JAK2-positive Polycythemia Vera Research Articles

Teaching NeuroImages: Intracranial extramedullary hematopoiesis

A 73-year-old woman with JAK-2–positive polycythemia vera, myelodysplastic/myeloproliferative overlap disorder, and biopsy-proven extramedullary hematopoiesis (EMH) involving pleura and paravertebral regions presented with 6 weeks of headaches and blurry vision. Examination showed bilateral papilledema, preserved acuity, and mild right pyramidal weakness. MRI brain showed homogenously enhancing dural lesions with no associated edema (figure). She underwent external beam radiation (10 fractions, 200 cGy/fraction) for symptomatic management.

Spectrum of Myeloid Mutations in Patients with Elevated Hemoglobin

▪Background: JAK2 V617F and exon 12 mutations are the characteristic driver mutations in polycythemia vera (PV), identified in more than 95% of patients. In addition, other genetic mutations have previously been described in JAK2-positive PV that appear to have prognostic significance (Tefferi et al., Blood 2016). The incidence of other driver mutations in unselected patients referred for elevated hemoglobin is less well studied. This study aims to characterize the genetic mutational landscape in a real-world population of patients referred for elevated hemoglobin using a targeted Next-Generation Sequencing (NGS)-based assay.Methods: We reviewed all patients referred for elevated hemoglobin levels (>160 g/L in females or >165 g/L in males) between 2018 and 2020 to hematology clinics at London Health Sciences Centre in Southwestern Ontario, Canada who underwent testing for genetic variants using the NGS-based Oncomine Myeloid Research Assay (ThermoFisher Scientific, MA, USA). This assay targets 40 key genes with diagnostic and prognostic implications in several myeloid malignancies (17 full genes and 23 genes with clinically relevant “hotspot” regions) and a panel of 29 fusion driver genes (>600 fusion partners). Patient demographics, laboratory data and final diagnosis were extracted from the electronic medical record. For all patients with genetic mutations, clinical diagnosis was confirmed by three independent reviewers.Results: A total of 529 patients underwent genetic testing for elevated hemoglobin levels: 389 (73.5%) were males (mean age 58; range 18-95) and 140 (26.5%) were female (mean age 60; range 24-85). JAK2 mutations were detected in 10.9% (58/529) of patients and a diagnosis of PV was confirmed. The majority of JAK2-mutated PV patients (n=57) were positive for JAK2 V617F, while one patient had an exon 12 mutation. Additional single myeloid mutations were detected in 34.5% (20/58) of JAK2-positive patients and involved the following genes: TET2 (11; 19%), DNMT3A (2; 3.4%), ASXL1 (2; 3.4%), SRSF2 (2; 3.4%), BCOR (1; 1.7%), TP53 (1; 1.7%) and ZRSR2 (1; 1.7%) (Figure 1A). JAK2 mutations were not detected in 89.0% (471/529) of our cohort. A diagnosis of PV was confirmed in 2 JAK2-negative patients based on clinical features and myeloid mutations were detected in both: SRSF2 and TET2 gene mutations in 1 patient and SRSF2, IDH2, ASXL1 gene mutations in the other patient. Three JAK2-negative patients tested positive for the BCR-ABL fusion and were diagnosed with chronic myeloid leukemia. The remaining 466 JAK2-negative patients were diagnosed with secondary erythrocytosis and myeloid mutations were found in 6% (28/466) of these cases. Mutations were detected in DNMT3A (12; 2.6%), TET2 (5; 1.1%), ASXL1 (5; 1.1%), TP53 (2; 0.4%), NF1 (2; 0.4%), KIT (1; 0.2%), U2AF1 (1; 0.2%) (Figure 1B). All patients with JAK2-negative secondary erythrocytosis had only one myeloid gene mutation detected.Conclusion: Additional myeloid mutations other than JAK2 mutations are frequently identified in patients referred for erythrocytosis, with the highest frequencies observed in the TET2, DNMT3A and ASXL1 genes. The spectrum of myeloid mutations and overall incidence in JAK2-negative patients with secondary erythrocytosis is similar to the reported incidence of Clonal Hematopoiesis of Indeterminate Potential (CHIP) (Jaiswal et al., NEJM 2014), and suggests that these may represent incidental age-related mutations. By contrast, among the JAK2-positive patients, 34.5% had at least one additional myeloid mutation supporting a pathogenic role in these patients with myeloproliferative neoplasms. While concomitant myeloid mutations in patients with PV are well-described, further research is required to elucidate the significance of variants identified in JAK2-negative patients classified as secondary erythrocytosis in order to determine whether these mutations contribute to clinical phenotype or represent background CHIP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cerebral Venous Sinus Thrombosis in a 40-year-old Lady with JAK2-positive Polycythemia Vera: A Case Report

