Alice Norton

Abnormalities in the myeloid progenitor compartment in Down syndrome fetal liver precede acquisition of GATA1 mutations

Down syndrome (DS) children have a high frequency of acute megakaryoblastic leukemia (AMKL) in early childhood. At least 2 in utero genetic events are required, although not sufficient, for DS-AMKL: trisomy 21 (T21) and N-terminal-truncating GATA1 mutations. To investigate the role of T21 in DS-AMKL, we compared second trimester hemopoiesis in DS without GATA1 mutations to gestation-matched normal controls. In all DS fetal livers (FLs), but not marrows, megakaryocyte-erythroid progenitor frequency was increased (55.9% +/- 4% vs 17.1% +/- 3%, CD34(+)CD38(+) cells; P < .001) with common myeloid progenitors (19.6% +/- 2% vs 44.0% +/- 7%) and granulocyte-monocyte (GM) progenitors (15.8% +/- 4% vs 34.5% +/- 9%) commensurately reduced. Clonogenicity of DS-FL versus normal FL CD34(+) cells was markedly increased (78% +/- 7% vs 15% +/- 3%) affecting megakaryocyte-erythroid ( approximately 7-fold higher) and GM and colony-forming unit-granulocyte, erythrocyte macrophage, megakaryocyte (CFU-GEMM) progenitors. Replating efficiency of CFU-GEMM was also markedly increased. These data indicate that T21 itself profoundly disturbs FL hemopoiesis and they provide a testable hypothesis to explain the increased susceptibility to GATA1 mutations in DS-AMKL and DS-associated transient myeloproliferative disorder.

Acute megakaryoblastic leukaemia (AMKL) and transient myeloproliferative disorder (TMD) in Down syndrome: a multi-step model of myeloid leukaemogenesis.

Children with Down syndrome (DS) have a marked increase in susceptibility to Acute Megakaryoblastic Leukaemia (DS‐AMKL) and the closely linked neonatal preleukaemic syndrome, Transient Myeloproliferative Disorder (DS‐TMD). The distinct stages of DS‐TMD and DS‐AMKL provide an excellent tractable model to study leukaemogenesis. This review focuses on recent studies describing clinical, haematological and biological features of DS‐AMKL and DS‐TMD. The findings from these studies suggest that mutations in the key haemopoietic regulator GATA1 (GATA binding protein 1) in DS‐AMKL and DS‐TMD may be useful in diagnosis and assessing minimal residual disease. These findings raise the possibility of population‐based screening strategies for DS‐TMD and the development of treatment to eliminate the preleukaemic TMD clone to prevent DS‐AMKL. Advances in our understanding of perturbed haemopoiesis in DS, the role of GATA1 and of cooperating mutations are also discussed. These findings have implications for leukaemia biology more broadly given the frequency of acquired trisomy in other human leukaemias.

Excellent outcome of matched unrelated donor transplantation in paediatric aplastic anaemia following failure with immunosuppressive therapy: a United Kingdom multicentre retrospective experience

We retrospectively analysed the outcome of consecutive children with idiopathic severe aplastic anaemia in the United Kingdom who received immunosuppressive therapy (IST) or matched unrelated donor (MUD) haematopoietic stem cell transplantation (HSCT). The 6‐month cumulative response rate following rabbit antithymocyte globulin (ATG)/ciclosporin (IST) was 32·5% (95% CI 19·3–46·6) (n = 43). The 5‐year estimated failure‐free survival (FFS) following IST was 13·3% (95% confidence interval [CI] 4·0–27·8). In contrast, in 44 successive children who received a 10‐antigen (HLA‐A, ‐B, ‐C, ‐DRB1, ‐DQB1) MUD HSCT there was an excellent estimated 5‐year FFS of 95·01% (95% CI 81·38–98·74). Forty of these children had failed IST previously. HSCT conditioning was a fludarabine, cyclophosphamide and alemtuzumab (FCC) regimen and did not include radiotherapy. There were no cases of graft failure. Median donor chimerism was 100% (range 88–100%). A conditioning regimen, such as FCC that avoids total body irradiation is ideally suited in children. Our data suggest that MUD HSCT following IST failure offers an excellent outcome and furthermore, if a suitable MUD can be found quickly, MUD HSCT may be a reasonable alternative to IST.

