Adam Cuker

MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia

Background The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR). Methods and Findings DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9–4.0 × 10 12/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis. Conclusions Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.

Predictive value of the 4Ts scoring system for heparin-induced thrombocytopenia: a systematic review and meta-analysis

The 4Ts is a pretest clinical scoring system for heparin-induced thrombocytopenia (HIT). Although widely used in clinical practice, its predictive value for HIT in diverse settings and patient populations is unknown. We performed a systematic review and meta-analysis to estimate the predictive value of the 4Ts in patients with suspected HIT. We searched PubMed, Cochrane Database, and ISI Web of Science for studies that included patients with suspected HIT, who were evaluated by both the 4Ts and a reference standard against which the 4Ts could be compared. Quality of eligible studies was assessed by QUADAS-2 criteria. Thirteen studies, collectively involving 3068 patients, fulfilled eligibility criteria. A total of 1712 (55.8%) patients were classified by 4Ts score as having a low probability of HIT. The negative predictive value of a low probability 4Ts score was 0.998 (95% CI, 0.970-1.000) and remained high irrespective of the party responsible for scoring, the prevalence of HIT, or the composition of the study population. The positive predictive value of an intermediate and high probability 4Ts score was 0.14 (0.09-0.22) and 0.64 (0.40-0.82), respectively. A low probability 4Ts score appears to be a robust means of excluding HIT. Patients with intermediate and high probability scores require further evaluation.

The HIT Expert Probability (HEP) Score: a novel pre‐test probability model for heparin‐induced thrombocytopenia based on broad expert opinion

Summary.  Background: The diagnosis of heparin‐induced thrombocytopenia (HIT) is challenging. Over‐diagnosis and over‐treatment are common. Objectives: To develop a pre‐test clinical scoring model for HIT based on broad expert opinion that may be useful in guiding clinical decisions regarding therapy. Patients/methods: A pre‐test model, the HIT Expert Probability (HEP) Score, was constructed based on the opinions of 26 HIT experts. Fifty patients referred to a reference laboratory for HIT testing comprised the validation cohort. Two hematology trainees scored each patient using the HEP Score and a previously published clinical scoring system (4 T’s). A panel of three independent experts adjudicated the 50 patients and rendered a diagnosis of HIT likely or unlikely. All subjects underwent HIT laboratory testing with a polyspecific HIT ELISA and serotonin release assay (SRA). Results: The HEP Score exhibited significantly greater interobserver agreement [intraclass correlation coefficient: 0.88 (95% CI 0.80–0.93) vs. 0.71 (0.54–0.83)], correlation with the results of HIT laboratory testing and concordance with the diagnosis of the expert panel (area under receiver‐operating curve: 0.91 vs. 0.74, P = 0.017) than the 4 T’s. The model was 100% sensitive and 60% specific for determining the presence of HIT as defined by the expert panel and would have allowed for a 41% reduction in the number of patients receiving a direct thrombin inhibitor (DTI). Conclusion: The HEP Score is the first pre‐test clinical scoring model for HIT based on broad expert opinion, exhibited favorable operating characteristics and may permit clinicians to confidently reduce use of alternative anticoagulants. Prospective multicenter validation is warranted.

Epigenetic abnormalities associated with a chromosome 18(q21-q22) inversion and a Gilles de la Tourette syndrome phenotype

Gilles de la Tourette syndrome (GTS) is a potentially debilitating neuropsychiatric disorder defined by the presence of both vocal and motor tics. Despite evidence that this and a related phenotypic spectrum, including chronic tics (CT) and Obsessive Compulsive Disorder (OCD), are genetically mediated, no gene involved in disease etiology has been identified. Chromosomal abnormalities have long been proposed to play a causative role in isolated cases of GTS spectrum phenomena, but confirmation of this hypothesis has yet to be forthcoming. We describe an i(18q21.1-q22.2) inversion in a patient with CT and OCD. We have fine mapped the telomeric aspect of the rearrangement to within 1 Mb of a previously reported 18q22 breakpoint that cosegregated in a family with GTS and related phenotypes. A comprehensive characterization of this genomic interval led to the identification of two transcripts, neither of which was found to be structurally disrupted. Analysis of the epigenetic characteristics of the region demonstrated a significant increase in replication asynchrony in the patient compared to controls, with the inverted chromosome showing delayed replication timing across at least a 500-kb interval. These findings are consistent with long-range functional dysregulation of one or more genes in the region. Our data support a link between chromosomal aberrations and epigenetic mechanisms in GTS and suggest that the study of the functional consequences of balanced chromosomal rearrangements is warranted in patients with phenotypes of interest, irrespective of the findings regarding structurally disrupted transcripts.