Cerebral venous sinus thrombosis (CVST) has a range of underlying cause. Here a case of CVST with an uncommon etiology is presented and discussed. A 40-year-old female presented with headache for 2 years and progressive visual loss for 4 months. She had conjunctival congestion and bilateral papilloedema with pale disc on right side. Computed tomography (CT) scans and contrast magnetic resonance imaging (MRI) of brain were normal but venography (MRV) revealed widespread thrombosis of dural sinuses including superior sagittal sinus. Cerebrospianal fluid (CSF) pressure was high (360 mm of H₂O) while other CSF parameters were normal. She was polycythemic (hemoglobin 18.1 g/dl, hematocrit 60.2%), Bone marrow study revealed pan-myeloid hyperplasia whereas trephine biopsy revealed hypercellular marrow with trilineage expansion suggestive of myeloproliferative disorder possibly polycythemia vera. JAK-2 mutation was also detected. CVST has different patterns of presentations including isolated intracranial hypertension leading to visual loss. It is essential to identify and treat underlying condition like polycythemia vera.

Teaching NeuroImages: Intracranial extramedullary hematopoiesis

A 73-year-old woman with JAK-2–positive polycythemia vera, myelodysplastic/myeloproliferative overlap disorder, and biopsy-proven extramedullary hematopoiesis (EMH) involving pleura and paravertebral regions presented with 6 weeks of headaches and blurry vision. Examination showed bilateral papilledema, preserved acuity, and mild right pyramidal weakness. MRI brain showed homogenously enhancing dural lesions with no associated edema (figure). She underwent external beam radiation (10 fractions, 200 cGy/fraction) for symptomatic management.

Beyond the Routine CBC: Research CBC Parameters Associated with Myelodysplastic Syndromes and Underlying Mutations

