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PURPOSE The aim of the randomized trial, UKALL2003, was to adjust treatment intensity on the basis of minimal residual disease (MRD) stratification for children and young adults with acute lymphoblastic leukemia. We analyzed the 10-year randomized outcomes and the time for patients to be considered cured (ClinicalTrials.gov identifier: NCT00222612 ). METHODS A total of 3,113 patients were analyzed including 1,054 patients who underwent random assignment (521 MRD low-risk and 533 MRD high-risk patients). Time to cure was defined as the point at which the chance of relapse was < 1%. The median follow-up time was 10.98 (interquartile range, 9.19-13.02) years, and survival rates are quoted at 10 years. RESULTS In the low-risk group, the event-free survival was 91.7% (95% CI, 87.4 to 94.6) with one course of delayed intensification versus 93.7% (95% CI, 89.9 to 96.1) with two delayed intensifications (adjusted hazard ratio, 0.73; 95% CI, 0.38 to 1.40; P = .3). In the high-risk group, the event-free survival was 82.1% (95% CI, 76.9 to 86.2) with standard therapy versus 87.1% (95% CI, 82.4 to 90.6) with augmented therapy (adjusted hazard ratio, 0.68; 95% CI, 0.44 to 1.06; P = .09). Cytogenetic high-risk patients treated on augmented therapy had a lower relapse risk (22.1%; 95% CI, 15.1 to 31.6) versus standard therapy (52.4%; 95% CI, 28.9 to 80.1; P = .016). The initial risk of relapse differed significantly by sex, age, MRD, and genetics, but the risk of relapse for all subgroups quickly coalesced at around 6 years after diagnosis. CONCLUSION Long-term outcomes of the UKALL2003 trial confirm that low-risk patients can safely de-escalate therapy, while intensified therapy benefits patients with high-risk cytogenetics. Regardless of prognosis, the time to cure is similar across risk groups. This will facilitate communication to patients and families who pose the question “When am I/is my child cured?”

Purpose:We investigated nilotinib exposure in pediatric patients with chronic myeloid leukemia (CML) or Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) resistant to, relapsed on, refractory to, or intolerant of previous treatment.Patients and Methods:Fifteen patients (aged 1–n = 11) or Ph+ ALL relapsed on or refractory to standard therapy (n = 4) enrolled in this phase I study. Nilotinib (230 mg/m2 twice daily; equivalent to the adult 400-mg twice-daily dose) was administered orally in 12 or 24 cycles of 28 days. The primary objective was to characterize the pharmacokinetics of nilotinib in pediatric patients.Results:The area under the concentration–time curve at steady state was slightly lower in pediatric patients versus adults (14,751.4 vs. 17,102.9 ng/h/mL); the geometric mean ratio (GMR; pediatric:adult) was 0.86 [90% confidence interval (CI), 0.70–1.06]. Body surface area–adjusted systemic clearance was slightly higher in pediatric versus adult patients (GMR, 1.30; 90% CI, 1.04–1.62). Nilotinib was generally well tolerated. The most common adverse events were headache, vomiting, increased blood bilirubin, and rash. Three patients with CML achieved major molecular response, and three with Ph+ ALL achieved complete remission.Conclusions:Nilotinib 230 mg/m2 twice daily in pediatric patients provided a pharmacokinetics and safety profile comparable with the adult reference dose; clinical activity was demonstrated in both CML and Ph+ ALL. This dose is recommended for further evaluation in pediatric patients. The safety profile was consistent with that in adults.

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Summary Background High hyperdiploidy is the most common genetic subtype of childhood acute lymphoblastic leukaemia and is associated with a good outcome. However, some patients relapse and, given its prevalence, patients with high hyperdiploidy account for a large proportion of all relapses. We aimed to evaluate putative risk factors and determine the optimal pattern of trisomies for predicting outcome. Methods We used discovery and validation cohorts from consecutive trials—UKALL97/99 (n=456) and UKALL2003 (n=725)—to develop the prognostic profile. UKALL97/99 recruited patients aged 1–18 years between Jan 1, 1997, and June 15, 2002, and UKALL2003 recruited children and young adults aged 1–24 years between Oct 1, 2003, and June 30, 2001, from the UK and Ireland who were newly diagnosed with acute lymphoblastic leukaemia. Cytogenetic and fluorescence in-situ hybridisation testing was performed on pre-treatment bone marrow samples by regional UK National Health Service genetic laboratories or centrally by the Leukaemia Research Cytogenetics Group, and results were reported using established nomenclature and definitions. We examined the prognostic effect of previously proposed genetic and non-genetic risk factors among patients with high hyperdiploid acute lymphoblastic leukaemia treated on UKALL2003. We used Bayesian information criterion, targeted projection pursuit, and multivariate analysis to identify the optimal number of trisomies, and best subset regression and multivariate analysis to identify the optimal combination. Survival analysis considered three endpoints, as follows: event-free survival, defined as time to relapse, second tumour, or death, censored at last contact; relapse rate, defined as time to relapse for those reaching complete remission, censored at death in remission or last contact; and overall survival, defined as time to death, censored at last contact. Findings The median follow-up time for UKALL97/99 was 10·59 years (IQR 9·25–12·06) and 9·40 years (8·00–11·55) for UKALL2003. UKALL97/99 included 208 female patients and 248 male patients, and UKALL2003 included 345 female patients and 380 male patients. We deduced that the trisomic status of four chromosomes provided the optimal information for predicting outcome. The good risk profile comprised karyotypes with +17 and +18 or +17 or +18 in the absence of +5 and +20. All remaining cases were classified in the poor risk profile. The ratio of patients with good risk and poor risk was 82:18 and 80:20 in the discovery and validation cohorts, respectively. In the validation cohort, patients with the high hyperdiploid good risk profile had an improved response to treatment compared with other patients with high hyperdiploidy at 10 years (relapse rate 5% [95% CI 3–7] vs 16% [10–23]; p

Treating refractory pediatric acute lymphoblastic leukemia (ALL) remains a challenge despite impressive remission rates (>90%) achieved in the last decade. The use of innovative immunotherapeutic approaches such as anti-CD19 chimeric antigen receptor T cells does not ensure durable remissions, because leukemia-propagating cells (LPCs) that lack expression of CD19 can cause relapse, which signifies the need to identify new markers of ALL. Here we investigated expression of CD58, CD97, and CD200, which were previously shown to be overexpressed in B-cell precursor ALL (BCP-ALL) in CD34+/CD19+, CD34+/CD19–, CD34–/CD19+, and CD34–/CD19– LPCs, to assess their potential as therapeutic targets. Whole-genome microarray and flow cytometric analyses showed significant overexpression of these molecules compared with normal controls. CD58 and CD97 were mainly co-expressed with CD19 and were not a prerequisite for leukemia engraftment in immune deficient mice. In contrast, expression of CD200 was essential for engraftment and serial transplantation of cells in measurable residual disease (MRD) low-risk patients. Moreover, these CD200+ LPCs could be targeted by using the monoclonal antibody TTI-CD200 in vitro and in vivo. Treating mice with established disease significantly reduced disease burden and extended survival. These findings demonstrate that CD200 could be an attractive target for treating low-risk ALL, with minimal off-tumor effects that beset current immunotherapeutic approaches.

