Primary Trombocythemia in Children and Adolescents Includes Different Subtypes Compared to Adult Essential Thrombocythemia

The recent discovery of various mutations of the CALR gene that are mutually exclusive with JAK2 and MPL mutations has allowed a correct diagnosis in about 90% of adult cases of essential thrombocythemia (ET). Moreover, the mutation status of JAK2 and CARL defines subtypes of ET in adults with a substantially different clinical course and outcome. Based on our experience, we suggested that primary thrombocythemia (PT) in children is characterized by subtypes that differ from those found in adult ET. The present study was carried out in children and adolescents with PT in order to (a) characterize the various subtypes of the disease and (b) analyze their clinical and biologic features, treatment approach and outcome. PT patients aged <20 years (yrs) at diagnosis (dx) were evaluated for mutations of JAK2, thrombopoietin (TPO) and its receptor (MPL) and CALR genes, and for clonal hematopoiesis (females). The presence of MPLS505A (confirmed on DNA from buccal swabs) defined a hereditary thrombocytosis (HT). ET was diagnosed according to WHO 2008 criteria. For wild type patients, an additional inclusion criteria was a follow-up >24 months. Among 58 PT patients (males: 23; females: 35; median age at dx: 14.4 yrs), 21 (36%) had HT due to MPLS505A, 14 were JAK2V617F-mutated (24%), 9 (16%) harbored CALR mutations and 14 (24%) were wild type for JAK2, CALR and MPL (Fig 1). JAK2- and CALR-mutated were older than those with wild type ET or with HT (median age, 17.6 and 16.1 vs 10.4 and 13.7 yrs, p .028). As to the hematologic findings, HT patients showed both hematocrit values (median, 36.3%) and leukocytes counts (median, 9.53 x109/L) significantly lower than ET patients, whatever the subtypes (median, 41.2% and 11.2 x109/L, p .006 and p .029, respectively). No differences were found with regard to platelets both between HT and ET and among the different ET subtypes. JAK2-mutated patients exhibited more frequently symptoms (69%) compared to CALR-mutated (22%), wild-type ET (14%) and HT (14%) patients (p. 0057). Splenomegaly at diagnosis was recorded more frequently in JAK2-mutated than in CALR-mutated or wild type-ET or HT (50%, 33% 21% and 14% , respectively, p .122). Antiplatelet agents, mostly acetylsalicylic acid (ASA), were started less frequently in HT than in ET patients, irrespective of the subtypes (57% vs 81%, p .05). The use of ASA progressively decreased over the time; at the last follow-up, 2 patients with HT, 2 CALR-mutated and 1 JAK2-mutated patients were still receiving ASA, while no wild type ET patient was on treatment. Cytoreductive agents, hydroxyurea and/or interferon and/or anagrelide, were used in a minority of HT patients (19%) in comparison with ET patients (65%), p .001, mainly with those wild-type (78%, p <.001). At the last observation, one HT patient was still receiving cytoreductive agents compared to 30% of ET patients whatever the subtypes (p .024). After a median follow-up of 196 months (similar in the different subtypes), all patients are alive. On the whole, 5 thrombotic events were recorded in 3 patients with HT and in 2 ET patients (1 JAK2-mutated and 1 JAK2 and CALR wild-type), without any significant thrombophilic abnormalities during treatment with ASA and/or cytoreductive agents. A progressive splenomegaly was recorded in 9 (15%) patients (2 HT, 4 JAK2-mutated, 3 CALR-mutated) and it was combined with grade ≥2 medullar fibrosis in 2/4 JAK2-mutated and in 2/3 CALR-mutated patients. None of the JAK2 and CALR wild-type patients had spleen enlargement or reticulin fibrosis (p .022). Two untreated patients (1 HT and JAK2 and CALR wild-type) developed malignancies. On the whole, these data emphasize that in young patients with PT, hereditary forms can be frequently observed. Thrombotic events, recorded mainly in HT patients despite treatment with ASA, were probably due to a MRP4 protein overexpression that was found in our MPLS505A HT. Moreover, our observations highlight that, in contrast to adult ET, more than one third of young ET patients have no JAK2 or CALR mutations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

MRP4 Expression and Platelet Activation in Children and Adolescents with Different Subtypes of Primary Thrombocythemia