Background: Myeloid neoplasms and reactive conditions cannot be readily distinguished by conventional CBC parameters. Given the time, expense and clinical expertise associated with the diagnostic workup of myeloid neoplasms, there is a clear need for a cost-effective screening laboratory test that can rapidly and accurately select for patients with cytopenias or cytoses potentially related to myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPN), respectively. Recent studies showed that incorporating NE-WX, a research CBC parameter related to neutrophil complexity, and IPF, an immature platelet fraction parameter, into a conventional CBC analysis helps identify patients with high likelihood of MDS (PMID 30406952) or JAK2-positive polycythemia vera (PMID 30601597), respectively. Our exploratory analysis was focused on using all research CBC parameters in combination with standard CBC parameters and mutational data to compare control patients to those with active myeloid disease, and MDS in particular. Methods: We measured standard and research CBC parameters (92 parameters overall) using the Sysmex XN series hematology analyzer in a cohort of 592 consecutive patients (pts), out of which 221 had abnormal CBC due to an underlying myeloid neoplasm (MDS 83 pts, MPN 54 pts, MDS/MPN 24 pts, acute myeloid leukemia 21 pts, clonal or non-clonal cytopenia or cytoses 33 and 6 pts, respectively) and 371 age-matched control patients with normal CBC. The former group was tested for the presence of somatic mutations in 95 genes associated with hematologic malignancies and included patients with active disease (n=127) and in remission (n=94). The MDS cohort included 59 patients with active disease and 24 patients in remission. We developed a variety of models (Linear and Logistic Regression, Decision Trees, Random Forests, Neural Networks, Support Vector Machines, Gradient Boosting) to predict the presence of an active myeloid neoplasm or active MDS using both CBC and molecular data, as well as CBC data alone. These models were compared using a leave one patient out cross-validation method and a champion model was chosen. Correlation matrix analysis was used for establishing relationships between MDS-associated mutations and CBC parameters, where a relevant association was considered at the levels of above 0.25 or below -0.25. Results: Table 1 lists the leave one out performance of the champion predictive models for the active myeloid and MDS cohorts based on different variables (all CBC parameters, mutations, age and gender), along with their corresponding sensitivity and specificity. Table 2 shows the most discriminatory research CBC parameters in the MDS cohort reflecting dysplastic changes in neutrophil morphology (NE-SFL, NE-WY, NE-WX, NE-FSC, NE-SSC) and degrees of cytopenia (increased IPF and decreased TNC). The standard parameters included Hgb, MCV, MCH, WBC and absolute neutrophil count. Several mutations showed a strong association with certain CBC parameters in the MDS cohort. ASXL1 was associated with lymphocyte complexity (LY-X; 0.38), TNC (0.28), WBC (0.28) and neutrophil (0.27) count and lower immature reticulocytes fraction (-0.67), RBC (-0.30) and reticulocyte (-0.31) counts, similar to the findings of the recently published mouse model expressing ASXL1 truncating protein (PMID 29113963), while DNMT3A and SF3B1 mutations showed higher platelet correlations (0.32). In addition, the latter was associated with lower neutrophil granularity (-0.32), cell size (-0.32) and nuclear complexity (-0.39). TP53 showed positive association with neutrophil and lymphocyte parameters reflecting population heterogeneity (0.26-0.40). Conclusions: Our exploratory study demonstrated that the Sysmex XN research parameters, which are measured with all routine CBCs, may be used in patients with abnormal CBC to build a predictive model with high specificity to rule out a clonal myeloid neoplasm, including MDS. The model's performance improves with the addition of the molecular status as a variable, while it appears to be independent of age and gender. In the entire MDS cohort, the most relevant research parameters in the predictive model are reflective of dysplastic neutrophil morphology and can be objectively measured. In addition, it appears that there may be unique CBC parameter signatures associated with an underlying mutation. Disclosures Pozdnyakova: Sysmex corporation of America: Research Funding. Niculescu:Sysmex America, Inc: Consultancy. Kroll:Sysmex America, Inc: Consultancy. Kim:Quanterix, Inc: Consultancy; Papgene, Inc: Consultancy; LabCorp, Inc: Consultancy.

47-Year-Old Man With Pruritus

47-year-old man with a medical history of headaches and seasonal allergies presented to the primary care clinic in late fall for evaluation of diffuse pruritus of 6 weeks’ duration that had occurred after initiating a new diet. The diet mostly restricted foods like dairy, soy, nuts, grains, and processed foods, but he stated that he had substantially increased his consumption of coconut and various spices including turmeric. The itching was present over most of his body but most prominent on the inner thighs and most notable after a hot shower. He provided picturesfromrecent episodes thatillustrated linear excoriations on the thighs. When he showered in cooler water, the diffuse pruritus was still present but minimally improved compared with a hot shower. He had been using the same body soap for years but tried a new soap without relief from the itching. In addition, he changed laundry detergent from one he had used for years with no relief. He had no occupational or habitual exposures that are suspected to contribute to pruritus. Overthe-counter hydrocortisone cream applied to his thighs provided minimal relief. His weight had been stable, and he reported no constitutional symptoms. On physical examination, the patient was a