Summary Relapse of paediatric acute lymphoblastic leukaemia (ALL) may occur due to persistence of resistant cells with leukaemia‐propagating ability (LPC). In leukaemia, the balance of B‐cell lymphoma‐2 (BCL‐2) family proteins is disrupted, promoting survival of malignant cells and possibly LPC. A direct comparison of BCL‐2 inhibitors, navitoclax and venetoclax, was undertaken on LPC subpopulations from B‐cell precursor (BCP) and T‐cell ALL (T‐ALL) cases in vitro and in vivo. Responses were compared to BCL‐2 levels detected by microarray analyses and Western blotting. In vitro, both drugs were effective against most BCP‐ALL LPC, except CD34−/CD19− cells. In contrast, only navitoclax was effective in T‐ALL and CD34−/CD7− LPC were resistant to both drugs. In vivo, navitoclax was more effective than venetoclax, significantly improving survival of mice engrafted with BCP‐ and T‐ALL samples. Venetoclax was not particularly effective against T‐ALL cases in vivo. The proportions of CD34+/CD19−, CD34−/CD19− BCP‐ALL cells and CD34−/CD7− T‐ALL cells increased significantly following in vivo treatment. Expression of pro‐apoptotic BCL‐2 genes was lower in these subpopulations, which may explain the lack of sensitivity. These data demonstrate that some LPC were resistant to BCL‐2 inhibitors and sustained remission will require their use in combination with other therapeutics.

Risk stratification is essential for the delivery of optimal treatment in childhood acute lymphoblastic leukemia. However, current risk stratification algorithms dichotomize variables and apply risk factors independently, which may incorrectly assume identical associations across biologically heterogeneous subsets and reduce statistical power. Accordingly, we developed and validated a prognostic index (PIUKALL) that integrates multiple risk factors and uses continuous data. We created discovery (n = 2405) and validation (n = 2313) cohorts using data from 4 recent trials (UKALL2003, COALL-03, DCOG-ALL10, and NOPHO-ALL2008). Using the discovery cohort, multivariate Cox regression modeling defined a minimal model including white cell count at diagnosis, pretreatment cytogenetics, and end-of-induction minimal residual disease. Using this model, we defined PIUKALL as a continuous variable that assigns personalized risk scores. PIUKALL correlated with risk of relapse and was validated in an independent cohort. Using PIUKALL to risk stratify patients improved the concordance index for all end points compared with traditional algorithms. We used PIUKALL to define 4 clinically relevant risk groups that had differential relapse rates at 5 years and were similar between the 2 cohorts (discovery: low, 3% [95% confidence interval (CI), 2%-4%]; standard, 8% [95% CI, 6%-10%]; intermediate, 17% [95% CI, 14%-21%]; and high, 48% [95% CI, 36%-60%; validation: low, 4% [95% CI, 3%-6%]; standard, 9% [95% CI, 6%-12%]; intermediate, 17% [95% CI, 14%-21%]; and high, 35% [95% CI, 24%-48%]). Analysis of the area under the curve confirmed the PIUKALL groups were significantly better at predicting outcome than algorithms employed in each trial. PIUKALL provides an accurate method for predicting outcome and more flexible method for defining risk groups in future studies.

Minimal residual disease (MRD) measured on end-of-induction bone marrow (BM) is the most important biomarker for guiding therapy in pediatric acute lymphoblastic leukemia (ALL). Due to limited sensitivity of current approaches, peripheral blood (PB) is not a reliable source for identifying patients needing treatment changes. We sought to determine if high-throughput sequencing (HTS) (next-generation sequencing) of rearranged immunoglobulin and T-cell receptor genes can overcome this and be used to measure MRD in PB.We employed a quantitative HTS approach to accurately measure MRD from one million cell equivalents of DNA from 17 PB samples collected at day 29 after induction therapy in patients with precursor B-cell ALL. We compared these results to the gold-standard real-time PCR result obtained from their paired BM samples, median follow-up 49 months.With the increased sensitivity, detecting up to one abnormal cell in a million normal cells, we were able to detect MRD in the PB by HTS in all those patients requiring treatment intensification (MRD ≥ 0.005% in BM).This is proof of principle that using the increased sensitivity of HTS, PB can be used to measure MRD and stratify children with ALL. The method is cost effective, rapid, accurate, and reproducible, with inherent advantages in children. Importantly, increasing the frequency testing by PB as opposed to intermittent BM sampling may allow extension of the dynamic range of MRD, giving a more complete picture of the kinetics of disease remission while improving relapse prediction and speed of detection.

Congenital sideroblastic anaemias (CSA) are a rare group of disorders characterized by the presence of pathologic iron deposits within the mitochondria of erythroid precursors (ring sideroblasts) in the bone marrow due to heterogenous germline mutations leading to defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Patients present with anaemia and relative reticulocytopenia, and systemic iron overload secondary to chronic ineffective erythropoiesis, leading to end-organ damage. The disease is heterogenous underlying the genetic variability and the variable response to treatment. Although a number of CSA patients have received a bone marrow transplant, the outcomes and toxicities are not known. This status makes it very difficult to understand the role of BMT in the management of CSA. A search in the EBMT database identified 28 patients receiving a HSCT for CSA between 1998 to 2018 by 24 participating centres. The median year of transplantation was 2014 (IQR 2004-2016). The distribution was equal between males (n=14) and females (n=14). The median age at transplantation was 7 years of age (3-10 years). Fifteen patients had a sibling HSCT (88%), one a family matched donor HSCT (6%) and one an unrelated matched (6%), the type of transplant being unknown in others (n=11). The source of stem cells was bone marrow in 20 cases (74%), peripheral blood in 4 cases (15%), cord blood in 2 (7%) and combined bone marrow and cord in one (4%). Five cases had a Bu/Cy based conditioning regimen, 4 had Bu/fludarabine based regimen and three fludarabine/treosulfan based conditioning with the rest having a variety of approaches. Eighty-six percent of cases had serotherapy with ATG or alemtuzumab. The median follow-up was 31.6 months (95% CI, 12.2-74.1%). The overall survival at 12 and 24 months was 88% (76-100) and 82% (66-99), respectively (figure 1). The median neutrophil engraftment was 18 (15-21) days and platelet engraftment >20 x 109/L was 29 (20-51) days, with a graft failure incidence of 7% (0-17) at 12 months. Two patients suffered from VOD. There were four deaths, three of which were related to transplant complications. The event free survival (survival without graft failure, relapse and second transplant) at 12 and 24 months was 85% (72-99) (figure 2). Six patients developed acute GvHD grade II and one case grade III; giving a grade II/III incidence of 28% (10-46). There was one case of limited and one of chronic GvHD, giving an incidence of 11% (0-26%) at 12 months and 24 months. In conclusion, whilst HSCT for CSA is a rare occurrence, these data demonstrate that HSCT for this condition is feasible and the outcomes are in keeping with those obtained for transplantation for transfusion dependent anaemias during the same time-period. Disclosures Handgretinger: Amgen: Honoraria. Moraleda:Gilead: Consultancy, Other: Travel Expenses; Jazz Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy, Other: Travel Expenses; Sandoz: Consultancy, Other: Travel Expenses; Takeda: Consultancy, Other: Travel Expenses. Risitano:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alnylam: Research Funding; Alexion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Jazz: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Amyndas: Consultancy; RA pharma: Research Funding; Biocryst: Membership on an entity's Board of Directors or advisory committees; Apellis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Achillion: Membership on an entity's Board of Directors or advisory committees; Pfizer: Speakers Bureau. Peffault De Latour:Amgen: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Apellis: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