Background and aims. Essential thrombocythemia (ET) rarely occurs in children and adolescents. In our experience, 40% of pediatric patients with primary thrombocythemia (PT) have JAK2 V617F or CALR mutations, 24% have a diagnosis of ET without any known molecular markers while 36% have hereditary thrombocytosis (HT) with a MPLS505A mutation. Thrombotic events, frequent in adult ET presenting high-risk factors and rare in pediatric PT, have been observed in pediatric HT patients with MPLS505A mutation in treatment with aspirin (ASA). The multidrug resistance protein-4 (MRP4) is an ATP-binding cassette transporter involved in the efflux of several pharmacological and physiological compounds. MRP4 has been identified as a modulator of ASA action in platelets; in addition, it also influences platelet activation. Recent studies have shown that MRP4 over-expression has a role in reducing the effect of ASA in patients who have undergone a bypass surgical procedure and that ASA induces platelet MRP4 upregulation. Aims of this study were to evaluate and correlate MRP4 expression and platelet function in children and adolescents aged <20 years at diagnosis with different subtypes of PT. Methods. MRP4 protein and mRNA expressions were evaluated in platelets obtained from healthy volunteers (HV) and from 41 PT patients: M/F ratio: 0.78; median age at diagnosis: 14.5 years; median platelet count at study: 671x109/L. Ten patients had MPLS505A mutation (HT), 10 were JAK2V617F-mutated (ET) and 6 harbored CALR mutations (ET) while 15 cases were JAK2, CALR and MPL wilde-type (ET). Expression of MRP4 protein and mRNA were analyzed by Western blot and RT-PCR, respectively, and the results were reported as ET/HV or HT/HV expression values. Platelet aggregation, using ADP at different concentration (0.8 to 2 μM) and collagen (1 μg/ml) as agonists, and platelet secretion, expressed as ATP release after U46619 + epinephrine (1 + 20 μM), using the luciferin-luciferase assay, were utilized as platelet function tests. This study was conducted in accordance with the Declaration of Helsinki. Results. Protein MRP4 expression was higher both in HT (4.23+/-1.88) and in ET patients with CALR mutations (4.27+/-2.60) compared to the values found in wild-type ET patients (3.55+/-1.52). In JAK2V617F-mutated ET patients, the MRP4 protein expression (2.03+/-1.46) was significantly lower compared to the values observed in all other ET and HT patients (p .05), whereas the MRP4 mRNA expression was significantly higher (ΔΔCt 0.021+0.003) compared both to HT patients (0.009+/-0.0039 ΔΔCt) (p .01) and CALR-mutated patients (ΔΔCt 0.014+/- 0.006). Patients with HT showed a significantly higher response to ADP 0.8 µM (83+/-29 Mx%) compared to all subgroups of ET patients who showed a similar response (60+/-34 Mx%; p .01). A significantly shorter lag-phase in response to collagen (1 µg/ml) was observed in HT compared to ET patients (33+/-3 sec vs 51+/-4 sec, p .005). Among the ET population, JAK2-mutated patients showed a significantly shorter lag-phase in response to collagen compared to wild-type patients (39+/-12.5 sec vs 50+/-15 sec), p .011. Platelet secretion was significantly higher in HT compared to ET patients, p .001. Conclusions. This study for the first time provides evidence that children and adolescents with MPLS505A-mutated HT show a higher platelet reactivity compared to age-matched patients with ET. Moreover, platelet reactivity correlates with MRP4 protein overexpression. These findings help to shed light into the thrombotic events observed in HT MPLS505A patients despite treatment with ASA. Disclosures No relevant conflicts of interest to declare.

Sub-Optimal Inhibition of Thrombus Formation by Aspirin (as measured by RTTP analysis) In Primary Thrombocythemia

Abstract 4218 Primary thrombocythemia (PT) is a myeloproliferative disease with a high incidence of thrombotic complications. First-line therapy is the inhibition of platelet aggregation with aspirin (via COX-1 inhibition) for the prevention of thromboembolic complications. However, this does not account for the potential for COX-2 expression in newly synthesized platelets. The purpose of the present study was 1) to determine whether aspirin reduces the thrombotic potential of PT patients who did not undergo cytoreductive therapy to the levels of those achieved in healthy volunteers (HV), and 2) to determine whether the presence of the JAK2V617F mutation is responsible for the elevated thrombotic potential in PT patients. We determined the thrombotic potential of a cohort of 16 PT patients and compared these values to those of 10 healthy volunteers all treated with 100mg aspirin QD. Thrombotic potential was determined using a custom built Real Time Thrombosis Profiler (RTTP). Whole blood anticoagulated with a Factor Xa inhibitor (to preserve physiological Ca++ concentration) with Rhodamine 6G-labeled platelets was perfused over a fibrillar collagen surface at shear rates approximating those in veins (100s-1), arteries (600 s-1) and moderately stenosed arteries (1600s-1). The deposition of fluorescently labeled platelets on the collagen surface was monitored in real time by fluorescence microscopy in the RTTP. Endpoint thrombosis (size of thrombi at t=300sec expressed as Mean Fluorescent Intensity/Area of coverage) and rate of thrombus growth (initial rate of platelet deposition) were recorded for each individual as measures of thrombotic potential and data are expressed as mean ± SEM (statistics performed using GraphPad Prism v4.03 using unpaired, 2-tailed Students t-test). Despite aspirin therapy, PT patients were characterized by significant increases in thrombotic potential at venous and arterial shear rates (Table 1) with a greater than 2-fold increase in the rate of platelet deposition. Interestingly only the size of thrombi formed but not the rate of formation was elevated in PT patients at moderately stenosed shear, likely a reflection of the increasing contribution of shear and decreasing dependence on absolute platelet count and other individual cellular factors in whole blood. The Disclosures: No relevant conflicts of interest to declare.