Lymphoblastic Leukemia In Children Research Articles

Prognostic relevance of the expression of genes associated with the P2RY8-CRLF2 fusion in acute lymphoblastic leukemia in childhood

The cytokine receptor-like factor 2 (CRLF2) gene is overexpressed in a subgroup of precursor B-cell acute lymphoblastic leukemia (pB-ALL) caused by different genetic aberrations. However, only the presence of the P2RY8-CRLF2 rearrangement is associated with a poor prognosis. Recently, we showed that the presence of the P2RY8-CRLF2 rearrangement is characterized by a distinct transcriptome profiling as detected by next generation sequencing (NGS) and we were interested whether gene identified here are of prognostic relevance also for childhood ALL irrespective of the CRLF2 status.

Arthritis as presenting manifestation of acute lymphoblastic leukaemia in children

BackgroundAt disease onset, children with acute lymphoblastic leukaemia (ALL) may present with arthralgia or even signs of arthritis. This might cause misdiagnosis and thereby lead to prolonged diagnostic delay. The...

Inherited coding variants at the CDKN2A locus influence susceptibility to acute lymphoblastic leukaemia in children.

There is increasing evidence from genome-wide association studies for a strong inherited genetic basis of susceptibility to acute lymphoblastic leukaemia (ALL) in children, yet the effects of protein-coding variants on ALL risk have not been systematically evaluated. Here we show a missense variant in CDKN2A associated with the development of ALL at genome-wide significance (rs3731249, P=9.4 × 10−23, odds ratio=2.23). Functional studies indicate that this hypomorphic variant results in reduced tumour suppressor function of p16INK4A, increases the susceptibility to leukaemic transformation of haematopoietic progenitor cells, and is preferentially retained in ALL tumour cells. Resequencing the CDKN2A–CDKN2B locus in 2,407 childhood ALL cases reveals 19 additional putative functional germline variants. These results provide direct functional evidence for the influence of inherited genetic variation on ALL risk, highlighting the important and complex roles of CDKN2A–CDKN2B tumour suppressors in leukaemogenesis.

Bone Marrow Recovery by Morphometry during Induction Chemotherapy for Acute Lymphoblastic Leukemia in Children.

Bone marrow architecture is grossly distorted at the diagnosis of ALL and details of the morphological changes that accompany response to Induction chemotherapy have not been reported before. While marrow aspirates are widely used to assess initial response to ALL therapy and provide some indications, we have enumerated marrow components using morphometric analysis of trephine samples with the aim of achieving a greater understanding of changes in bone marrow niches. Morphometric analyses were carried out in the bone marrow trephine samples of 44 children with ALL, using a NanoZoomer HT digital scanner. Diagnostic samples were compared to those of 32 control patients with solid tumors but without marrow involvement. Samples from patients with ALL had significantly increased fibrosis and the area occupied by bony trabeculae was lower than in controls. Cellularity was higher in ALL samples due to leukemic infiltration while the percentage of normal elements such as megakaryocytes, adipocytes, osteoblasts and osteoclasts were all significantly lower. During the course of Induction therapy, there was a decrease in the cellularity of ALL samples at day 15 of therapy with a further decrease at the end of Induction and an increase in the area occupied by adipocytes and the width of sinusoids. Reticulin fibrosis decreased throughout Induction. Megakaryocytes increased, osteoblasts and osteoclasts remained unchanged. No correlation was found between clinical presentation, early response to treatment and morphological changes. Our results provide a morphological background to further studies of bone marrow stroma in ALL.

Acute lymphoblastic leukemia in children and adolescents: prognostic factors and analysis of survival.