10001 Background: Approximately 15-20% of pediatric pts with ALL or LL will be refractory to/relapse after frontline treatment; relapsed disease is associated with poor outcomes. In a phase 2 study, 2 of 7 (28.6%) pts with T-cell ALL in first relapse achieved a complete response (CR) using the vincristine, prednisone, PEG-asparaginase, and doxorubicin (VPLD) reinduction backbone. DARA, a human IgGκ monoclonal antibody targeting CD38 approved for treating multiple myeloma, has shown preclinical efficacy in ALL models. We report the initial results of DARA plus VPLD in pediatric and young adult pts with relapsed/refractory T-cell ALL or LL enrolled in the phase 2, open-label DELPHINUS study. Methods: Eligible pts were aged 1-30 y, had T-cell ALL or LL in first relapse or refractory to 1 prior induction/consolidation regimen, and had a performance status ≥70. DARA (16 mg/kg IV QW) was given with VPLD in Cycle 1 and with methotrexate, cyclophosphamide, cytarabine, and 6-mercaptopurine in Cycle 2. Pts received age/risk-adjusted intrathecal therapy. Response was measured at the end of each cycle by local bone marrow morphology. The primary endpoint for ALL pts was CR rate in pediatric pts at the end of Cycle 1. Pts achieving CR after Cycles 1 or 2 could proceed to allogeneic HSCT off study. Overall response rate (ORR) was defined as CR or CR with incomplete hematological recovery (CRi) at any time before start of subsequent therapy or HSCT. Minimal residual disease (MRD) negativity (< 0.01%) at any time before disease progression, start of subsequent therapy, or HSCT was centrally reviewed by flow cytometry. Results: Twenty-four pediatric (age 1-17 y) and 5 young adult (age 18-30 y) ALL pts and 10 LL pts (age 1-30 y) received ≥1 DARA dose. Median (range) age was 10.0 (2-25) y (ALL) and 14.5 (5-22) y (LL); median (range) time from initial diagnosis to first study treatment was 2.0 (0.1-6.1) y (ALL) and 0.8 (0.5-6.0) y (LL). Pediatric ALL pts received a median (range) of 2 (1-3) treatment cycles; young adult ALL pts and LL pts each received 2 (1-2). Among pediatric ALL pts, 10 (41.7%) pts (90% CI, 24.6-60.3) achieved CR at the end of Cycle 1. ORR was 83.3% (CR, 13 [54.2%] pts; CRi, 7 [29.2%] pts) in pediatric and 60.0% (all CR) in young adult ALL pts and 40.0% (all CR) in LL pts. Ten (41.7%) pediatric ALL pts achieved MRD negativity. All pediatric ALL pts had a grade 3/4 TEAE. No pediatric ALL pt discontinued DARA primarily due to AEs and 1 (4.2%) died due to TEAEs (brain edema and hepatic failure) attributed to study treatment but unrelated to DARA. Conclusions: The addition of DARA to VPLD in pediatric and young adult pts with relapsed/refractory T-cell ALL or LL showed initial activity, generating improved response rates compared to those achieved with backbone therapy alone, with a manageable safety profile. Clinical trial information: NCT03384654.

Patients with an ABL-class fusion have a high risk of relapse on standard chemotherapy but are sensitive to tyrosine kinase inhibitors (TKI). In UKALL2011, we screened patients with post-induction MRD ≥1% and positive patients (12%) received adjuvant TKI. As the intervention started during UKALL2011, not all eligible patients were screened prospectively. Retrospective screening of eligible patients allowed the outcome of equivalent ABL-class patients who did and did not receive a TKI in first remission to be compared. ABL-class patients who received a TKI in first remission had a reduced risk of relapse/refractory disease: 0% vs. 63% at four years (P = 0·009).

Background In a multicenter phase 1 study of children with relapsed/refractory acute lymphoblastic leukemia (ALL), moxetumomab pasudotox, an anti-CD22 immunotoxin, demonstrated a manageable safety profile and preliminary evidence of clinical activity. A phase 2 study further evaluated efficacy. Procedure This international, multicenter, phase 2 study enrolled children with relapsed/refractory B-cell precursor ALL who received moxetumomab pasudotox 40 µg/kg intravenously every other day, for six doses per 21-day cycle. The primary objective was to evaluate the complete response (CR) rate. Secondary objectives included safety, pharmacokinetics, and immunogenicity evaluations. Results Thirty-two patients (median age, 10 years) were enrolled at 16 sites; 30 received study drug and were evaluable for safety; 28 were evaluable for response. The objective response rate was 28.6%, with three patients (10.7%) achieving morphologic CR, and five patients (17.9%) achieving partial response. Disease progression occurred in 11 patients (39.3%). Ten patients (33.3%) experienced at least one treatment-related serious adverse event, including capillary leak syndrome (CLS; n = 6), hemolytic uremic syndrome (HUS; n = 4), and treatment-related death (n = 1) from pulmonary edema. No differences were observed in inflammatory markers in patients who did or did not develop CLS or HUS. Conclusions Despite a signal for clinical activity, this phase 2 study was terminated at interim analysis for a CR rate that did not reach the stage 1 target. Preclinical data suggest enhanced efficacy of moxetumomab pasudotox via continuous infusion or in combination regimens; thus, further studies designed to optimize the efficacy and safety of moxetumomab pasudotox in pediatric ALL may be warranted.

Current therapies for childhood T-cell acute lymphoblastic leukemia have increased survival rates to above 85% in developed C countries. Unfortunately, some patients fail to respond to therapy and many suffer from serious side effects, highlighting the need to investigate other agents to treat this disease. Parthenolide, a nuclear factor kappa (κ)B inhibitor and reactive oxygen species inducer, has been shown to have excellent anti-cancer activity in pediatric leukemia xenografts, with minimal effects on normal hemopoietic cells. However, some leukemia initiating cell populations remain resistant to parthenolide. This study examined mechanisms for this resistance, including protective effects conferred by bone marrow stromal components. T-cell acute leukemia cells co-cultured with mesenchymal stem cells demonstrated significantly enhanced survival against parthenolide (73±11%) compared to cells treated without mesenchymal stem cell support (11±9%). Direct cell contact between mesenchymal cells and leukemia cells was not required to afford protection from parthenolide. Mesenchymal stem cells released thiols and protected leukemia cells from reactive oxygen species stress, which is associated with parthenolide cytotoxicity. Blocking cystine uptake by mesenchymal stem cells, using a small molecule inhibitor, prevented thiol release and significantly reduced leukemia cell resistance to parthenolide. These data indicate it may be possible to achieve greater toxicity to childhood T-cell acute lymphoblastic leukemia by combining parthenolide with inhibitors of cystine uptake.