Analysis of GATA1 mutations in Down syndrome transient myeloproliferative disorder and myeloid leukemia

Children with Down syndrome (DS) up to the age of 4 years are at a 150-fold excess risk of developing myeloid leukemia (ML-DS). Approximately 4%-5% of newborns with DS develop transient myeloproliferative disorder (TMD). Blast cell structure and immunophenotype are similar in TMD and ML-DS. A mutation in the hematopoietic transcription factor GATA1 is present in almost all cases. Here, we show that simple techniques detect GATA1 mutations in the largest series of TMD (n = 134; 88%) and ML-DS (n = 103; 85%) cases tested. Furthermore, no significant difference in the mutational spectrum between the 2 disorders was seen. Thus, the type of GATA1 sequence mutation is not a reliable tool and is not prognostic of which patients with TMD are probable to develop ML-DS.

Severe TMD/AMKL with GATA1 mutation in a stillborn fetus with Down syndrome

Background A 34-year-old woman was referred for evaluation of a recent stillborn male fetus, gestational age 27 6/7 weeks, found to have congenital myeloid leukemia at autopsy. Autopsy findings included high weight for gestational age, hepatomegaly, and extensive intravascular leukemic infiltrates in the placenta, heart, liver, thymus, lung, kidneys, and brain. Genetic consultation and examination of photographs of the fetus revealed dysmorphic features.Investigations Immunoperoxidase staining of placental tissue, fluorescence in situ hybridization of paraffin-embedded sections of the placenta using probes for t(12;21)(p13;q22), t(8;21)(q22;q22) and t/del(11q23), cytogenetic analysis of fetal tissue (tendon), sequence analysis of GATA1 in placental leukemic cells, and parental chromosome studies.Diagnosis Down syndrome with in utero onset of GATA1 mutation-positive severe transient myeloproliferative disorder/acute megakaryoblastic leukemia.Management Genetic counseling for the recurrence risk of Down syndrome on the basis of maternal age.

Characterization of leukemias with ETV6-ABL1 fusion

To characterize the incidence, clinical features and genetics of ETV6-ABL1 leukemias, representing targetable kinase-activating lesions, we analyzed 44 new and published cases of ETV6-ABL1-positive hematologic malignancies [22 cases of acute lymphoblastic leukemia (13 children, 9 adults) and 22 myeloid malignancies (18 myeloproliferative neoplasms, 4 acute myeloid leukemias)]. The presence of the ETV6-ABL1 fusion was ascertained by cytogenetics, fluorescence in-situ hybridization, reverse transcriptase-polymerase chain reaction and RNA sequencing. Genomic and gene expression profiling was performed by single nucleotide polymorphism and expression arrays. Systematic screening of more than 4,500 cases revealed that in acute lymphoblastic leukemia ETV6-ABL1 is rare in childhood (0.17% cases) and slightly more common in adults (0.38%). There is no systematic screening of myeloproliferative neoplasms; however, the number of ETV6-ABL1-positive cases and the relative incidence of acute lymphoblastic leukemia and myeloproliferative neoplasms suggest that in adulthood ETV6-ABL1 is more common in BCR-ABL1-negative chronic myeloid leukemia-like myeloproliferations than in acute lymphoblastic leukemia. The genomic profile of ETV6-ABL1 acute lymphoblastic leukemia resembled that of BCR-ABL1 and BCR-ABL1-like cases with 80% of patients having concurrent CDKN2A/B and IKZF1 deletions. In the gene expression profiling all the ETV6-ABL1-positive samples clustered in close vicinity to BCR-ABL1 cases. All but one of the cases of ETV6-ABL1 acute lymphoblastic leukemia were classified as BCR-ABL1-like by a standardized assay. Over 60% of patients died, irrespectively of the disease or age subgroup examined. In conclusion, ETV6-ABL1 fusion occurs in both lymphoid and myeloid leukemias; the genomic profile and clinical behavior resemble BCR-ABL1-positive malignancies, including the unfavorable prognosis, particularly of acute leukemias. The poor outcome suggests that treatment with tyrosine kinase inhibitors should be considered for patients with this fusion.