Cardiovascular Events and Intensity of Treatment in Polycythemia Vera

BackgroundCurrent treatment recommendations for patients with polycythemia vera call for maintaining a hematocrit of less than 45%, but this therapeutic strategy has not been tested in a randomized clinical trial.MethodsWe randomly assigned 365 adults with JAK2-positive polycythemia vera who were being treated with phlebotomy, hydroxyurea, or both to receive either more intensive treatment (target hematocrit, <45%) (low-hematocrit group) or less intensive treatment (target hematocrit, 45 to 50%) (high-hematocrit group). The primary composite end point was the time until death from cardiovascular causes or major thrombotic events. The secondary end points were cardiovascular events, cardiovascular hospitalizations, incidence of cancer, progression to myelofibrosis, myelodysplasia or leukemic transformation, and hemorrhage. An intention-to-treat analysis was performed.ResultsAfter a median follow-up of 31 months, the primary end point was recorded in 5 of 182 patients in the low-hematocrit group (2.7%) and 18 of 183 patients in the high-hematocrit group (9.8%) (hazard ratio in the high-hematocrit group, 3.91; 95% confidence interval [CI], 1.45 to 10.53; P=0.007). The primary end point plus superficial-vein thrombosis occurred in 4.4% of patients in the low-hematocrit group, as compared with 10.9% in the high-hematocrit group (hazard ratio, 2.69; 95% CI, 1.19 to 6.12; P=0.02). Progression to myelofibrosis, myelodysplasia or leukemic transformation, and bleeding were observed in 6, 2, and 2 patients, respectively, in the low-hematocrit group, as compared with 2, 1, and 5 patients, respectively, in the high-hematocrit group. There was no significant between-group difference in the rate of adverse events.ConclusionsIn patients with polycythemia vera, those with a hematocrit target of less than 45% had a significantly lower rate of cardiovascular death and major thrombosis than did those with a hematocrit target of 45 to 50%. (Funded by the Italian Medicines Agency and others; ClinicalTrials.gov number, NCT01645124, and EudraCT number, 2007–006694-91.)

Minimal Residual Disease and Normalization of the Bone Marrow after Long-Term Treatment with Alpha-Interferon2b in Polycythemia Vera. A Report on Seven Patients in Sustained Complete Hematological Remission with Major Molecular Responses.

Background : Polycythemia vera (PV) is a clonal myeloproliferative disorder characterized by the presence of the JAK2V617 mutation in virtually all patients. Recently several studies have shown that the JAK2V617F mutational load decreases during treatment with alpha-interferon2 (1–6).Aim: To report on molecular and histomorphological bone marrow responses in seven PV patients with complete molecular remissions during and after long-term treatment with alpha-interferon 2b.Patients: Seven patients treated with alpha-interferon2b for a median of 84 months (range 31–120) are reported. In four of the patients alpha-interferon2b was started at the time of diagnosis and in three patients 9, 36 and 42 months from the time of diagnosis, respectively.Methods: The mutation was determined by allele specific PCR (n=2 only) (7) and quantitative PCR (qPCR) (n=5) (8). In three out of these patients qPCR JAK2V617F was performed on archived bone marrow from diagnosis (2 patients) and on peripheral blood (one patient) prior to treatment with alpha-interferon2b. A complete molecular remission (CMoR) was defined by less than 2 % JAK2 V617F mutated alleles (7).Results: Molecular Responses. All patients obtained a CMoR after a median of 84 months (29–120 months) of treatment with alpha-interferon2b. Subsequently all patients have discontinued alpha-interferon with a follow-up period of median 10 months (range 4–30 months) and sustained complete hematological remission. Furthermore, in three patients molecular responses have recently been updated – April and May 2008 - showing CMoRs in all (1,2 %, 0,9 % 0,1 % mutated alleles, respectively).Bone Marrow Responses. Follow-up bone marrow biopsies were available in five patients. Complete normalization of the bone marrow was seen in three patients after treatment with alpha-interferon2b for 84, 132 and 132 months, respectively. In the bone marrow from the patient being treated with alpha-interferon for 132 months a qPCR JAK2V617 analysis was performed detecting the mutation at a very low level (0,5 % mutated alleles). In two other patients, being treated with alpha-interferon2b for 24 and 120 months, respectively, and having obtained a CmoR in peripheral blood the bone marrow histomorphology showed marked regression of PV-features but in both patients still with focal areas displaying an increased number of morphologically abnormal megakaryocytes. Updated histomorphological and molecular response patterns will be presented.Discussion and Conclusion : Previous studies on the molecular response during alpha-interferon2a treatment have shown that a substantial proportion of patients achieve a significant molecular response after 12 months with a continuous decrease in the JAK2V617F mutation load at 24 and 36 months (1,5,6). This report confirms and extends preliminary data, showing that long-term treatment with alpha-interferon 2b in a subgroup of PV-patients is able to induce complete molecular remissions with normalization of the bone marrow morphology, which may even be sustained after discontinuation of alpha-interferon2b for up to 20 months (5). Prolonged treatment for several years seems necessary to induce such sustained responses, since treatment for only a few months has been reported to be followed by rapid recurrence of clonal hematopoiesis (9). In conclusion, a state of “minimal residual disease” may be achieved in PV by long-term immune therapy using alpha-interferon 2. Our observations call for large prospective clinical studies in which treatment with alpha-interferon is initiated up-front in patients with JAK2-positive PV and allied diseases. These studies should also aim at exploring the minimal dose of alpha-interferon needed to elicit complete molecular responses in order to minimize side effects of the drug and accordingly diminish the high drop-out rates reported in most previous studies.