Introduction The use of dexamethasone in acute lymphoblastic leukaemia therapy contributes to short- and long-term toxicities. The UKALL 2011 randomised trial investigated whether a more intense dexamethasone dose (10 mg/m2/d x 14d, short vs 6 mg/m2/d x 28d, standard) would lead to a more rapid cytoreduction and reduced adverse effects associated with longer durations of steroids in induction. The impact of dose and duration on dexamethasone pharmacokinetics was investigated. Methods Blood samples were obtained on one of the first three and last three days of induction dexamethasone dosing at time points up to 8 h after oral administration. Plasma dexamethasone levels were quantified in 1084 plasma samples obtained from 174 children and a population pharmacokinetic model developed. Results Drug exposure varied significantly between patients, with a >12-fold variation in AUC0–12h values and a marked overlap in dexamethasone exposures between dose levels. Intuitively, AUC0–12h was significantly higher with short dosing (10 mg/m2/d), but cumulative exposure was significantly higher with standard dosing over 28 days, after a higher cumulative dose. Concomitant rasburicase administration was associated with a 60% higher dexamethasone clearance. Day 8 bone marrow response was comparable between dosing arms, but those with 5%. No statistical differences were observed between arms in terms of steroid-related toxicity or minimal residual disease at the end of induction. Conclusion The potential significance of dexamethasone AUC0–12h on early response and higher cumulative exposure on the standard arm suggest that duration of therapy and exposure may be more important factors than absolute dose from a clinical pharmacology perspective.

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 EBF1-PDGFRB gene fusion accounts for 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.

Microsatellite analyses show that self-reported ethnicity often correlates poorly with true genetic ancestry. As unknown ancestral differences could potentially have an impact on transplant outcome, we developed an average allele length discrepancy (AALD) score to assess allele length discrepancy between donor/recipient (D/R) using microsatellites analysed routinely in post-transplant chimeric assessment. This was then compared with outcome in a homogeneously treated cohort of pediatric patients undergoing high-resolution sibling or matched unrelated donor transplantation for acute lymphoblastic leukemia (ALL). AALD scores formed a numeric continuum ranging from 0 to 1.4 (median 0.76) for sibling pairs and 0.8–2.17 (median 1.6) for high-resolution matched unrelated donor (HR-MUD) pairs. There was a trend for worse OS with increasing AALD score, which reached statistical significance above a threshold of 1.7 for OS. Patients whose transplants had an AALD score of ⩾1.8 had a risk of non-relapse mortality 4.9 times greater (P=0.025) and relapse risk three times greater (P=0.058) than those scoring

Purpose Minimal residual disease (MRD) and genetic abnormalities are important risk factors for outcome in acute lymphoblastic leukemia. Current risk algorithms dichotomize MRD data and do not assimilate genetics when assigning MRD risk, which reduces predictive accuracy. The aim of our study was to exploit the full power of MRD by examining it as a continuous variable and to integrate it with genetics. Patients and Methods We used a population-based cohort of 3,113 patients who were treated in UKALL2003, with a median follow-up of 7 years. MRD was evaluated by polymerase chain reaction analysis of Ig/TCR gene rearrangements, and patients were assigned to a genetic subtype on the basis of immunophenotype, cytogenetics, and fluorescence in situ hybridization. To examine response kinetics at the end of induction, we log-transformed the absolute MRD value and examined its distribution across subgroups. Results MRD was log normally distributed at the end of induction. MRD distributions of patients with distinct genetic subtypes were different ( P.001). Patients with good-risk cytogenetics demonstrated the fastest disease clearance, whereas patients with high-risk genetics and T-cell acute lymphoblastic leukemia responded more slowly. The risk of relapse was correlated with MRD kinetics, and each log reduction in disease level reduced the risk by 20% (hazard ratio, 0.80; 95% CI, 0.77 to 0.83; P.001). Although the risk of relapse was directly proportional to the MRD level within each genetic risk group, absolute relapse rate that was associated with a specific MRD value or category varied significantly by genetic subtype. Integration of genetic subtype-specific MRD values allowed more refined risk group stratification. Conclusion A single threshold for assigning patients to an MRD risk group does not reflect the response kinetics of the different genetic subtypes. Future risk algorithms should integrate genetics with MRD to accurately identify patients with the lowest and highest risk of relapse.

Whilst there is a classic triad of symptoms (pallor, fever and easy bruising) are present in a majority of patients with ALL, a significant minority have a more varied presentation. Bone pain and the signs and symptoms of mediastinal and central nervous system involvement are of particular relevance. Time to presentation for medical attention is related to presenting symptoms. In this chapter, the clinical and laboratory features of ALL at presentation are discussed, as are the essentialdiagnostic investigations. Finally, clinical and laboratory prognostic variables and their interactions are reviewed.

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.

Glucose-6-phosphate dehydrogenase deficiency affects approximately 400 million people worldwide and is an X-linked disorder most commonly found in individuals of African, Asian, Mediterranean, and Middle Eastern descent. It can present with acute hemolysis in response to certain drugs, infections, or fava beans, and affected individuals may not be aware that they have glucose-6-phosphate dehydrogenase deficiency. This case illustrates the importance of those working in the acute and urgent care sector having an awareness of the condition and the value of a full set of vital signs in an unwell child.

A rare case of a low incidence red cell antigen causing severe fetal allo-immune red cell disease is presented. Discussion of how this can be diagnosed and successfully managed antenatally using middle cerebral artery Doppler ultrasound and maternal antibody titre levels for fetal surveillance and timing of intervention with intrauterine transfusion.

[no abstract]

Objective/Aim: To evaluate the safety of transfusing pooled, whole blood-derived granulocytes in additive solution and plasma (GASP) in 30 recipients. Background: Demand for granulocytes in England has increased five-fold. With the advantages of reduced red cell, plasma and overall volume, GASP maintains function in vitro. Methods and Materials: Observations were recorded prior to and post transfusion. Increments were recorded at 1 h and the following morning. Leucocyte antibody screening was undertaken prior to and at 1–6 months following transfusion. Results: Thirty patients aged between 8 months and 68 years received 221 GASP in 148 transfusion episodes. GASP contained an average of 1·0 ×1010 granulocytes in 207 mL. Adults usually received two packs and children 10–20 mL kg–1. Children and adults received a median [interquartile range (IQR)] dose of 12·5 (9·1–25·3) and 19·7 (12·0–25·8) ×109 granulocytes per transfusion, respectively. There was one episode of transfusion-associated circulatory overload (TACO) in a patient with chronic cardiac failure following 600 mL of unpooled granulocytes, other fluids and one GASP. New leucocyte alloimmunisation occurred in 3/30 recipients 10%. No other significant reactions were reported. Median peripheral blood neutrophil increments at 1 h post transfusion were 0·06 (IQR, 0·01–0·17) in children and (0·03) (IQR, 0–0·16) in adults. Conclusion: GASP has a similar safety profile to other sources of granulocytes for patients with refractory infection or in need of secondary prophylactic transfusion. Further studies are required to clarify the role of GASP in the treatment of neutropenic patients.

