Rukhmi Bhat

Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies

Disease alleles that activate signal transduction are common in myeloid malignancies; however, there are additional unidentified mutations that contribute to myeloid transformation. Based on the recent identification of TET2 mutations, we evaluated the mutational status of TET1, TET2, and TET3 in myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Sequencing of TET2 in 408 paired tumor/normal samples distinguished between 68 somatic mutations and 6 novel single nucleotide polymorphisms and identified TET2 mutations in MPN (27 of 354, 7.6%), CMML (29 of 69, 42%), AML (11 of 91, 12%), and M7 AML (1 of 28, 3.6%) samples. We did not identify somatic TET1 or TET3 mutations or TET2 promoter hypermethylation in MPNs. TET2 mutations did not cluster in genetically defined MPN, CMML, or AML subsets but were associated with decreased overall survival in AML (P = .029). These data indicate that TET2 mutations are observed in different myeloid malignancies and may be important in AML prognosis.

Evaluation of a post-operative thrombin inhibitor replacement protocol to reduce haemorrhagic and thrombotic complications after paediatric liver transplantation.

INTRODUCTION Bleeding and thrombotic complications contribute to morbidity and mortality following paediatric orthotopic liver transplantation (OLT). However, the pathophysiology of haemostasis during paediatric OLT is not well understood. This report consists of two complimentary studies examining the frequency of haemostatic complications before and after the introduction of a post-operative thrombin inhibitor replacement therapy protocol at a single institution. MATERIALS AND METHODS A retrospective study of 40 patients who underwent 43 liver transplants between July 1992 and July 2002, identified bleeding to be the most frequent complication associated with OLT (30%), however thrombotic complications were also common (12.5%). In 2003, following a detailed analysis of haemostatic profiles of children undergoing OLT, a thrombin inhibitor replacement protocol was introduced. A prospective clinical outcome audit was undertaken from April 2003 to September 2008 to determine the effect of the new protocol on haemostasis. RESULTS Commencement of the thrombin inhibitor replacement protocol significantly reduced the incidence of thrombosis (from 5 to 1, p<0.05), graft loss (from 4 to none, p<0.05), mortality due to thrombosis or bleeding (from 3 to none, p<0.05) and was associated with a 50% reduction in frequency of major bleeding. CONCLUSION In conclusion, the introduction of a post-operative thrombin inhibitor replacement therapy protocol following paediatric OLT significantly improved haemostasis-related morbidity and mortality outcomes in children.

The preterm infant with thrombosis

In paediatrics, sick preterm infants are at highest risk of developing thrombotic complications. Haemostasis is in a fine balance in the neonatal period, despite age-related differences in coagulation proteins. However, both inherited and acquired risk factors can disrupt this balance and can lead to thrombosis. Important risk factors are sepsis, asphyxia, dehydration, central venous lines and inherited and acquired thrombophilia. Among various treatment modalities, anticoagulation is primarily used in the management of thrombosis. Different agents, dosages and durations of treatment in this age group are extrapolated from adult data. The article reviews the epidemiology, pathophysiology, risk factors, diagnosis and treatment of thrombotic disorders in preterm infants.

Mutational analysis of candidate tumor-associated genes in acute megakaryoblastic leukemia