Reversal of novel oral anticoagulants in patients with major bleeding

Novel oral anticoagulants (NOACs) that directly inhibit thrombin (dabigatran) or factor Xa (rivaroxaban, apixaban, edoxaban) are effective therapies for the prevention and treatment of thromboembolism with reduced bleeding complications compared with warfarin for some indications. However, specific antidotes to reverse the anticoagulant activity of NOACs in the event of major bleeding are not available. Evidence supporting non-specific prohemostatic therapies (prothrombin complex concentrate [PCC], activated prothrombin complex concentrate [aPCC], recombinant factor VIIa) in this setting is limited to healthy human volunteers, animal models, and in vitro studies. Clinical outcome data are lacking. Administration of PCC or aPCC may be considered in addition to supportive measures for patients with severe or life-threatening bleeding. Clinical studies are needed to establish the efficacy and safety of these treatments. Target-specific antidotes are in development and hold promise for NOAC reversal, but require further investigation.

A distinctive form of immune thrombocytopenia in a phase 2 study of alemtuzumab for the treatment of relapsing-remitting multiple sclerosis

In a phase 2 clinical trial of annual alemtuzumab for treatment of relapsing-remitting multiple sclerosis, 6 of 216 patients (2.8%) developed immune thrombocytopenia (ITP). Over mean follow-up of 4.5 years, the incidence rate of ITP was 6.2 (95% confidence interval, 2.3-13.3) per 1000 person-years. Median times from initial and last alemtuzumab exposure to ITP diagnosis were 24.5 and 10.5 months, respectively. Five patients developed severe thrombocytopenia. Four were symptomatic, including fatal intracranial hemorrhage in the index case. Four patients received standard first-line ITP therapy, all of whom responded to treatment within 1 week. All 5 surviving patients achieved complete remission and remained in complete remission without need for ongoing ITP therapy for a median duration of 34 months at last follow-up. A monitoring plan for the early detection of ITP, implemented after presentation of the index case, identified all 5 subsequent cases before serious hemorrhagic morbidity or mortality occurred. In conclusion, we describe a distinctive form of ITP associated with alemtuzumab treatment characterized by delayed presentation after drug exposure, responsiveness to conventional ITP therapies, and prolonged remission. Clinicians should maintain a high level of vigilance and consider routine monitoring for ITP in patients treated with this agent. This trial was registered at www.clinicaltrials.gov as #NCT00050778.

Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant

Background The prevention of bleeding with adequately sustained levels of clotting factor, after a single therapeutic intervention and without the need for further medical intervention, represents an important goal in the treatment of hemophilia. Methods We infused a single‐stranded adeno‐associated viral (AAV) vector consisting of a bioengineered capsid, liver‐specific promoter and factor IX Padua (factor IX–R338L) transgene at a dose of 5×1011 vector genomes per kilogram of body weight in 10 men with hemophilia B who had factor IX coagulant activity of 2% or less of the normal value. Laboratory values, bleeding frequency, and consumption of factor IX concentrate were prospectively evaluated after vector infusion and were compared with baseline values. Results No serious adverse events occurred during or after vector infusion. Vector‐derived factor IX coagulant activity was sustained in all the participants, with a mean (±SD) steady‐state factor IX coagulant activity of 33.7±18.5% (range, 14 to 81). On cumulative follow‐up of 492 weeks among all the participants (range of follow‐up in individual participants, 28 to 78 weeks), the annualized bleeding rate was significantly reduced (mean rate, 11.1 events per year [range, 0 to 48] before vector administration vs. 0.4 events per year [range, 0 to 4] after administration; P=0.02), as was factor use (mean dose, 2908 IU per kilogram [range, 0 to 8090] before vector administration vs. 49.3 IU per kilogram [range, 0 to 376] after administration; P=0.004). A total of 8 of 10 participants did not use factor, and 9 of 10 did not have bleeds after vector administration. An asymptomatic increase in liver‐enzyme levels developed in 2 participants and resolved with short‐term prednisone treatment. One participant, who had substantial, advanced arthropathy at baseline, administered factor for bleeding but overall used 91% less factor than before vector infusion. Conclusions We found sustained therapeutic expression of factor IX coagulant activity after gene transfer in 10 participants with hemophilia who received the same vector dose. Transgene‐derived factor IX coagulant activity enabled the termination of baseline prophylaxis and the near elimination of bleeding and factor use. (Funded by Spark Therapeutics and Pfizer; ClinicalTrials.gov number, NCT02484092.)

