Mark Velangi

Exome Sequencing Identifies Autosomal-Dominant SRP72 Mutations Associated with Familial Aplasia and Myelodysplasia

Aplastic anemia (AA) and myelodysplasia (MDS) are forms of bone marrow failure that are often part of the same progressive underlying disorder. While most cases are simplex and idiopathic, some show a clear pattern of inheritance; therefore, elucidating the underlying genetic cause could lead to a greater understanding of this spectrum of disorders. We used a combination of exome sequencing and SNP haplotype analysis to identify causative mutations in a family with a history of autosomal-dominant AA/MDS. We identified a heterozygous mutation in SRP72, a component of the signal recognition particle (SRP) that is responsible for the translocation of nascent membrane-bound and excreted proteins to the endoplasmic reticulum. A subsequent screen revealed another autosomal-dominant family with an inherited heterozygous SRP72 mutation. Transfection of these sequences into mammalian cells suggested that these proteins localize incorrectly within the cell. Furthermore, coimmunoprecipitation of epitope-tagged SRP72 indicated that the essential RNA component of the SRP did not fully associate with one of the SRP72 variants. These results suggest that inherited mutations in a component of the SRP have a role in the pathophysiology of AA/MDS, identifying a third pathway for developing these disorders alongside transcription factor and telomerase mutations.

Similar outcome of upfront-unrelated and matched sibling stem cell transplantation in idiopathic paediatric aplastic anaemia. A study on behalf of the UK Paediatric BMT Working Party, Paediatric Diseases Working Party and Severe Aplastic Anaemia Working Party of EBMT

We explored the feasibility of unrelated donor haematopoietic stem cell transplant (HSCT) upfront without prior immunosuppressive therapy (IST) in paediatric idiopathic severe aplastic anaemia (SAA). This cohort was then compared to matched historical controls who had undergone first‐line therapy with a matched sibling/family donor (MSD) HSCT (n = 87) or IST with horse antithymocyte globulin and ciclosporin (n = 58) or second‐line therapy with unrelated donor HSCT post‐failed IST (n = 24). The 2‐year overall survival in the upfront cohort was 96 ± 4% compared to 91 ± 3% in the MSD controls (P = 0·30) and 94 ± 3% in the IST controls (P = 0·68) and 74 ± 9% in the unrelated donor HSCT post‐IST failure controls (P = 0·02).The 2‐year event‐free survival in the upfront cohort was 92 ± 5% compared to 87 ± 4% in MSD controls (P = 0·37), 40 ± 7% in IST controls (P = 0·0001) and 74 ± 9% in the unrelated donor HSCT post‐IST failure controls (n = 24) (P = 0·02). Outcomes for upfront‐unrelated donor HSCT in paediatric idiopathic SAA were similar to MSD HSCT and superior to IST and unrelated donor HSCT post‐IST failure. Front‐line therapy with matched unrelated donor HSCT is a novel treatment approach and could be considered as first‐line therapy in selected paediatric patients who lack a MSD.

NUP98-NSD1 Fusion in Association with FLT3-ITD Mutation Identifies a Prognostically Relevant Subgroup of Pediatric Acute Myeloid Leukemia Patients Suitable for Monitoring by Real Time Quantitative PCR

The cytogenetically cryptic t(5;11)(q35;p15) leading to the NUP98‐NSD1 fusion is a rare but recurrent gene rearrangement recently reported to identify a group of young AML patients with poor prognosis. We used reverse transcription polymerase chain reaction (PCR) to screen retrospectively diagnostic samples from 54 unselected pediatric AML patients and designed a real time quantitative PCR assay to track individual patient response to treatment. Four positive cases (7%) were identified; three arising de novo and one therapy related AML. All had intermediate risk cytogenetic markers and a concurrent FLT3‐ITD but lacked NPM1 and CEBPA mutations. The patients had a poor response to therapy and all proceeded to hematopoietic stem cell transplant. These data lend support to the adoption of screening for NUP98‐NSD1 in pediatric AML without otherwise favorable genetic markers. The role of quantitative PCR is also highlighted as a potential tool for managing NUP98‐NSD1 positive patients post‐treatment.

Less than half of patients aged 65 years or under with myeloma proceed to transplantation: results of a two region population‐based survey*

In this population‐based survey covering two geographically distinct UK regions, we evaluated the number of myeloma patients aged ≤65 years who have not undergone transplantation. The combined data from both of these regions showed that 57% of age‐eligible patients were not transplanted. While early death and comorbidity accounted for nearly half of the non‐transplanted patients, we examined the other reasons for non‐transplantation within each region, assessed regional variations in reasons for non‐transplant and looked at possible strategies aimed at increasing the transplantation rate.