Introduction Reported outcomes for children with Downs syndrome (DS) and acute lymphoblastic leukaemia (ALL) treated on our previous national study, UKALL 2003, were inferior compared to non-DS children. 5 year event free survival (EFS) for DS children was 65.6% vs. 87.7% for non-DS children and 5 year overall survival (OS) was 70.0% vs. 92.2% (British Journal of Haematology 2014; 165: 552-555). Excess treatment related mortality (TRM, 21.6% vs. 3.3% at 5 years) in children with DS-ALL was primarily due to infection. Similar results have been reported by other study groups. In our successor study, UKALL 2011, we employed several steps of de-escalation for DS-ALL patients compared to standard therapy. Here, we report that de-escalated therapy has resulted in a dramatic reduction in TRM for children with DS-ALL treated in the UK. Methods DS-ALL patients treated on UKALL 2011 had their treatment modified in the following respects: 1)NCI high risk patients did not receive anthracycline in induction unless they had a slow early response at day 15; 2) two years maintenance was given for both boys and girls; 3) no pulses were given in maintenance for MRD low risk patients. In addition, we recommended use of prophylactic antibiotics during induction and intensive phases of treatment. Fisher's exact test was used to compare induction mortality in the DS and non-DS pts. OS was defined as time from registration to death. EFS was defined as time from registration to first event (induction failure, relapse, second malignancy or death from any cause). The Kaplan-Meier method was used for survival estimation. Results The UKALL 2011 trial opened to recruitment on 26th April 2012 and has recruited 2362 patients, of whom 50 have Down syndrome, in the period up to 22nd February 2018. The study is open in 41 centres in UK and Ireland and included patients with both ALL and lymphoblastic lymphoma (LBL). The baseline characteristics of the patients recruited up to this date are shown in the table below. For DS-ALL, Day 29 MRD was available for 48 patients and was low risk in 25 cases (50%), risk in 19 (38 %) and no result was obtained in 4 (8 %). The therapy was generally well-tolerated, there have been no cases of therapy curtailment due to toxicity and only one patient due to receive intensified therapy per MRD risk assessment was unable to escalate due to toxicity. At a median follow-up of 32.7 months, there have been 4 events. These include two relapses (relapse rate: 4.1% (1.1-15.5) at 3 years) and two TRMs (one TRM in induction and the other in consolidation; both due to infection). Three year EFS is: 92.9% (95% CI: 79.1 - 97.7) with a three year OS of 95.9% (84.5 - 99.0). The remarkably low induction TRM for DS children on UKALL 2011 (1 death out of 50 patients, 2%), is comparable to that of non-DS children treated on the same protocol with a 3 drug induction (12 out of 1174 children, 1% induction TRM; induction TRM in DS-ALL vs non-DS-ALL p=0.42). It is also comparable to the induction TRM in DS-ALL noted in the latter half of the UKALL 2003 study (1/40, 2.5%) when the aforementioned treatment modifications were first implemented in the UK. Importantly, this TRM is a quarter of that when an historic cohort of DS-ALL children were treated with a 4-drug induction on our previous study (UKALL 2003, DS-ALL TRM 4/46 patients i.e. 8 %, p=0.19). Conclusion These preliminary results suggest that de-escalation of treatment is successful in reducing treatment related mortality for children with DS-ALL without increasing the risk of early relapse, however longer term follow-up is needed. Disclosures Ghorashian: Celgene: Other: travel support; Novartis: Honoraria. Hough:University College London Hospital's NHS Foundation Trust: Employment. Kearns:Pfizer: Consultancy, Research Funding; Galen: Research Funding. Vora:Jazz: Other: Advisory board; Medac: Other: Advisory board; Pfizer: Other: Advisory board; Amgen: Other: Advisory board; Novartis: Other: Advisory board.

BackgroundMinimal residual disease (MRD) is defined as the presence of sub-microscopic levels of leukaemia. Measurement of MRD from bone marrow at the end of induction chemotherapy (day 28) for childhood acute lymphoblastic leukaemia (ALL) can highlight a large group of patients (>40%) with an excellent (>90%) short-term event-free survival (EFS). However, follow-up in recent published trials is relatively short, raising concerns about using this result to infer the safety of further therapy reduction in the future.MethodsWe examined MRD data on 225 patients treated on one of three UKALL trials between 1997 and 2003 to assess the long-term (>10 years follow-up) outcome of those patients who had low-risk MRD (ResultsOur pilot data define a cohort of 53% of children with MRD ConclusionsThe excellent outcome for childhood ALL in patients with MRD

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.

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.

‘Stroke-like syndrome’ caused by intrathecal methotrexate in patients treated during the UKALL 2003 trial

