Key Points: Measurement of platelet diameters in 376 patients resulted in a new classification of inherited thrombocytopenias based on platelet size. Measurement of platelet diameters is a useful tool for differential diagnosis of inherited thrombocytopenias.

ETV6 -related thrombocytopenia ( ETV6 -RT) is an autosomal dominant thrombocytopenia that has been recently identified in a few families and has been suspected to predispose to hematological malignancies. To gain further information on this disorder, we searched for ETV6 mutations in the 130 families with inherited thrombocytopenia of unknown origin from our cohort of 274 consecutive pedigrees with familial thrombocytopenia. We identified 20 ETV6 -RT patients from 7 pedigrees. They have 5 different ETV6 variants, including three novel mutations affecting the highly conserved E26 transformation-specific domain. The relative frequency of ETV6 -RT resulted 2.6% in the whole case series and 4.6% among the families with known forms of inherited thrombocytopenia. The degree of thrombocytopenia and bleeding tendency of ETV6 -RT patients were mild, but 4 subjects developed B-cell acute lymphoblastic leukemia during childhood, resulting in a significantly increased incidence compared to the general population. Clinical and laboratory findings did not identify any peculiar defects that can be used to suspect this disorder by routine diagnostic workup. However, at variance with most inherited thrombocytopenias, platelet size was not enlarged. In vitro studies revealed that patients megakaryocytes have defective maturation and impaired proplatelet formation. Moreover, ETV6 -RT platelets have reduced ability to spread on fibrinogen. Since also the dominant thrombocytopenias due to mutations in RUNX1 and ANKRD26 are characterized by normal platelet size and predispose to hematological malignancies, we suggest that mutation screening of ETV6 , RUNX1 and ANKRD26 should be performed in all the subjects with autosomal dominant thrombocytopenia and normal platelet size.

Patients with inherited thrombocytopenias often require platelet transfusions to raise platelet count before surgery or other invasive procedures; moreover, subjects presenting clinically significant spontaneous bleeding may benefit from an enduring improvement of thrombocytopenia. The hypothesis that thrombopoietin-mimetics can increase platelet count in inherited thrombocytopenias is appealing, but evidence is scarce. We conducted a prospective, phase 2 clinical trial to investigate the efficacy of the oral thrombopoietin-mimetic eltrombopag in different forms of inherited thrombocytopenia. We enrolled 24 patients affected with MYH9-related disease, ANKRD26-related thrombocytopenia, X-linked thrombocytopenia/Wiskott-Aldrich syndrome, monoallelic Bernard-Soulier syndrome, or ITGB3-related thrombocytopenia. Average pre-treatment platelet count was 40.4 x10⁹/L. Patients received a 3- to 6-week course of eltrombopag in a dose-escalation manner. Of 23 patients evaluable for response, 11 (47.8%) achieved a major response (platelet count >100 x10⁹/L), 10 (43.5%) a minor response (platelet count at least twice than baseline), whereas 2 patients (8.7%) did not respond. Average increase of platelet count compared to baseline was 64.5 x10⁹/L (p<0.001). Four patients who presented clinically significant spontaneous bleeding were admitted to a long-term eltrombopag administration (16 additional weeks): all of them obtained remission of mucosal hemorrhages that persisted throughout the treatment period. Treatment was globally well tolerated: 5 patients reported mild adverse events and one patient a moderate adverse event. In conclusion, eltrombopag was safe and effective in increasing platelet count and reducing bleeding symptoms in different forms of inherited thrombocytopenia. Despite these encouraging results, caution is recommended when using thrombopoietin-mimetics in inherited thrombocytopenias predisposing to leukemia. ClinicalTrials.gov identifier: NCT02422394.

Congenital amegakaryocytic thrombocytopenia (CAMT) is an inherited disorder characterized at birth by thrombocytopenia with reduced megakaryocytes, which evolves into generalized bone marrow aplasia during childhood. Although CAMT is genetically heterogeneous, mutations of MPL, the gene encoding for the receptor of thrombopoietin (THPO), are the only known disease‐causing alterations. We identified a family with three children affected with CAMT caused by a homozygous mutation (p.R119C) of the THPO gene. Functional studies showed that p.R119C affects not only ability of the cytokine to stimulate MPL but also its release, which is consistent with the relatively low serum THPO levels measured in patients. In all the three affected children, treatment with the THPO‐mimetic romiplostim induced trilineage hematological responses, remission of bleeding and infections, and transfusion independence, which were maintained after up to 6.5 years of observation. Recognizing patients with THPO mutations among those with juvenile bone marrow failure is essential to provide them with appropriate substitutive therapy and prevent the use of invasive and unnecessary treatments, such as hematopoietic stem cell transplantation or immunosuppression. Synopsis Congenital amegakaryocytic thrombocytopenia (CAMT) is a fatal inherited juvenile bone marrow failure syndrome, unless children are treated with hematopoietic stem cell transplantation (HSCT). The only known cause of CAMT is mutations in the MPL gene. A novel THPO mutation is here described. We identified a family affected with CAMT that is caused by a novel homozygous missense mutation (p.R119C) in THPO, the gene encoding for thrombopoietin (THPO). Functional studies in vitro showed that p.R119C significantly impairs secretion of THPO, consistent with the relatively low serum THPO levels measured in patients. The mutation also affects interaction of THPO with MPL, resulting in defective signalling downstream of the receptor, which is reduced by about 50% compared with wild‐type THPO. In three affected children, administration of the THPO‐mimetic romiplostim induced sustained trilineage improvement of blood counts and remission of bleeding and infections, maintained after an up to 6.5‐year follow‐up. THPO mutations must be considered in patients presenting with the phenotype of CAMT: recognition of this disorder (disease variant) is essential to avoid unnecessary HSCT and give appropriate substitutive therapy with MPL agonists.

ANKRD26-related thrombocytopenia (ANKRD26-RT) is an autosomaldominant thrombocytopenia caused by mutations in the 5’UTR of the ANKRD26 gene. ANKRD26-RT is characterised by dysmegakaryopoiesis and an increased risk of leukaemia. PaCSs are novel particulate cytoplasmic structures with selective immunoreactivity for polyubiquitinated proteins and proteasome that have been detected in a number of solid cancers, in the epithelia of Helicobacter pylori gastritis and related preneoplastic lesions, and in the neutrophils of Schwachman- Diamond syndrome, a genetic disease with neutropenia and increased leukaemia risk. We searched for PaCSs in blood cells from 14 consecutive patients with ANKRD26-RT. Electron microscopy combined with immunogold staining for polyubiquitinated proteins, 20S and 19S proteasome showed PaCSs in most ANKRD26-RT platelets, as in a restricted minority of platelets from healthy controls and from subjects with other inherited or immune thrombocytopenias. In ANKRD26-RT platelets, the PaCS amount exceeded that of control platelets by a factor of 5 (p<0.0001). Immunoblotting showed that the higher PaCS number was associated with increased amounts of polyubiquitinated proteins and proteasome in ANKRD26-RT platelets. PaCSs were also extensively represented in ANKRD26-RT megakaryocytes, but not in healthy control megakaryocytes, and were absent in other ANKRD26-RT and control blood cells. Therefore, large amounts of PaCSs are a characteristic feature of ANKRD26-RT platelets and megakaryocytes, although these novel cell components are also present in a small subpopulation of normal platelets. The widespread presence of PaCSs in inherited diseases with increased leukaemia risk, as well as in solid neoplasms and their preneoplastic lesions, suggests a link of these structures with oncogenesis.