Prenatal therapy in transient abnormal myelopoiesis: a systematic review

Objective To systematically review current evidence regarding prenatal diagnosis and management of transient abnormal myelopoiesis (TAM) in fetuses with trisomy 21. A novel case of GATA1-positive TAM, in which following serial in utero blood transfusion clinical improvement and postnatal remission were observed, is included. Search strategy and data collection A systematic search of electronic databases (inception to October 2014) and reference lists, hand-searching of journals and expert contact. All confirmed cases of prenatal TAM were included for analysis. Data on study characteristics, design and quality were obtained. Results Of 73 potentially relevant citations identified, 22 studies were included, describing 39 fetuses. All studies included comprised single case or small cohort studies; overall quality was ‘very low’. Fetal/neonatal outcome was poor; 12 stillbirths (30.8%), 4 neonatal deaths (10.2%) and 7 infant deaths (17.9%). In two cases, the pregnancy was terminated (5.1%). TAM was primarily detected in the third trimester (79.4%), and in 14 a retrospective diagnosis was made postpartum. Ultrasound features indicative of TAM included hepatomegaly±splenomegaly (79.5%), hydrops fetalis (30.8%), pericardial effusion (23.1%) and aberrant liquor volume (15.4%). When performed, liver function tests were abnormal in 91.6% of cases. Conclusions Prenatal TAM presents a challenging diagnosis, and prognosis is poor, with consistently high mortality. A low threshold to measure haematological and biochemical markers is advised when clinical features typical of TAM are detected in the context of trisomy 21. Larger prospective studies are warranted to accurately ascertain the role of GATA1 analysis and potential value of prenatal therapy.

G371 Defining transient abnormal myelopoiesis (TAM) and silent tam in neonates with down syndrome

Background and aims Children with Down syndrome (DS) have a 150-fold-increased risk of acute myeloid leukaemia (ML-DS) in the first 5y of life (peak age 12–15 months). ML-DS is preceded by Transient Abnormal Myelopoiesis (TAM), a neonatal pre-leukaemic disorder unique to DS. Recent studies of clinically-diagnosed TAM show acquired mutations in the GATA1 gene in all cases. However, the true clinical spectrum of TAM is unknown since previous retrospective reports have not systematically evaluated blood films or GATA1 mutation status. The purpose of our study was to prospectively determine the clinical, haematological, molecular features and natural history of TAM. Methods Neonates with karyotypically-confirmed DS were prospectively enrolled to the Oxford-Imperial DS Cohort Study (OIDSCS) from October 2006. Detailed clinical and FBC/blood film data were matched to GATA1 mutational analysis by Sanger sequencing/Direct High Performance Liquid Chromatography (Ss/DHPLC). Targeted next-generation-sequencing (NGS) was used to determine clone size and/or detect small (<5%) mutant GATA1 clones. TAM was prospectively defined as: >10% peripheral blood blasts and GATA1 mutation(s) detected by Ss/DHPLC. Silent TAM was defined as: blasts <10% and GATA1 mutation (s) detected by Ss/DHPLC or NGS. Results Of 382 neonates recruited to OIDSCS by June 2014, 39 (10.2%) had TAM. Although no clinical features were specific for TAM, hepatosplenomegaly, pericardial/pleural effusion and skin rash were more common in TAM (p < 0.0001, p < 0.01, p < 0.05) than DS neonates without GATA1 mutations. The only haematological features specific for TAM were blasts >20% and WBC >45 × 109/L. Ten neonates with TAM and 8 without TAM had 11–20% blasts. In 163 DS neonates with blasts <10% screened by NGS, 33(20.2%) had small GATA1 clones (Silent TAM); their clinical and haematological features were indistinguishable from 130/163 without mutations. 4 neonates with TAM received low-dose chemotherapy and 1 died. ML-DS has developed in 4/39 TAM, 1/33 Silent TAM and no DS neonates without GATA1 mutations (median follow-up 57 months, range 18- >60). Conclusion In neonates with DS, acquired mutations in the GATA1 gene are common, often clinically and haematologically silent and confer a risk of ML-DS in TAM and Silent TAM.

Genome instability is a consequence of transcription deficiency in patients with bone marrow failure harboring biallelic ERCC6L2 variants

Significance Bone marrow failure (BMF) is an inherited life-threatening condition characterized by defective hematopoiesis, developmental abnormalities, and predisposition to cancer. BMF caused by ERCC6L2 mutations is considered to be a genome instability syndrome, because DNA repair is compromised in patient cells. In this study, we report BMF cases with biallelic disease-causing variants and provide evidence from patients’ cells that transcription deficiency can explain the genome instability. Specifically, we demonstrate that ERCC6L2 participates in RNA polymerase II-mediated transcription via interaction with DNA-dependent protein kinase (DNA-PK) and resolves DNA–RNA hybrids (R loops). Collectively, our data point to a causal mechanism in BMF in which patients with ERCC6L2 mutations are defective in the repair of transcription-associated DNA damage. Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients’ lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients’ LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients’ LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA–RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

Single dose Rasburicase is a clinically effective pharmacoeconomic approach for preventing tumour lysis syndrome in children with high tumour burden