Non-specific amplification of background DNA may cause false positive MRD results using conventional Ig/TCR gene rearrangement RQ-PCR; non-amplification control (NAC) samples taken from pooled buffy coat lymphocytes are thus routinely analysed in parallel to test samples. In contrast NGS-based MRD relies solely on DNA sequence specificity to avoid false positives. However at certain alleles gene rearrangement diversity is limited and false positive results remain a risk (the same DNA sequence from normal not leukaemic lymphocytes). We therefore assessed the relevant parameters of Ig/TCR gene rearrangement (5'/3' gene selection, deletion/insertion size) across all loci screened by the Biomed-2 PCR panel, in pooled buffy coat (24 donors), single blood donor cones and patient samples at end of induction (EOI). This subsequently allows us to determine an 'e-score', the probability that a given sequence is generated by chance. This allows investigators to avoid selecting gene rearrangements with poor diversity for MRD analysis in a systematic manner. In a 1st round PCR DNA was amplified with primers covering 6 Ig/TCR loci (IgH, inc. IgH, IgK, TRB, TRD and TRG) containing partial MiSeq adaptor sequences. The resulting amplicons were purified using Agencourt AMPure XP beads (Beckman Coulter, Jersey City, NJ). The amplicons acted as templates in a 2nd round PCR, using primers containing index and full MiSeq adaptor sequences. Samples were purified, then quantitated by Qubit (InvitroGen, Carlsbad, CA) and TapeStation analysis (Agilent, Santa Clara, CA). This data was used to normalise DNA and construct the final sequencing library which was quantified using the Kapa Library Quantification Kit (Kapa Biosystems, Wilmington, MA) to ensure ideal cluster density. PhiX Sequencing Control v3 (Illumina, San Diego, CA) was added to the sequencing library at 8% to ensure early cycle high sequence diversity. Bi-directional sequencing was completed using Illumina MiSeq 500 cycle cartridge and Nano Reagent Kit (Illumina, San Diego, CA). The Vidjil bioinformatic pipeline (Bonsai team, CRIStAL, Lille, France) was used for data analysis. As illustrated in Fig 1 for TRG, 5' and 3' allele selection was found to be biased at all loci. However, the biases were stable across NAC, cone and importantly EOI samples. EOI samples were somewhat more oligoclonal than those of normal individuals (146 vs 212 vs 255 clones/1000 reads for EOI, NAC and cone respectively) leading to slightly greater variance around mean allele usage. Mean 5' and 3' deletion lengths and N insertion size varied both across loci and between alleles of a given gene. The patterns were stable across NAC, cone and EOI samples (fig 2). As the data from NAC samples is representative of background DNA in typical end of induction test samples, an e-score for defining rearrangement probability is therefore calculable (fig 3). Example e-scores for typical rearrangements are: - VH2.5-DH3.22-JH4.02 (-4/7/-7)(-5/9/-6) = 2.03x10-11 DD2-DD3 (-3/5/-3) = 1.06x10-4 TRBV6-2*01-TRBD2*01-TRBJ2-3*01 (-3/7/-4)(-2/10/-5) = 2.05x10-11 VKINT-KDE (-5/6/-1) = 9.94x10-5 IGKV3-20*01 (-3/5/-6) KDE = 4.32x10-6 The worst e-score would be found for rearrangements with poor diversity including IGK-KDE rearrangements (VKInt-KDE 0/0/0 e = 1.7x10-01) and certain TRG rearrangements (V8*01/J1*02 0/0/0 e=1.3x10-03). In contrast the worst e-score for IgH (V3-23*01-D6-13-J4*02(0/0/0)(0/0/-4) is 3.5x10-07. Incorporating a 1 log margin of safety discrimination of specific amplification over background amplification as per Ig/TCR RQ-PCR MRD criteria would mean that an e-score of In conclusion the e-score provides a standardised methodology for robust target selection that is useful for quality control purposes and one that should be incorporated into clinical NGS-MRD pipelines. Disclosures Moppett: Jazz Pharmaceuticals: Honoraria.

Paediatric ALL is heterogeneous in terms of clinical presentation, response to therapy and underlying genetic mechanisms. The application of treatment stratification algorithms has resulted in survival rates exceeding 90%. However, current algorithms apply risk factors as binary variables using arbitrary thresholds and in an independent manner. This approach potentially distorts biological heterogeneity resulting in loss of prediction accuracy. Therefore, we sought to determine whether treating clinical risk factors and MRD response as continuous variables and integrating genetic features can refine prognostication. A total of 2542 patients, aged 1-24 years old, recruited to UKALL2003 were available for analysis. NCI standard/high patients were initially assigned to regimen A/B. Sow early responders and those with HR genetics were transferred to the more intensive regimen C. Minimal residual disease (MRD) was evaluated at the end of induction (EOI) by real-time quantitative PCR analysis of Ig/TCR rearrangements. Patients with EOI MRD >0.01% were randomised to stay on regimen A/B or move to C whereas patients with EOI MRD In order to examine MRD as a continuous variable, we log transformed the raw EOI MRD value assuming a minimum detection level of 0.00001 and assigned cases with undetectable MRD a value of one log below this threshold. The transformed MRD variable [t(MRD)] followed a log normal distribution (Figure 1). Importantly, this log normal distribution was distinct across the genetic subtypes (Figure 1, p Next we combined multiple significant risk factors from univariate analysis using forward selection modelling to fit a multivariate prognostic model. The final model included log(WCC) and t(MRD) as continuous variables and two binary genetic variables: GRcyto and HRcyto. Using the coefficients from this model, we constructed a linear model which enabled the calculation of individual risk scores. Figure 2 shows the correlation between an individual's risk score and their RR. To investigate the clinical relevance of this risk score, we generated five risk groups based on ascending RR (Table 2) which have significant differential outcomes (all p25% and together with the standard risk (SR) group capture >70% relapses and, importantly, >80% of HR relapses (very early/early relapses). Among the 136 VLR/LR patients who had MRD >0.01%, there was no difference in EFS between patients randomised to regimen A/B v C (97% v 99%, p=0.5). Equally among 470 VLR/LR patients with MRD In conclusion, we have showed that EOI MRD is log normally distributed but that the shape of the distribution depends on genetic subtype; suggesting differential MRD kinetics. In addition, we have shown that using a single MRD threshold does not reflect the biological heterogeneity that is a fundamental feature of the disease. Finally, we demonstrate that integrating clinical risk factors as continuous variables with genetics can better define prognostic subgroups. Disclosures No relevant conflicts of interest to declare.

PCR bias is a potential confounder for PCR-based NGS-MRD quantitation. A method was published using artificial DNA constructs (gBlocks, IDT Technologies, Coralville, IO) to assess PCR primer bias and correct for it(1). We sought to confirm those findings by assessing PCR bias of the Biomed-2 TRG primer set. 36 synthetic DNA templates of 495bp length gBlocks combining each TRG V (V 2,3,4,5,7,8,9,10,11) & J gene (JG1-01, JG1-02, JP1, JP2) amplified by the Biomed-2 primer set were synthesised containing external MiSeq flow cell adaptor sequence (universal primer (UP)) binding sites, VG and JG sequence and junctional regions. Barcodes inserted internal to the universal primer sites and centrally enabled accurate template identification (fig 1). Individual gBlocks supplied at a nominal concentration of 10ng/uL (30nM) were quantitated by TapeStation (Agilent, Santa Clara, CA) and KAPA (Kapa Biosystems, Wilmington, MA) methodologies and pooled (unadjusted). gBlockswithin the pool were further quantitated by both 6 and 8 cycles of PCR with universal MiSeq adaptor primers. Next the gBlock pool was amplified in triplicate in a 2-stage PCR process: 1) Using Biomed-2 TRG primers that contained partial MiSeq adaptor sequences. The resulting products were purified with Agencourt AMPure XP beads (Beckman Coulter, Jersey City, NJ). 2) These amplicons were further amplified by primers containing indices and full MiSeq adaptor sequences. After purification, amplicons were Qubit (InvitroGen, Carlsbad, CA) and TapeStation analysed for normalisation and to construct the sequencing library, which was KAPA quantitated to ensure ideal cluster density. Bi-directional sequencing was performed using an Illumina MiSeq 500 cycle cartridge and Nano Reagent Kit (Illumina, San Diego, CA). Bioinformatics analysis was performed using the Vidjil platform (Bonsai team, CRIStAL, Lille, France). TapeStation quantitation showed a mean concentration of 3.3ng/uL (10nM) (range 1.1-6.3ug/uL). KAPA quantitation by contrast showed low quantities of all gBlocks (2nM, 0.67ug/uL) with the exception of all VG9 and most VG10 constructs (16nM, 5ug/uL)(fig 2a). Comparison of 6 vs 8 cycles of amplification using universal primers showed good correlation (R2=0.991) confirming that PCR cycle length does not affect the UP amplification (fig 2b), but there was an 8.5-fold variation in gBlock quantitation by UP PCR. There was no correlation between NGS quantitation and with either TapeStation or KAPA quantitation. Family specific V & J gene amplification efficiency for the Biomed-2 primers is shown in fig 3. Amplification efficiencies of V gene primers were This study has shown that there are clear PCR efficiency differences between the V primers in the Biomed-2 primer set, with VG9 and 10 being 5x more efficient. This could be adjusted for by reducing the concentrations of the VG9/10 primers. However, the variable results of the 3 methods used to quantitate the artificial templates raises significant questions about the rationale. TapeStation quantitation is not PCR based and produced broadly equivalent (but lower than anticipated) gBlock concentrations for the individual constructs. PCR based KAPA showed very low concentrations in all but the VG9/VG10 templates. It is notable that this exactly parallels the results for the locus specific PCR. The most plausible explanation is that unexpected secondary structure reduces the amplifiability of the templates in PCR reactions. The near 10-fold variation in template concentration as assessed by UP NGS-PCR is concerning, suggesting significant secondary structure effects, though not dissimilar to the 5-fold difference seen in the published method(1). It is impossible to know if the postulated secondary structure effect that we see is replicated in vivo. In that regard it is reassuring that published results of NGS-MRD appear to correlate well with RQ-PCR results. In conclusion, secondary structure issues potentially affect the in vitro method for eliminating PCR bias. Within the EuroClonality consortium, studies at other loci are underway to confirm these preliminary findings, and to design primer sets with minimal risk of PCR bias. 1. Carlson CS, Emerson RO, Sherwood AM et al. Nat Commun. 2013;4:2680. Disclosures Moppett: Jazz Pharmaceuticals: Honoraria.

