH2, and EZH2; combined prevalence was 15% and 15%, respectively. Adverse variants/mutations were associated with inferior survival in both PV (median, 7.7 vs 16.9 years) and ET (median, 9 vs 22 years) and the effect was independent of conventional prognostic models with respective hazard ratio (95% confidence interval) of 2.8 (1.5-5.1) and 2.6 (1.4-4.8); these observations were validated in 215 Italian patients with PV and 174 with ET. In both Mayo Clinic and Italian cohorts, leukemic or fibrotic progression was also predicted by adverse variants/mutations. Number of mutations did not provide additional prognostic information. We conclude that targeted deep sequencing in PV and ET allows for genetic risk stratification that is independent of clinically derived prognostic models.

An accurate, simple prognostic model consisting of age, JAK2, CALR, and MPL mutation status for patients with primary myelofibrosis.

In most patients with primary myelofibrosis, one of three mutually exclusive somatic mutations is detected. In approximately 60% of patients, the Janus kinase 2 gene is mutated, in 20%, the calreticulin gene is mutated, and in 5%, the myeloproliferative leukemia virus gene is mutated. Although patients with mutated calreticulin or myeloproliferative leukemia genes have a favorable outcome, and those with none of these mutations have an unfavorable outcome, prognostication based on mutation status is challenging due to the heterogeneous survival of patients with mutated Janus kinase 2. To develop a prognostic model based on mutation status, we screened primary myelofibrosis patients seen at the MD Anderson Cancer Center, Houston, USA, between 2000 and 2013 for the presence of Janus kinase 2, calreticulin, and myeloproliferative leukemia mutations. Of 344 primary myelofibrosis patients, Janus kinase 2V617F was detected in 226 (66%), calreticulin mutation in 43 (12%), and myeloproliferative leukemia mutation in 16 (5%); 59 patients (17%) were triple-negatives. A 50% cut-off dichotomized Janus kinase 2-mutated patients into those with high Janus kinase 2V617F allele burden and favorable survival and those with low Janus kinase 2V617F allele burden and unfavorable survival. Patients with a favorable mutation status (high Janus kinase 2V617F allele burden/myeloproliferative leukemia/calreticulin mutation) and aged 65 years or under had a median survival of 126 months. Patients with one risk factor (low Janus kinase 2V617F allele burden/triple-negative or age >65 years) had an intermediate survival duration, and patients aged over 65 years with an adverse mutation status (low Janus kinase 2V617F allele burden or triple-negative) had a median survival of only 35 months. Our simple and easily applied age- and mutation status-based scoring system accurately predicted the survival of patients with primary myelofibrosis.

BLVRB redox mutation defines heme degradation in a metabolic pathway of enhanced thrombopoiesis in humans

AbstractHuman blood cell counts are tightly maintained within narrow physiologic ranges, largely controlled by cytokine-integrated signaling and transcriptional circuits that regulate multilineage hematopoietic specification. Known genetic loci influencing blood cell production account for <10% of platelet and red blood cell variability, and thrombopoietin/cellular myeloproliferative leukemia virus liganding is dispensable for definitive thrombopoiesis, establishing that fundamentally important modifier loci remain unelucidated. In this study, platelet transcriptome sequencing and extended thrombocytosis cohort analyses identified a single loss-of-function mutation (BLVRBS111L) causally associated with clonal and nonclonal disorders of enhanced platelet production. BLVRBS111L encompassed within the substrate/cofactor [α/β dinucleotide NAD(P)H] binding fold is a functionally defective redox coupler using flavin and biliverdin (BV) IXβ tetrapyrrole(s) and results in exaggerated reactive oxygen species accumulation as a putative metabolic signal leading to differential hematopoietic lineage commitment and enhanced thrombopoiesis. These data define the first physiologically relevant function of BLVRB and implicate its activity and/or heme-regulated BV tetrapyrrole(s) in a unique redox-regulated bioenergetic pathway governing terminal megakaryocytopoiesis; these observations also define a mechanistically restricted drug target retaining potential for enhancing human platelet counts.