ObjectiveTo describe the clinical and laboratory features of children and adolescents with acute lymphoblastic leukemia treated at three referral centers in Ceará and evaluate prognostic factors for survival, including age, gender, presenting white blood cell count, immunophenotype, DNA index and early response to treatment.MethodsSeventy-six under 19-year-old patients with newly diagnosed acute lymphoblastic leukemia treated with the Grupo Brasileiro de Tratamento de Leucemia da Infância – acute lymphoblastic leukemia-93 and -99 protocols between September 2007 and December 2009 were analyzed. The diagnosis was based on cytological, immunophenotypic and cytogenetic criteria. Associations between variables, prognostic factors and response to treatment were analyzed using the chi-square test and Fisher's exact test. Overall and event-free survival were estimated by Kaplan–Meier analysis and compared using the log-rank test. A Cox proportional hazards model was used to identify independent prognostic factors.ResultsThe average age at diagnosis was 6.3 ± 0.5 years and males were predominant (65%). The most frequently observed clinical features were hepatomegaly, splenomegaly and lymphadenopathy. Central nervous system involvement and mediastinal enlargement occurred in 6.6% and 11.8%, respectively. B-acute lymphoblastic leukemia was more common (89.5%) than T-acute lymphoblastic leukemia. A DNA index >1.16 was found in 19% of patients and was associated with favorable prognosis. On Day 8 of induction therapy, 95% of the patients had lymphoblast counts <1000/μL and white blood cell counts <5.0 × 109/L. The remission induction rate was 95%, the induction mortality rate was 2.6% and overall survival was 72%.ConclusionThe prognostic factors identified are compatible with the literature. The 5-year overall and event-free survival rates were lower than those reported for developed countries. As shown by the multivariate analysis, age and baseline white blood cell count were independent prognostic factors.

Health Status and Quality of Life of Long-term Survivors of Childhood Acute Leukemia

We evaluated the impact of central nervous system irradiation (CNSI) on long-term health status and quality of life (QoL) of childhood lymphoblastic leukemia survivors included in the French L.E.A. (Childhood and Adolescent Leukemia) multicentric cohort. QoL was self-reported in adults and assessed by parents in children and adolescents, using adapted questionnaires. From 2004 to 2009, 630 nongrafted patients were assessed after 11.8±6.3 years from diagnosis. Patients receiving CNSI (18.6%) or chemotherapy alone (81.4%) were compared. The risk of having long-term physical effects was increased with CNSI (odds ratio=3.3; 95% confidence interval, 1.8-5.9), especially regarding growth failure, second tumor, cataract, and overweight. QoL did not differ significantly according to the treatment received, despite a tendency toward lower scores with CNSI in children and adolescents (summary score 63.6±13.3 vs. 71.7±12.4, P=0.14). Compared with French norms, adult survivors had an impaired QoL, especially in mental domains (mental composite score 45.2±9.8 vs. 47.9±2.1, P<0.001). In pediatric survivors, QoL was not impaired and even tended to be higher than population norms (summary score 71.7±12.4 vs. 70.0±4.2, P=0.054), mainly in social and relational domains. In conclusion, QoL seems to be impaired by the trauma of a life-threatening illness in childhood, as well as by the treatment received.

Body composition and bone health in long-term survivors of acute lymphoblastic leukaemia in childhood and adolescence: the protocol for a cross-sectional cohort study

IntroductionSuccess in the treatment of young people with cancer, as measured conventionally by survival rates, is mitigated by late effects of therapy that impose a burden of morbidity and limit...

Clinical, immunophenotype and cytogenetic profile of acute lymphoblastic leukemia in children at tertiary health care centre in India