Over nine years, 33 children with neonatal neuroblastoma were registered with the UKCCSG (United Kingdom Children's Cancer Study Group). Tumours of all stages were found, but stage 4S disease predominated. Five tumours were detected prenatally by ultrasonography. Treatment varied according to tumour stage. The overall survival of the group was 91%. Ten children have had long term complications as a result of their disease, usually as a result of spinal tumour involvement. The good overall prognosis in this age group is encouraging, but the poor neurological outcome of patients with intraspinal extension is of concern.

BACKGROUND Malignant melanoma (MM) is one of the least common types of childhood cancer, accounting for less than 1% of all pediatric malignancies. Neurocutaneous melanosis (NCM) is a rare phakomatosis consisting of congenital abnormal pigmentation of the skin and meninges. The meningeal lesions are particularly prone to malignant change. METHODS The authors describe 5 patients with NCM and 1 with primary leptomeningeal melanoma (LMM) seen at 2 treatment centers in the north of England over a 13-year period (1984–1997). RESULTS The clinical features, progress, radiological findings, and treatment of these patients are discussed. All six died within eight months of their diagnosis, illustrating the difficulties faced in treating patients with these conditions. The authors reviewed the published literature on NCM, concentrating on the various therapeutic strategies that have been tried. Very little consistency in approach was found. Malignant skin lesions in NCM may be less responsive than primary malignant melanoma, but the small number of patients with primary LMM or brain metastases of MM make comparisons with NCM difficult. The authors' own series illustrates well the piecemeal nature of therapy for patients with these rare conditions. CONCLUSIONS The rate of incidence of MM melanoma in the U.K. is increasing, and it will represent an increasing proportion of the pediatric oncologist's workload. A consistent approach to the therapy of patients with metastatic MM and NCM is needed if we are to have any hope of offering more than palliative therapy to these children in the future. Cancer 1999;86:878–86. © 1999 American Cancer Society.

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.

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

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.

Background: Overall survival (OS) for childhood ALL treated on contemporary protocols is now > 90%. For low risk patients the risk of treatment related mortality (TRM) is now similar to the risk of death due to disease relapse. The only way to improve TRM and morbidity in this group is to identify even lower risk patients and reduce therapy. We analysed outcomes for patients with low risk MRD at end of induction (EOI) therapy treated on UKALL 2003, to identify a very low risk group who could potentially benefit from protocols that further reduce TRM. Methods: UKALL 2003 trial recruited 3126 patients aged 1-25 years with Philadelphia-negative ALL between Oct 1, 2003, and June 30, 2011. Treatment was initially stratified based on NCI risk and cytogenetic results. Subsequent treatment was directed by MRD from bone marrow (BM) measured using EuroMRD approved real-time quantitative PCR method for immunoglobulin and T-cell receptor gene rearrangements at EOI therapy. Analysis was performed on the 2666 patients with available MRD results and focused on patients with low risk MRD combined with other patient characteristics - cytogenetic risk group, white cell count (WCC) and age to identify those subgroups with extremely good event free survival (EFS). Low risk MRD at EOI was defined a level Results: Overall 52% (n=1391) of patients had MRD < 0.005% with the remaining 48% (n=1275) of patients with MRD ≥ 0.005%. 5yr OS 97.6 (95% CI 96.6-98.3) vs. 89.1% (87.2-90.7, p50 / age>10yrs). Long term survival remained excellent with 10 year EFS 93.9% (OS 98.2%). There were 32 relapses in the low risk MRD/ good risk cytogenetics group (relapse sites: 11 BM, 7 combined BM and central nervous system (CNS), 3 BM + other site, 6 isolated CNS and 5 other site, non BM/CNS), giving an overall relapse rate of 3.8% with a salvage rate of 81%. The relapse death rate was 0.7% (n=6) with TRM of 0.8% (n=7). The low risk group can be further sub divided into a group of 442 patients (16.6% of whole trial) with undetectable MRD at EOI and good risk cytogenetics who have a 5yr EFS of 97.4% (5yr OS 99.5%). Conclusion: This excellent long term outcome for a third of all patients with childhood ALL, low risk MRD and good risk cytogenetics in a large multi-center randomized controlled trial supports exploration of further reduction of therapy in this group to reduce TRM. This strategy is further supported by the observation that the rate of TRM is now similar the relapse death rate in this group, coupled with the very high salvage rates for those patients who do relapse. The superior OS for patients with undetectable MRD and good risk cytogenetics further highlights the additive strength of MRD to accurately predict outcome in the low risk cytogenetic group. Disclosures No relevant conflicts of interest to declare.