BRAF gene is not mutated in mismatch repair-proficient or -deficient plasma cell dyscrasias

BRAF gene is not mutated in mismatch repair-proficient or -deficient plasma cell dyscrasias

Donor lymphocyte infusions for post‐transplant relapse of refractory anemia with excess blasts and monosomy 7

An 8‐year‐old male relapsed with refractory anemia with excess blasts (RAEB) and monosomy 7 and mixed chimerism (MC) 21 months after HLA‐matched unrelated donor bone marrow transplant (BMT). He received three donor lymphocyte infusions (DLI) using an escalating dose schedule. He developed grade II acute graft‐versus‐host disease (GVHD) 9 days after the third DLI, but continued to deteriorate for 2 months with decreasing marrow cellularity but persisting blasts, MC, and monosomy 7, before exhibiting a delayed but complete response which has persisted for 5 years. This case suggests that DLI and graft‐versus‐myelodysplasia (GVMDS) may be beneficial in post‐transplant relapse of pediatric myelodysplasia. Pediatr Blood Cancer 2008;50:670–672.

Detecting Mismatch Repair Defects in Myeloma

Defects in the mismatch repair system are associated with a microsatellite unstable phenotype. In this chapter, we describe the preparation of purified plasma cells using CD138 magnetic microbeads as a source of tumor DNA. We also describe a robust, sensitive method for comparing microsatellite repeat units of tumor to constitutive DNA using polymerase chain reaction and laser scanning of fluorescently labeled amplicons in an automated sequencer in order to assess microsatellite instability in myeloma.

UK experience of unrelated cord blood transplantation in paediatric patients

Cord blood is a valuable alternative source of cells for haematopoietic stem cell transplantation when suitable human leucocyte antigen (HLA)-matched sibling or unrelated donors are unavailable. The use of unrelated cord blood transplantation (UCBT) has been widely adopted in North America (Kurtzberg et al, 2008), but uptake in the UK had been slow. Following recommendations for incorporating UCBT into standard transplant practice within the UK (Shaw et al, 2009), the use of UCBT in children has increased significantly. British Society of Blood and Marrow Transplantation and Eurocord data revealed that 335 children (0 1–17 9 years) underwent UCBT in the UK between 1998 and 2012 (Table I). The median follow-up of survivors was 4 4 years (0 4–14 6). Amongst patients with malignant disease (MD) (n = 167), the median age was 6 5 years (0 3–17 7), most had acute leukaemia (n = 128) and the majority were cytomegalovirus (CMV) seronegative (n = 93). Most patients received myeloablative conditioning (n = 138), with a single cord blood unit (CBU) (n = 138) matched for 5/6 HLA antigens (n = 82). The median collected total nucleated cell (TNC) and CD34 cell dose was 6 7 9 10/kg (range 1 4–62 3) and 2 7 9 10/kg (range 0 1–41 7), respectively. Amongst patients with non-malignant diseases (NMD), the median age was younger at 1 year (range 0 1–17 9), and the most frequent diagnosis was primary immune deficiency (PID) (n = 74). In contrast to MD, the majority of patients received reduced intensity conditioning (n = 92), a better HLA-matched CBU (6/6 n = 77; 5/6 n = 65) and higher TNC and CD34 cell doses: 14 4 9 10/kg (2 9–65 1) and 5 3 9 10/kg (0 5–59 7). Engraftment was achieved in 149/167 (89%) patients with MD and 136/162 (84%) with NMD. The median time to neutrophil recovery >0 5 9 10/l was 23 days (7–77) and 19 days (9–80) in patients with MD NMD, respectively. Platelet recovery was also slower in MD patients at a median of 40 days (8–151) versus 33 days (13–152), presumably reflecting the lower TNC and CD34 dose. The cumulative incidence of neutrophil and platelet recovery by day 100 was 90 9% [95% confidence interval (CI): 85 2–94 4] and 75 8% (95% CI: 68 1–82 0) for MD and 84 4% (95% CI: 77 8–89 3) and 72 9% (95% CI: 65 1–79 2) for NMD patients. The cumulative incidence of graft-versus-host disease (GvHD), non-relapse mortality and relapse are given in Appendix S1. Overall survival (OS) at 1 and 4 years was 64 6% (95% CI: 57 6–72 3) and 51 8% (95% CI: 44 4–60 4) for the group of patients with MD (Fig 1a). Univariate analysis (Table I) showed improved OS for patients aged <6 5 years, with the use of myeloablative conditioning, a TNC and CD34 collected cell dose >median (Fig 1b), and CBUs matched for 6/6 HLA antigens (Fig 1c). There was a trend to improved OS with patient CMV-negative serology and a single CBU. There was significantly worse survival in patients with lymphoproliferative disease. Surprisingly, there was no impact of disease status on outcome, so acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) patients were analysed separately. There remained no impact of disease status on outcome for AML patients (P = 0 52), but for ALL there was a trend to improved survival in CR1 (P = 0 07). For the NMD group, OS was 85 2% (95% CI: 79 9–90 8) at 100 days, 75 2% (95% CI: 68 8–82 1) at 1 year and 74 5% (68 1–81 6) at 4 years (Fig 1d). Univariate analysis showed a trend to improved OS with 6/6 HLA-matched CBU (Fig 1f), a significantly poorer outcome with a collected TNC dose in the lowest quartile (Fig 1e), and a trend to poorer outcome in haemophagocytic lymphohistiocytosis (HLH) (Table I). Multivariate analyses are given in Appendix S2. This report provides a comprehensive picture of the use of UCBT in the UK paediatric population from 1998 to 2012, reflecting approximately 16% of all unrelated transplants performed during this time. Although similar numbers MD and NMD patients were transplanted throughout this period, use of UCBT significantly increased after 2009 in both groups. Despite the relative reluctance to use UCBT in the UK, the outcomes have been comparable to UCBT performed elsewhere. Eapen et al (2007) examined the outcomes of 503 children with leukaemia undergoing UCBT in the USA, determining 5-year probabilities of leukaemia-free survival of 60%, 45% and 33% after 6/6, 5/6 and 4/6 HLA-matched CBT, respectively. The equivalent figures from this UK study were similar, at 75%, 48% and 33%, respectively (Table I). Treatment-related mortality (TRM) and relapse rates for leukaemia patients undergoing UCBT were also broadly similar between the two series: 37 8% and 23 5% respectively in the USA; 24% and 26 5%, respectively in the UK. Eurocord examined the outcome of 279 children with NMD undergoing UCBT (Rocha & Gluckman, 2009). There was better engraftment in this UK study compared to the Eurocord study, with a cumulative incidence of neutrophil and platelet recovery of 84% and 73% versus 69% and 50%, reflecting a larger proportion of children with marrow failure, lower cell doses infused and greater HLA-mismatching in the Eurocord cohort. Acute GvHD (II–IV) and chronic GvHD rates were very similar at 32% and 24% for Eurocord and 31% and 17% for the UK cohort. The 49% OS at 100 days in the Eurocord cohort was influenced by cell dose