Tumour lysis syndrome (TLS) is an oncological metabolic emergency caused by the rapid lysis of tumour cells and subsequent release of large amounts of potassium, phosphate and nucleic acids into the systemic circulation. Catabolism of nucleic acids can lead to a rapid increase of uric acid excretion and a precipitation of uric acid in the renal tubules. Both high levels of uric acid and phosphate can result in kidney impairment and significant morbidity and mortality (Davidson et al, 2004). The main prophylactic strategies are intravenous hydration and the use of anti-hyperuricaemic agents, such as Allopurinol and Rasburicase. Prophylaxis is selected according to individual risk stratification, assessed by the disease burden and the treatment chosen. Rasburicase is a recommended strategy for prevention of TLS in adults and children with high tumour burden. It is a recombinant form of the enzyme urate oxidase, which is present in most mammals except humans (Wu et al, 1989). Rasburicase converts uric acid to thewater-soluble compound, allantoin, promoting its renal excretion. It reduces the uric acid levels much lower than the normal range (Coiffier et al, 2003). In adults, a single dose of Rasburicase has been shown to adequately control the toxic effects of TLS (Feng et al, 2013; Vadhan-Raj et al, 2012). However, there is lack of evidence regarding the effectiveness of single dose Rasburicase in paediatrics. Rasburicase effectively reduces the uric acid levels in children with high tumour burden, but it is not known whether a single dose of 0 2 mg/kg is effective. The British Committee for Standards in Haematology suggests that single dose Rasburicase is adequate in presenting children at risk for TLS (Jones et al, 2015). However, the most up-to-date evidence recommends five doses of Rasburicase and therefore paediatric haematology-oncology physicians use single or multiple doses of Rasburicase at their discretion (Goldman et al, 2001). We recently conducted an email survey of tertiary paediatric centres in England, and found that seven out of eight centres use multiple doses of Rasburicase for highrisk patients. Rasburicase is expensive; hence multiple doses also result in significant financial toxicity. A pharmacoeconomic approach is required to identify potential ways of reducing the cost of cancer care by appropriate usage of high cost drugs. It is therefore important to determine whether a single dose of Rasburicase is clinically effective in children with high tumour burden. We retrospectively evaluated the use of Rasburicase in children presenting with haematological malignancy or neuroblastoma to determine the effectiveness of single dose Rasburicase in normalising uric acid and renal function. Schwartz formula was used to assess the renal function by calculating the Glomerular filtration rate (GFR). All patients with haematological malignancies that received Rasburicase between October 2012 and October 2015 at Birmingham Children’s Hospital were included. These patients were identified through the pharmacy database. Laboratory results and clinical information were retrieved using the hospital’s electronic system. Fifty-three children with a median age of 7 3 years (range: 4 months to 16 years; ≤ 5 years: n = 24; 6–12 years: n = 17; 13–16 years: n = 12) received Rasburicase for the following indications: acute myeloid leukaemia (n = 7), acute lymphoblastic leukaemia (n = 33), Hodgkin lymphoma (n = 2), non-Hodgkin lymphoma (n = 9) and neuroblastoma (n = 2). The tumour burden of white blood cell (WBC) count > 100 9 10/l, Stage 3 or 4 lymphoma or high tumour burden neuroblastoma at presentation were classified as appropriate indications for using Rasburicase. Rasburicase was indicated in 44 out of 53 (83%) children. Twenty-six met the criteria of WBC >100 9 10/l and 18 met the criteria of stage 3 or 4 lymphoma or high burden disease (neuroblastoma). In the remaining 11 children, the tumour burden was low and hence, Rasburicase was not indicated. Laboratory tumour lysis, as per the criteria of Cairo and Bishop (2004), was observed in three patients’ prior receiving single dose and in two patients receiving multiple doses. Nineteen (43%) of 44 children received single dose Rasburicase. The remaining 25 children received between two to five daily doses of Rasburicase. Twenty-four hours after receiving single dose of Rasburicase, normalisation of uric acid was observed in all patients. A sustained normalisation of uric acid was observed in 15 of 19 children receiving a single dose of Rasburicase (Fig 1). In the remaining four children, a rebound of uric acid >400 lmol/l between 48 and 96 h after first dose of Rasburicase was observed. These children were appropriately given second dose of Rasburicase. Thus, moderately elevated uric acid levels were normalised in majority of children after single dose of Rasburicase. A higher uric acid level of >600 lmol/l at presentation was Correspondence