Low Prevalence of JAK2 Val617Phe Mutation in Patients with Idiopathic Erythrocytosis.

Idiopathic Erythrocytosis (IE) is a frequent clinical condition (estimated prevalence 1.1 cases every 1,000 normal subjects), characterized by an absolute increase of red blood cell mass, without the diagnostic features of polycythemia vera (PV), secondary polycythemias and congenital erythrocytosis. The clinical course of IE is marked by a lower rate of thrombotic complications than observed in PV and a very low, if any, spontaneous transition to acute leukemia. The differential diagnosis between IE and PV may be sometimes difficult but it is clinically relevant, also because cytotoxic drugs, frequently used in PV, should be avoided in IE. The recent description of a Val617Phe mutation in the exon 12 of JAK2 gene in 74–97% of patients with PV may represent a reliable molecular marker to differentiate PV from IE. However, the prevalence of JAK2 mutation in patients with IE is presently unknown. For this reason, we evaluated JAK2 mutation on granulocyte DNA or RNA using an allele-specific PCR in 31 patients with IE (29 males, 2 females, median age 46 years, range 19–74) and results were compared with those of 31 patients with overt PV (16 males and 15 females, median age 56 years, range 34–74). PV was diagnosed according to the WHO criteria. At variance, cases with IE were identified by increased hematocrit (median 54%, range 50–61%) and red blood cell mass (>25% above mean normal predicted value), but normal leukocyte and platelet counts, normal arterial O2 saturation and chest X-ray, no splenomegaly by abdominal ultrasound scanning and normal erythropoietin level and PRV-1 expression on peripheral blood granulocytes. Both IE and PV patients were either newly diagnosed or established cases in follow-up. Median follow-up was 5.5 years (range 0–20) in IE and 4.1 years (range 0–17) in PV patients respectively. Twenty-eight patients with PV (90%) but only 3 with IE (10%) showed the JAK2 Val617Phe mutation (p<0.001). In 3 patients with PV (10%) but none with IE, the JAK2 mutation was homozygous. The annual incidence of thrombotic complications was 3.2% in PV vs. 0.85% patient-year in IE (p<0.05). Our data confirm the hypothesis that JAK2 mutation is an useful molecular test to distinguish IE from PV in the great majority of patients with absolute erythrocytosis. Patients with “JAK2negative IE” require a different therapeutic approach than those with the classical “JAK2 positive PV”, since the incidence of thrombosis appears to be different. The few patients (about 10%) with discordant molecular and clinical diagnosis (i.e. “JAK2positive IE” or “JAK2 negative PV”) deserve particular attention. The long period of observation of our “JAK2positive IE” patients (3, 8 and 20 years, respectively) seems to exclude that these cases may represent an early phase of PV. However, whether this molecular marker confers a more aggressive or different clinical behaviour in a small subgroup of IE patients remains to be demonstrated in larger prospective studies.