Hybrid chemotherapy in two children with acute leukemia of ambiguous lineage.

To the Editor: We report two children who presented with acute leukemia at the ages of 18months and 7years. The bone marrow aspirate showed a heterogeneous blast population indicative of myeloid and lymphoid morphology. Cytochemistry showed PAS block positivity in small blasts and Sudan Black positivity in larger blasts. No adverse cytogenetics were found. Immunophenotyping showed positivity for myeloid and lymphoid antigens. The WHO classification of acute leukemias contains a subgroup of ‘ambiguous lineage.’ Our respective cases score 2.5 and 3.5 for B lymphoid and 4 each for myeloid features, fulfilling the criteria (score >2 for each lineage) for ‘ambiguous lineage’ [1]. Initial treatment consisted ofALL induction therapy for 28 days and intrathecal methotrexate. AML consolidation chemotherapy was given as per the current AML 15 trial. One case proceeded to a sibling allograft and the other maintenance ALL chemotherapy. Aberrant antigen expression in acute leukemia is well documented. Even in 1998, when our first case presented, it was apparent that ‘aberrant’ myeloid antigen expression by lymphoblasts and vice versa in children was a common finding that did not in itself have an important impact on prognosis. These cases were not eligible for therapy in ALL or AML trials and, in the absence of poor prognostic cytogenetic features, decisions about therapeutic options were based on the assumption that biphenotypic leukemia was biologically different from either classical ALL or AML and perhaps, erroneously, that the outcome would be poorer than for average cases. In both our cases, a complete remission has been achieved and sustained. Studies have shown a higher rate of Philadelphia chromosome positivity and CD34 expression in this subtype of leukemia and this suggests stem cell involvement in their pathogenesis [2,3]. The largest published group of acute leukemia, diagnosed using the EGIL scoring system [4], within the UK comprised eight children [5] and all would fit into the current WHO criteria for acute leukemia of ambiguous lineage. Three received standard ALL therapy and the remainder hybrid combinations. As further consolidation, one had an autologous, one a sibling, and two unrelated donor stem cell transplants. Two were Philadelphia positive (one died from transplant-related problems, the other relapsed post-transplant) and one had 11q23 rearrangement (a survivor). Overall 5/6 of the Philadelphia negative cases remained alive inCR at the time of publication and no difference in overall survival between the eight cases and matched controls was observed. There is still no formal clinical trial for collecting outcome data of acute leukemia of ambiguous lineagewithin the UK. Although the WHO classification provides uniformity of diagnostic criteria, each case still constitutes a unique therapeutic dilemma. It seems likely that others may also go through a similar therapeutic decision-making process. Because of the established infrastructure for collating outcome data in childhood leukemia, data collection should be simpler for children than adults, provided cases like these that do not fit into current therapeutic trials, are reported into a standard registry.