Acute megakaryoblastic leukemia (AMKL) is a biologically and cytogenetically heterogeneous subtype of acute myeloid leukemia. AMKL either presents de novo or as therapy-related leukemia after chemotherapy or as a leukemic transformation of chronic myeloproliferative neoplasms. The incidence of AMKL in adult de novo acute myeloid leukemia patients is estimated to be about 1–2%, whereas the incidence in the pediatric population is as high as 5–7%, largely because of its association with Down’s syndrome (DS). DS-AMKL and nonDS-AMKL have vastly different genetic abnormalities and outcomes. Although patients with DS-AMKL have a favorable prognosis, with a 5-year event-free survival rate between 70 and 80%, children with non-DS-AMKL fare much worse, with a 5-year event-free survival of 22–28%. Similarly, the prognosis of adult AMKL is dismal, with a median survival of B10 months. With respect to the molecular basis for megakaryocytic subtypes of acute myeloid leukemia, DS-AMKL blasts uniformly harbor trisomy 21 and somatic mutations in the X-linked hematopoietic transcription factor, GATA1. Distinct abnormalities, such as t(1;22)(p13;q13), which leads to expression of the OTT-MAL (RBM15-MLK1) fusion protein, t(10;11), t(9;11) or þ 8 are seen in cases of non-DS pediatric AMKL. However, very little is known about the etiology of adult AMKL, as no specific chromosomal rearrangements or genetic mutations have been described, apart from a rare detection of mutations in c-MPL, c-KIT, JAK2, JAK3 or FLT3. Leukemia is caused through a multistep process involving genetic alterations that lead to both a proliferative advantage and a block in terminal differentiation. With respect to DS-AMKL, it is likely that trisomy 21 provides a proliferative advantage to fetal hematopoietic progenitors that harbor GATA1 mutations and that these two alterations are sufficient enough to cause TMD (transient myeloproliferative disorder), a preleukemia that is characterized by an aberrant expansion of megakaryoblasts in the fetal liver and peripheral blood. However, trisomy 21 and GATA1 mutations alone are not sufficient to cause DS-AMKL. Consistent with this model, recent studies have shown that DS-AMKL blasts harbor rare activating mutations in FLT3, JAK2, JAK3 and c-MPL. Despite these new insights, the specific genetic events that cooperate with GATA1 and trisomy 21 in the evolution of AMKL are largely unknown. Moreover, common recurrent genetic abnormalities present in pediatric non-DS AMKL and adult AMKL have not been defined. To identify novel mutations that are associated with AMKL, we systematically analyzed a cohort of 16 adult non-DS-AMKL (diagnosed by immunophenotype and/or French American British (FAB) criteria) patient samples (Table 1) by nucleotide sequence analysis for mutations in genes that are associated with hematopoietic disorders (FLT3, PTEN, c-MPL, PTPN11 and JAK1) and genes that are implicated in normal megakaryocytic differentiation (NOTCH1, GATA2, ETS2, ERG, MEF2C, GABPA, TEL and FLI1). We assayed all the coding regions of these genes by sequencing with the following exceptions: for NOTCH1, we analyzed sequences that encode the heterodimerization and peptide signal rich in proline, glutamic acid, serine, threonine

Risk Factors for Neonatal Venous and Arterial Thromboembolism in the Neonatal Intensive Care Unit-A Case Control Study

Objective To identify risk factors associated with venous and arterial thrombosis in sick neonates admitted to the neonatal intensive care unit. Study design A case‐control study was conducted at 2 centers between January 2010 and March 2014 using the Childrens Hospital Neonatal Database dataset. Cases were neonates diagnosed with either arterial or venous thrombosis during their neonatal intensive care unit stay; controls were matched in a 1:4 ratio by gestational age and presence or absence of central access devices. Bivariable and conditional logistic regression analyses for venous and arterial thrombosis were performed separately. Results The overall incidence of neonatal thrombosis was 15.0 per 1000 admissions. A higher proportion of neonates with thrombosis had presence of central vascular access devices (75% vs 49%; P < .01) were of extremely preterm gestational age (22–27 weeks; 26% vs 15.0%; P < .05) and stayed ≥31 days in the neonatal intensive care unit (53% vs 32.9%; P < .01), when compared with neonates without thrombosis. A final group of 64 eligible patients with thrombosis and 4623 controls were analyzed. In a conditional multivariable logistic regression model, venous thrombosis was significantly associated with male sex (AOR, 2.12; 95% CI, 1.03–4.35; P = .04) and blood stream infection (AOR, 3.47; 95% CI, 1.30–9.24; P = .01). Conclusions The incidence of thrombosis was higher in our neonatal population than in previous reports. After matching for central vascular access device and gestational age, male sex and blood stream infection represent independent risk factors of neonatal venous thrombosis. A larger cohort gleaned from multicenter data should be used to confirm the study results and to develop thrombosis prevention strategies.

Thrombosis in Childhood Cancer

Over the past five decades, the survival rate for children diagnosed with oncologic disease has dramatically increased from less than 20% to almost 80%. Accordingly, an area of growing interest and concern is the long-term effects of the patients’ disease and therapy. Thromboembolic events (TEE) occur with greater frequency in children and adults with cancer and have been extensively studied in the latter population. However, given the relative infrequency of TEE in the general pediatric population and paucity of coordinated efforts, information on the incidence, nature and impact of thrombosis in children with cancer has been lacking until recently. In this chapter, we address the salient features of TEEs in the pediatric cancer patient.