Objective: Data on acute lymphoblastic leukemia (ALL) in Indian children being limited, we analyzed the clinical, immunophenotype and cytogenetic profile of ALL in our pediatric patients. Materials and Methods: This was a prospective and retrospective observational study in which data of 103 children with ALL, aged three months to 18 years were analyzed. Results: Definite male preponderance (70.87%) was observed, 58.25% patients were aged between two to five years. Common clinical features included fever (92.33%), pallor (87.38%), hepatomegaly (85.44%) and splenomegaly (83.50). central nervous system involvement was seen in 6.80% and tumor lysis syndrome in 32.04% patients. Hemoglobin was 50,000 in 20.39% patients. Platelet count was <50,000 cells/hpf in 48.54%. Blast cells in peripheral smear present in 67.96% patients of 97 in whom immunophenotyping was done, 82.44% were Pre B cell ALL. Pre T and T cell ALL was seen in 15/97 (15.46%) patients. Karyotyping revealed hyperdiploidy in 9/89, hypodiploidy in 2/89, miscellaneous changes in 35 patients. FISH analysis in 63 patients revealed Philadelphia chromosome positivity in seven (11.11%). MLL gene was positive in 12.70%. TELAML was positive in 13.64% (3/22) patients. 97.06% patients went in to remission post induction. Conclusion: Most of our patients come under standard risk by their age, WBC count, immunophenotyping and cytogentic analysis. We suggest larger multi-centric studies even in developing countries to understand the clinical profile and RT-PCR technique in cytogenic analysis for better risk stratification to help in risk adopted therapy.

DENTAL CONSIDERATIONS FOR LEUKEMIC PEDIATRIC PATIENTS: AN UPDATED REVIEW FOR GENERAL DENTAL PRACTITIONER.

The early signs of leukemia can usually manifest in the oral cavity due to infiltration of leukemic cells or due to associated decline in normal marrow elements, especially in the acute phase of leukemia, as common lesions at this stage of the disease can be screened and diagnosed by the dentist. Therefore, the dental community should be aware of the oral manifestations of leukemia and oral complications of anticancer treatment. This can eliminate the oral symptoms of the disease and to improve quality of life for these patients. An extensive search in PubMed line using a combination of terms like “leukemia, children, dental, Acute lymphoblastic leukemia, pediatric” for last ten years was made. Reviews and case reports concerned about acute lymphoblastic leukemia in children were all collected and analyzed and data were extracted. Accordingly, the aim of this review is to highlight on the oral presentations of leukemia in children attending dental clinics and the management of its undesirable side effects.

Emerging Immunotherapies for Cancer and Their Potential for Application in Pediatric Oncology.

After decades of basic research, immune-based therapeutics for the treatment of cancer are showing evidence of efficacy in clinical trials; several immunotherapeutics already incorporated into standard treatment regimens. Intensive research is underway to improve the efficacy of immunotherapeutics and to expand the application of immunotherapy to a wider array of cancers. The therapeutic options that comprise immunotherapy for cancer are vast and span monoclonal antibodies, tumor vaccines, adoptive cellular therapies, as well as therapies aimed at reversing immunosuppression and enhancing immune reactivity globally and/or locally within the tumor microenvironment. In pediatric cancer, monoclonal antibodies have demonstrated efficacy in hematologic malignancies, and neuroblastoma and bispecific antibodies that activate resident T cells, as well as adoptive cell therapy, have shown recent exciting results for the treatment of acute lymphoblastic leukemia in childhood. This review discusses the basic principles of tumor immunology driving clinical development of new immunotherapies, describes immunotherapeutics with demonstrated efficacy and several currently in clinical trials, and highlight agents that seem to be most promising for the treatment of pediatric cancer.

Bacterial recombinant L-asparaginases: properties, structure and anti-proliferative activity

For more than 40 years L-asparaginases are used in combined therapy of acute lymphoblastic leukemia in children and the range of tumors sensitive to these enzymes constantly extends. This review summarizes results of studies aimed at creation of new systems for heterological expression of bacterial L-asparaginases as Erwinia carotovora (EwA), Helicobacter pylori (HpA), Yersinia pseudotuberculosis (YpA) and Rhodospirillum rubrum (RrA); special attention is paid to isolation of purified enzymes and their crystallization, modification by chitosan/polyethylene, physicochemical, kinetic and structural properties characterization, and the study of the cytotoxic or anti-proliferative activity of new recombinant L-asparaginases on cell cultures in vitro. The resultant recombinant L-asparaginases (EwA, YpA, HpA и RrA) exhibit reasonable cytotoxic action on the human leukemia cells comparable to the pharmacologically available L-asparaginase EcA and represent practical interest in respect to creation, on their basis, new effective antineoplastic remedies. Further prospects of researches on bacterial L-asparaginases are associated with development of analogs of Rhodospirillum rubrum L-asparaginase (RrA) by means of directed changes of the protein structure using genetic engineering, development of chito-PEGylation for receiving L-asparaginase preparations with improved pharmacokinetic characteristics.

