John Moppett

Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD)

Mutations in genes encoding proteins that are involved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturation of both nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1 homolog, CCA1, we confirm that the patient-associated TRNT1 mutations result in partial loss of function of TRNT1 and lead to metabolic defects in both the mitochondria and cytosol, which can account for the phenotypic pleiotropy.

Minimal residual disease prior to stem cell transplant for childhood acute lymphoblastic leukaemia

Summary. Allogeneic stem cell transplantation (SCT) is a highly effective therapy for childhood acute lymphoblastic leukaemia (ALL). Concerns about unnecessary toxicity and expense mean that SCT is currently largely reserved for children who cannot be cured with chemotherapy. Not surprisingly, many such children also fail SCT. Retrospective studies have shown that a single analysis of minimal residual disease (MRD) pre‐SCT identified those at highest risk of relapse. It is now appropriate to call for the universal incorporation of standardized MRD testing into SCT protocols as the next step to maximize the clinical impact of this technology in ALL.

EBF1-PDGFRB fusion in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL): genetic profile and clinical implications

The EBF1-PDGFRB gene fusion accounts for <1% of B-cell precursor acute lymphoblastic leukemia (ALL) cases and occurs within the Philadelphia-like ALL subtype. We report 15 EBF1-PDGFRB-positive patients from childhood ALL treatment trials (ALL 97/99, UKALL 2003, UKALL 2011) in the United Kingdom. The fusion arose from interstitial deletion of 5q33 (n = 11), balanced rearrangement (n = 2), or complex rearrangement (n = 2). There was a predominance of females (n = 11), median age of 12 years, and median white blood cell count of 48.8 × 10(9)/L. Among 12 patients who achieved complete remission on earlier trials (ALL 97/99 and UKALL 2003), 10 were positive for minimal residual disease (MRD) at the end of induction, and 7 relapsed 18 to 59 months after diagnosis. The majority (9 of 12) remained alive 6 to 9 years after diagnosis. There are reports of EBF1-PDGFRB-positive patients who are refractory to conventional chemotherapy who achieve complete response when treated with the tyrosine kinase inhibitor imatinib. These findings have prompted screening for EBF1-PDGFRB in patients entered onto the current UKALL 2011 trial for whom induction therapy failed, who did not achieve remission by day 29, or who remained MRD positive (>0.5%) at week 14. Two UKALL 2011 patients, positive for EBF1-PDGFRB, received imatinib; 1 died 6 months after a matched unrelated bone marrow transplant as a result of undefined encephalopathy, and the other remained in remission 10 months after diagnosis.

Inhibition affecting RQ-PCR-based assessment of minimal residual disease in acute lymphoblastic leukemia: reversal by addition of bovine serum albumin

Inhibition affecting RQ-PCR-based assessment of minimal residual disease in acute lymphoblastic leukemia: reversal by addition of bovine serum albumin

The clinical relevance of detection of minimal residual disease in childhood acute lymphoblastic leukaemia

Risk directed treatment forms a central component of modern protocols for childhood acute lymphoblastic leukaemia (ALL). A review of recent studies of minimal residual disease (MRD) analysis shows that it is a powerful prognostic factor in both first line and relapse treatment. However, the value of MRD analysis is both time point and protocol specific, and the threshold for MRD detection of the technique used impacts upon the results obtained. MRD analysis does have a useful role to play in the risk directed treatment of childhood ALL, and this is currently being investigated in large prospective studies.

Parthenolide eliminates leukemia initiating cell populations and improves survival in xenografts of childhood acute lymphoblastic leukemia

Approximately 20% of children with acute lymphoblastic leukemia (ALL) relapse because of failure to eradicate the disease. Current drug efficacy studies focus on reducing leukemia cell burden. However, if drugs have limited effects on leukemia-initiating cells (LICs), then these cells may expand and eventually cause relapse. Parthenolide (PTL) has been shown to cause apoptosis of LIC in acute myeloid leukemia. In the present study, we assessed the effects of PTL on LIC populations in childhood ALL. Apoptosis assays demonstrated that PTL was effective against bulk B- and T-ALL cells, whereas the CD34(+)/CD19(-), CD34(+)/CD7(-), and CD34(-) subpopulations were more resistant. However, functional analyses revealed that PTL treatment prevented engraftment of multiple LIC populations in NOD/LtSz-scid IL-2Rγ(c)-null mice. PTL treatment of mice with established leukemias from low- and high-risk patients resulted in survival and restoration of normal murine hemopoiesis. In only 3 cases, disease progression was significantly slowed in mice engrafted with CD34(+)/CD19(-) or CD34(+)/CD7(-) and CD34(-) cells, but was not prevented, demonstrating that individual LIC populations within patients have different responses to therapy. These observations indicate that PTL may have therapeutic potential in childhood ALL and provide a basis for developing effective therapies that eradicate all LIC populations to prevent disease progression and reduce relapse.

Use of Minimal Residual Disease Assessment to Redefine Induction Failure in Pediatric Acute Lymphoblastic Leukemia.