IFNɣ Block, Treosulfan Conditioning and αβ T Cell Deplete PBSCT for XIAP-Deficient HLH

The outcomes of hematopoietic stem cell transplantation (HSCT) for refractory hemophagocytic lymphohistiocytosis (HLH) in X-linked inhibitor of apoptosis protein (XIAP) deficiency are poor [1]. Amongst other functions, XIAP regulates apoptosis in hepatocytes and other cells and its deficiency may result in increased sensitivity to chemotherapy. A high rate of transplant-related mortality (TRM) is observed following both myeloablative and reduced-intensity conditioning regimens. Treatment-related mortality is attributed to uncontrolled HLH and increased sensitivity to chemotherapeutic agents. Hence, there is a need for both novel agents to control HLH and reduced-toxicity transplantation techniques. We herein present the first case reported to date of refractory HLH and XIAP deficiency treated with interferon gamma (IFNɣ) blockade (NI-0501, an anti-IFNγ human monoclonal antibody) followed by HSCT using a reduced-toxicity treosulfan-based conditioning regimen and ex vivo alpha beta T cell and CD19 depletion of the graft. The patient presented on the first day of life with hepatosplenomegaly, fevers, jaundice and poor feeding and was found to be pancytopenic with ferritin >250,000 μg/L. Six out of eight clinical criteria for HLH were met. X-linked inhibitor of apoptosis deficiency was diagnosed by the absence of protein expression and a c.894_898del hemizygous mutation was subsequently detected. Central nervous system involvement was excluded by lumbar puncture and cerebral imaging and infectious screens were negative. The patient was commenced on the HLH 2004 protocol on day ten of life (twice weekly etoposide, dexamethasone and cyclosporin). Inflammatory markers showed a slow partial response to initial therapy but on steroid taper, fevers reemerged and ferritin rapidly increased. HLH 2004 protocol was complicated by Staphylococcus epidermidis bacteremia, Enterococcus growth from urine and respiratory syncytial virus infection all of which responded promptly to antimicrobial treatment. At 3 months, after two unsuccessful steroid tapers, the patient was taken off protocol. Dexamethasone was continued and salvage therapy with NI-0501 was initiated. NI0501 was provided for the patient on compassionate-use grounds and parental consent was obtained. NI-0501 was administered intravenously at a dose of 1 mg/kg with subsequent dosing intervals guided by pharmacokinetic monitoring. Nine doses were given over 1 month. During this period, ferritin levels declined to 1278 μg/L, fibrinogen and triglycerides normalised, fevers, splenomegaly and pancytopenia resolved and dexamethasone could be discontinued (Fig. 1). Rhinovirus caused transient fever during NI-0501 treatment. At 5 months of age, the patient proceeded to 9/10, HLA-C mismatched unrelated donor peripheral blood stem cell transplantation. Ex vivo alpha beta T cell and CD19 depletion was performed (CliniMACS; Mitenyi Biotec, Bergisch Gladbach, Germany) [2]. The CD34 dose reinfused was 10.7 × 10/kg. The conditioning regimen comprised a total dose of alemtuzumab 1 mg/kg given over days –5 to –1, treosulfan 36 mg/m, fludarabine 160 mg/m and thiotepa 7 mg/kg. Defibrotide and ursodeoxycholic acid were used to prevent veno-occlusive disease (VOD). Mycophenolate mofetil was given as additional graft-versus-host disease (GvHD) prophylaxis for 60 days as the alpha beta T cell dose in the product was >2.5 × 10/kg (3.46 × 10/kg). The conditioning was well tolerated. The transplant course was complicated by Streptococcus mitis bacteremia and * Ciara O’Rafferty orafferc@gmail.com