Hematopoietic stem cell transplantation following unsuccessful salvage treatment for relapsed acute lymphoblastic leukemia in children.

For children who experience a re-induction failure or multiple recurrences following the first relapse of acute lymphoblastic leukemia (ALL), it is uncertain whether additional intensive chemotherapy aimed at hematopoietic stem cell transplantation (SCT) in complete remission (CR) or immediate SCT even in non-CR should be performed. This study aimed to investigate the impact of disease status at SCT on the outcomes of SCT for these children, whose prognosis is considered unquestionably poor even with SCT. The medical records of 55 children with ALL who underwent SCT following the experience of re-induction failure (n = 25) or multiple relapses (n = 30) were retrospectively analyzed. Twenty-one patients underwent SCT in CR (delayed CR2, CR3, and CR4) and 34 in non-CR (first or subsequent relapse). The probability of overall survival of patients with CR and with non-CR at SCT was 42.9% and 23.5% (P = 0.15), leukemia-free survival was 38.1% and 20.6% (P = 0.18), and the cumulative incidence of relapse at 2 years was 23.8% and 50%, respectively (P = 0.05). In multivariate analysis, non-CR at SCT was a significant risk factor for higher relapse incidence and male sex was a significant risk factor for lower survival. Our results indicated that in case of tolerable patient condition, further re-induction chemotherapy might be reasonable so that SCT could be performed in CR, which might result in a low incidence of relapse after SCT. Novel approaches are required to induce CR for the treatment of children with relapsed/refractory ALL.

Follow-up of post-transplant minimal residual disease and chimerism in childhood lymphoblastic leukaemia: 90 d to react.

Relapse after transplantation is a major cause of treatment failure in paediatric acute lymphoblastic leukaemia (ALL). Here, we report the findings of a prospective national study designed to investigate the feasibility of immune intervention in children in first or subsequent remission following myeloablative conditioning. This study included 133 children who received a transplant for ALL between 2005 and 2008. Minimal Residual Disease (MRD) based on T cell receptor/immunoglobulin gene rearrangements was measured on days -30, 30, 90 and 150 post-transplantation. Ciclosporin treatment was rapidly discontinued and donor lymphocyte infusions (DLI) were programmed for patients with a pre- or post-transplant MRD status ≥10(-3) . Only nine patients received DLI. Pre- and post-transplant MRD status, and the duration of ciclosporin were independently associated with 5-year overall survival (OS), which was 62·07% for the whole cohort. OS was substantially higher in patients cleared of MRD than in those with persistent MRD (52·3% vs. 14·3%, respectively). Only pre-transplant MRD status (Hazard Ratio 2·57, P=0·04) and duration of ciclosporin treatment (P<0·001) were independently associated with relapse. The kinetics of chimerism were not useful for predicting relapse, whereas MRD monitoring up to 90d post-transplantation was a valuable prognostic tool to guide therapeutic intervention.

Genome-Wide Mapping of Binding Sites and Networks of Potential Target Genes of the Fusion Oncogene ETV6/RUNX1 in Acute Lymphoblastic Leukemia in Childhood