Purpose Our aim was to determine the role of end-of-induction (EOI) minimal residual disease (MRD) assessment in the identification and stratification of induction failure in patients with pediatric acute lymphoblastic leukemia (ALL) and to identify genetic abnormalities that drive disease in these patients. Patients and Methods Analysis included 3,113 patients who were treated in the Medical Research Council UKALL2003 multicenter randomized trial (NCT00222612) between 2003 and 2011. MRD was measured by using standardized real-time quantitative PCR. Median follow-up was 5 years 9 months. Results Fifty-nine patients (1.9%) had morphologic induction failure with 5-year event-free survival (EFS) of 50.7% (95% CI, 37.4 to 64.0) and 5-year overall survival of 57.7% (95% CI, 44.2 to 71.2). Of these, a small proportion of patients with M2 marrow (6 of 44) and a low EOI MRD level (< 0.01%) had 5-year EFS of 100%. Conversely, among patients with morphologic remission 2.3% (61 of 2,633) had high MRD (≥ 5%) and 5-year EFS of 47.0% (95% CI, 32.9 to 61.1), which was similar to those with morphologic induction failure. Redefining induction failure to include morphologic induction failure and/or MRD ≥ 5% identified 3.9% (120 of 3,133 patients) of the trial cohort with 5-year EFS of 48.0% (95% CI, 39.3 to 58.6). Induction failure (morphologic or MRD ≥ 5%) occurred most frequently in T-ALL (10.1%; 39 of 386 T-ALL cases) and B-other ALL, that is, lacking established chromosomal abnormalities (5.6%; 43 of 772 B-other cases). Genetic testing within the B-other group revealed the presence of PDGFRB gene fusions, particularly EBF1-PDGFRB, in almost one third of B-other ALL cases. Conclusion Integration of EOI MRD level with morphology identifies induction failure more precisely than morphology alone. Prevalence of EBF1-PDGFRB fusions in this group highlights the importance of genetic screening to identify abnormalities that may be targets for novel agents.

Improved survival in matched unrelated donor transplant for childhood ALL since the introduction of high-resolution matching at HLA class I and II

We present the first detailed study analysing OS in BMT for paediatric ALL following the introduction of high-resolution (HR) HLA matching. A total of 356 consecutive paediatric ALL stem cell transplants performed between 1988 and 2007 were reviewed; 80 of them were performed following the introduction of HR HLA class I and class II matching to the transplant programme in 2002. Comparisons of matched unrelated donor (MUD) transplant outcomes before and after this period were made. Matching at the HR level for HLA-A, -B, -C, -DRB1 and -DQB1 (HR-MUD) correlated with a greater than 25% improvement in 2- and 5-year OS in paediatric ALL patients transplanted with MUDs (P=0.009, P=0.005, respectively). Two-year OS for contemporaneous HLA-matched sibling transplants (80.8%) and HR-MUD transplants (78.8%) was equivalent. At 6%, non-relapse mortality (NRM) in MUD transplants since 2002 was significantly reduced compared with previous epochs. Changes in treatment and epoch-dependent improvements in outcome were reviewed for possible confounders to the influence of HR typing using univariate and multivariate analysis.

Accurate Sample Assignment in a Multiplexed, Ultrasensitive, High-Throughput Sequencing Assay for Minimal Residual Disease

High-throughput sequencing (HTS) (next-generation sequencing) of the rearranged Ig and T-cell receptor genes promises to be less expensive and more sensitive than current methods of monitoring minimal residual disease (MRD) in patients with acute lymphoblastic leukemia. However, the adoption of new approaches by clinical laboratories requires careful evaluation of all potential sources of error and the development of strategies to ensure the highest accuracy. Timely and efficient clinical use of HTS platforms will depend on combining multiple samples (multiplexing) in each sequencing run. Here we examine the Ig heavy-chain gene HTS on the Illumina MiSeq platform for MRD. We identify errors associated with multiplexing that could potentially impact the accuracy of MRD analysis. We optimize a strategy that combines high-purity, sequence-optimized oligonucleotides, dual indexing, and an error-aware demultiplexing approach to minimize errors and maximize sensitivity. We present a probability-based, demultiplexing pipeline Error-Aware Demultiplexer that is suitable for all MiSeq strategies and accurately assigns samples to the correct identifier without excessive loss of data. Finally, using controls quantified by digital PCR, we show that HTS-MRD can accurately detect as few as 1 in 10(6) copies of specific leukemic MRD.

The impact of hyperglycemia on risk of infection and early death during induction therapy for acute lymphoblastic leukemia (ALL)

Hyperglycemia during induction chemotherapy for childhood acute lymphoblastic leukemia (ALL) has been inconsistently associated with risk of infection. We investigated the incidence of hyperglycemia during induction for childhood ALL in a retrospective cohort study of 144 patients treated on a single national protocol (UKALL2003) and explored its association with infection. All patients received dexamethasone. Overt hyperglycemia was seen in 36% and proven bacterial or fungal infection was most common in this group (OR 4.1 (1.1–15.6), P = 0.039). Both hyperglycaemia and infection were particularly common in patients with Down Syndrome. Pediatr Blood Cancer 2013;60:E157–E159.