Background: Overall survival (OS) for children with ALL treated on contemporary protocols is now > 90%. To further improve outcomes, stratification must direct intensive treatment to those patients with the highest risk of relapse. Whilst minimal residual disease (MRD) has emerged as the most powerful predictor of outcome, its use in isolation is insufficient to identify those patients with the greatest risk. To address this, we performed a detailed analysis of the entire patient cohort from the recently reported large multi-center randomized controlled trial, UKALL 2003. Integration of end of induction (EOI) MRD with other predictive factors - cytogenetics, age and white cell count (WCC) - allowed the identification of very high risk groups that are likely to benefit from early treatment intensification or novel therapies. Methods: The UKALL 2003 trial recruited 3126 patients aged 1-25 years with Philadelphia-negative ALL between Oct 1, 2003, and June 30, 2011. Treatment was initially stratified based on NCI risk and cytogenetic results. Subsequent treatment was directed by EOI MRD measured using EuroMRD approved real-time quantitative PCR for immunoglobulin and T-cell receptor gene rearrangements. Analysis was performed on 2666 patients with available MRD results and focused on the systematic integration of MRD results with other patient characteristics to identify subgroups with a very poor OS ( Results: Both MRD and cytogenetic risk group were independently predictive of survival. In particular, patients with high risk (HR) genetic abnormalities - low hypodiploidy, near haploidy, KMT2A (MLL) rearranged, iAMP21 and t(17;19)(q23;p13) (n=100) had a poor outcome with 5 yr OS of 76.0%. Combining these factors showed that MRD could stratify the HR cytogenetic subgroup with a significant difference in survival between those with low risk MRD and positive MRD (5yr OS 93.0 vs. 63.1%, p=0.002, n=43 vs. 57). Crucially, this demonstrates that HR cytogenetics are only truly poor risk in the context of poor treatment response. Importantly, there were almost no survivors following relapse in the HR cytogenetic/MRD positive group, whilst 50% of those with low risk MRD could be salvaged after relapse. In addition, whilst t(1;19)(q23;p13)was not considered a HR cytogenetic factor in this trial, combination with positive EOI MRD identified an additional subgroup of 20 patients with a 5yr OS of only 74.7% (vs. 96.7% in MRD low risk patients, p=0.003). Whilst WCC at presentation was also predictive of outcome, this difference was significant only in those with B-ALL and not T-ALL. In B-ALL, WCC>100 x109/L was associated with significantly poorer survival (5yr OS 95.1 vs. 84.0%, p200 x109/L (5yr OS 90.2 vs. 86.7%, p=0.283). Integration of MRD results allowed the identification of a very high risk group with B-ALL, WCC>100 and positive EOI MRD (n=80) which had a 5yr OS of only 71.3%. Further breakdown by cytogenetic risk group showed those patients with good risk cytogenetics (ETV6-RUNX1, high hyperdiploidy) (n=19) had a remarkably good survival despite WCC>100 (5yr OS 94%). In contrast, those with intermediate (n=34) or HR cytogenetics (n=15) had a very poor 5yr OS (65.7% and 60.0% respectively). Older age (≥10yr) was also associated with poorer outcomes compared to those Conclusions: Through the integration of EOI MRD with other risk factors in a large pediatric ALL cohort we have identified a population of patients with a very poor OS. When combined, these criteria select only 6% of all patients, but capture 35% of all deaths suggesting current treatment is suboptimal in this group. This information is essential for the planning of future trials to ensure that treatment intensification and novel therapies are targeted at those patients at greatest risk, whilst avoiding unnecessary treatment in those patients that already experience good outcomes. Disclosures No relevant conflicts of interest to declare.

Minimal residual disease (MRD) assessment via next generation sequencing (NGS) of immunoglobulin (Ig) and T-cell receptor (TR) gene rearrangements for lymphoid malignancies is currently under extensive development. NGS MRD has a potential to overcome the limitations of current techniques; laboriousness and difficult interpretation of qPCR for Ig/TR and low sensitivity of flow cytometry. However, amplicon-based NGS MRD has potential pitfalls that have to be addressed before it can be safely introduced for clinical decision making. Multi-center concordance in the experimental setting, quality control and interpretation of the results need to be achieved in order to surpass the advantages of qPCR, which is currently rigorously standardized within the EuroMRD consortium. Our aim was to test the stability and reproducibility of an optimized Ig heavy chain (IGH) based NGS approach for MRD assessment in a multi-center setting within the EuroClonality NGS Consortium on two different sequencing platforms. A one-step PCR library preparation approach was tested in seven institutions (Kiel, Salamanca, Milano, Bristol, London, Prague, Torino). Serial dilutions (10-1 to 10-5) of diagnostic DNA into polyclonal DNA as well as follow-up samples of 30 B-cell precursor ALLs with known complete IGH rearrangements were sequenced on the MiSeq. Serial dilutions of five different diagnostic ALL samples and libraries from polyclonal control were sequenced in parallel on both the MiSeq and Ion Torrent platforms. All samples were spiked with pre-defined copy numbers of five reference IGH sequences as a calibrator. FR2 primers, harboring platform-specific sequencing adapters, were used during the one-step PCR with 500ng of DNA per sample (75,000 copies). Negative and positive controls (27 pooled B-cell lines) were used for testing assay stability and reproducibility among the labs. Purpose-built bioinformatics methods were applied to analyze data. MRD results were compared to results of EuroMRD-based qPCR results. A total of 333 libraries were sequenced in 29 deep sequencing runs producing 194 million reads. The IGH gene rearrangements of all 27 pooled positive B-cell line controls were identified in all centers. NGS MRD analysis in 116 ALL follow-up samples revealed MRD positivity in 69/116 samples vs. 66/116 samples in qPCR, with discrepancies concerning samples with low MRD (R2=0.81). The dilution experiments gave similar results for both platforms, with a minimum sensitivity of 10-4 (as currently required by most treatment protocols using qPCR) for all tested assays. The correlation between MRD levels obtained by the two NGS platforms was good (R2=0.84). Ratios of reads containing reference IGH sequences were highly consistent in intra- and inter-laboratory analyses, independent of the total number of reads in the sample. When comparing platforms, in 10-1 dilution samples sequenced on MiSeq the ratio of reads harboring reference sequences was 2.1 to 2.7 times lower than in remaining dilutions, while on the Ion Torrent it was only 0.9 to 1.3 times, reflecting the competition with the leukemic clone. The correlation of the amounts of spiked-in sequences with the representation of reads harboring these sequences was slightly better for the Ion Torrent (R2=0.88) than for the MiSeq (R2=0.79). Amplification efficiency of each primer was checked by analyzing libraries from healthy polyclonal control. All primer sequences were present in all samples on both platforms, however, the differences between four libraries prepared from the same sample sequenced on the MiSeq were 2.6 times higher than in one library from this sample sequenced in five replicates on the Ion Torrent. The newly developed IGH assay shows robust intra and inter-laboratory reproducibility, which is the first step towards the safe use of this new MRD technique in a multi-center setting. The distribution of reference sequences and sequences of primers confirmed that the main source of differences between platform strategies is the library preparation and not the platform itself. Using the same amount of DNA, the sensitivity of the method is similar to qPCR. The performance and costs of the assay are similar for both the MiSeq and Ion Torrent. MRD analysis via NGS has therefore a great potential to replace qPCR as the gold standard for MRD-guided therapy in ALL, provided that thorough standardization can be achieved. Support: NV15-30626A, GBP302/12/G101. Disclosures Langerak: Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam); DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics..

Stenotrophomonas maltophilia bacteraemia in 40 haematology patients: risk factors, therapy and outcome