Heparin-induced thrombocytopenia (HIT) in 2011: An epidemic of overdiagnosis

Heparin-induced thrombocytopenia (HIT) is a prothrombotic and potentially fatal iatrogenic disorder mediated by IgG antibodies that target multimolecular complexes of platelet factor 4 (PF4) and heparin (1). Although the first description of the disorder dates back more than 50 years (2), it was not until the 1990s that the target antigen in HIT was identified and the now widely used anti-PF4/heparin ELISA was developed (1), a detailed clinical characterisation of the disorder emerged, and the first effective therapies for HIT gained regulatory approval. Little wonder, then, that a major thrust of medical training, and the literature of the day was to raise clinicians’ awareness of this nascent disorder. Papers entitled “Think of HIT” and “Don’t miss HIT” exhorted physicians to recognise the disease early in its course and initiate therapy promptly (3–4). Perhaps owing to the success of this educational campaign, the last several years have witnessed a sea change in the diagnostic landscape. The major clinical problem in 2011 is no longer under-recognition of HIT, but over-diagnosis and over-treatment. Among a recently reported cohort of patients managed with a direct thrombin inhibitor (DTI) for HIT at the University of Pennsylvania, 41% received treatment despite a very low clinical probability of disease as judged post hoc by a panel of expert adjudicators (5). Over a recent 12-month interval at the same institution, 91 of 100 DTItreated patients were ultimately determined to have a negative result by serotonin release assay (SRA), the gold standard laboratory test for HIT (unpublished data). Over-diagnosis is particularly rampant in the intensive care units and the post-cardiac surgery setting, where the frequency of alternative anticoagulant use outstrips the incidence of HIT by an order of magnitude or more (6–8). With exposure of large numbers of thrombocytopenic patients to costly alternative anticoagulants and their attendant 10–20% risk of major bleeding, the clinical and economic impact of such over-diagnosis and over-treatment is likely to be substantial. Thrombotic complications may also arise in this population as a result of inappropriate withholding of heparin (9). Why is over-diagnosis of HIT so pervasive? The answer likely lies in the poor specificity of clinical diagnosis and the anti-PF4/heparin ELISA, the diagnostic tools most readily available to clinicians. The cardinal clinical manifestation of HIT, a fall in the platelet count in the setting of a proximate heparin exposure, is an exceedingly common finding among hospitalised patients for which alternative explanations often exist. In a multicenter registry of 2,420 hospitalised patients receiving heparin for four or more consecutive days, fully 36.4% developed thrombocytopenia during their hospital course, though the incidence of HIT in this largely medical population was likely on the order of only 1–2% (10). Scoring models lend a degree of standardisation to clinical diagnosis, but also suffer from limited specificity (5, 11). The anti-PF4/heparin ELISA fares little better than clinical diagnosis in this regard. False-positive results are common and may result from detection of non-pathogenic antibodies or antiphospholipid antibodies directed against PF4 (12, 13). A review of one institution’s experience suggested that the ELISA was associated with the potential to over-diagnose HIT by as much as 100% (12). Use of an IgG-specific ELISA, a higher optical density cut-off, or the high heparin confirmatory procedure improves specificity, but false-positive results remain common and false-negative results may also occur (14–16). Moreover, many institutions perform the ELISA in batch once or twice a week, often leaving clinicians to make initial management decisions without the benefit of laboratory results. Turnaround time is an even greater problem for more specific functional assays such as the SRA, which are performed at a small number of reference laboratories around the world and are only available as send-out tests to the vast majority of providers. In light of the limitations of available diagnostic tools, the novel anti-PF4/polyanion IgG-specific lateral-flow immunoassay (LFI) reported by Sachs et al. in this issue of Thrombosis and Haemostasis offers a ray of hope (17). The authors compared the LFI to two IgG-specific ELISAs and a particle gel immunoassay (PGIA) using samples from 452 patients consecutively referred to a reference laboratory for HIT testing. Clinical information was supplied by the referring providers and scored using the 4Ts system (11) by two investigators, who were blinded to the results of laboratory testing. HIT was defined as an intermediate or high clinical probability 4Ts score in conjunction with a positive heparin-induced platelet activation (HIPA) assay, a functional test with favourable operating characteristics similar to those of the SRA. Of the 34 patients that met the protocol definition for HIT, all tested positive by LFI using a visual inspection endpoint as well as by the two IgG-specific ELISAs. Three HIT-positive patients were missed by the PGIA. In addition, the LFI was associated with a significantly smaller number of false-positive results than the ELISAs and PGIA (29 vs. 45–55). While these observa-

Clinical and laboratory diagnosis of heparin-induced thrombocytopenia: an integrated approach.

Heparin-induced thrombocytopenia (HIT) is a clinicopathologic disorder that predisposes to thrombosis. Diagnosis rests on a compatible clinical picture and laboratory evidence of antiplatelet factor 4 (PF4)/heparin antibodies that activate platelets in a heparin-dependent manner. Rapid and accurate diagnosis is paramount to avoid the perils of misdiagnosis. Clinical evaluation may be guided by scoring systems such as the 4Ts and HIT Expert Probability (HEP) score. Laboratory tests include immunoassays, such as the PF4/heparin enzyme-linked immunosorbent assay (ELISA) and functional tests such as the 14C-serotonin release assay and heparin-induced platelet activation assay. Clinical scoring systems and commercially available immunoassays have high sensitivity but modest specificity. Functional assays are more specific, but they are technically demanding. Novel laboratory assays with faster turnaround times, greater specificity, and lesser technical complexity are in development. A Bayesian approach that combines the 4T score and the PF4/heparin ELISA result may be used to estimate the probability of HIT and guide clinical decision making.

Pathogenesis of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by antibody-mediated platelet destruction. The platelet, as an accessible target, has made ITP an attractive disorder in the study of autoimmunity. However, the pathogenesis of ITP has proven complex with diverse pre-existing challenges to the immune system in the form of infection, genetic predisposition, underlying autoimmune repertoire, inhibition of platelet production, perturbations of cell mediated affector and effector pathways, sequestered harbors within lymphoid organs, and responsiveness to intervention. This chapter surveys key new insights into the pathogenesis of ITP and attempts to integrate them into a model that may serve as a template for future investigation.