Acute lymphoblastic leukemia (ALL) in childhood, a clinically and biologically heterogeneous disease, represents the most common malignant disease in childhood. Approximately 20-25% of B-cell precursor ALL (BCP-ALL) carry the cryptic chromosomal translocation t(12;21)(p13;q22), the most common reciprocal chromosomal translocation in childhood ALL. This translocation combines two transcription factors and essential regulators of normal hematopoiesis, ETV6 and RUNX1, into the fusion oncogene ETV6/RUNX1 (E/R; synonym TEL/AML1).Recent studies in various animal models have strengthened the view that E/R positive cells give rise to preleukemic clones with a differentiation block in the pro/pre-B stage of B cell development that, after acquisition of additional mutations, may transform into full malignancy.Regarding the molecular mechanism by which the chimeric fusion protein E/R causes gene expression changes, it is assumed that E/R binds with the runt homology domain of RUNX1 (RHD, DNA-binding domain) to RUNX1 target sequences of gene promoters and recruits corepressors and histone deacetylases through its ETV6 portion, leading to chromatin condensation and transcriptional repression. Thus, E/R appears to act mainly as an epigenetic repressor of genes that are normally activated by RUNX1. However, the precise mechanism of cellular transformation and the identity of E/R target genes are largely unknown.Therefore, we used chromatin immunoprecipitation (ChIP), followed by next generation sequencing (ChIP-Seq) to identify E/R target genes in the E/R positive BCP-ALL cell lines REH and UoC-B6 as well as in primary patient material from children with relapsed E/R positive ALL. We were able to detect a core gene set of 335 candidate target genes common to all samples analyzed. Those genes could be assigned to 15 significantly overrepresented KEGG pathways (e.g. cell cycle, pathways in cancer, hematopoietic cell lineage and B cell receptor signaling pathway).The results show, besides target genes already reported in the literature such as EPOR, MPO and IGLL1, numerous not previously described candidate E/R target genes, such as LEF1, E2F2, FLT3, FGFR1 and RUNX1 that are potentially important in the pathogenesis of E/R positive ALL and may lead to new treatment options. DisclosuresNo relevant conflicts of interest to declare.

Early T Acute Lymphoblastic Leukemia in Childhood: Prevalence and Clinical Characteristics

Background: Early T precursor ALL has been defined as a poor prognosis subtype, characterized phenotypically by absence of CD1a and CD8, CD5 weak expression and presence of at least one myeloid and progenitor cell marker.Objectives: Our aims were to identify Early T-ALL among our T ALL cases, based on the characteristic phenotype and thus evaluate the prevalence, biological characteristics and outcome of this particular subset of T-ALLMethods: From April ’94 to December ’13, 197 T-ALL cases were diagnosed and 20 of them showed the typical Early T marker profile by flow cytometry (FC). Conventional cytogenetics, FISH and RT-PCR studies were performed according to standard techniques. Clonality assessment for TRG, TRB, TRD, IGH and IGK was performed by PCR (Biomed-2), heteroduplex and sequencing.Results: Sex distribution was: 16 males and 4 females; median of age: 7.9 (range: 0.3-16.6) years; median WBC: 14.9 (range: 0.6-254.0) x109/L. All but one case expressed CD34, 70% HLA-DR, 61% CD117 and 50% TdT. The most frequently expressed myeloid markers were: CD33 (85%) and CD13 (55%). Three cases expressed g/d TCR. Chromosomal abnormalities were detected in 14 of 16 evaluated cases, in 5 of them compromising 12p13 region. TCR/IgH rearrangements were detected in 63% (10/16). FLT3-ITD mutations were found in 14 % (2/14).Patients were treated with BFM-based ALL schedules and were stratified as Intermediate Risk ALL (n: 7) and High Risk ALL (n: 13) according to protocol criteria. Early response to treatment was poor in 16 cases: 6 presented poor response to prednisone (day 8), 3 MRD >10%, 5 presented bone marrow M2-M3 and 2 non response. Seventeen cases (85%) achieved CR on day 33 and 2 achieved CR later. Five patients underwent HSCT in first CR. Three patients relapsed at 5, 6 and 65 months from diagnosis and one showed lineage switch to M5-AML at 12 months from CR. Three pts died in CR (2 after HSCT and 1 patient with primary immunodeficiency due to pneumonia) and one is in palliative care. Twelve patients remain in CR with a median follow-up of 54 (r: 8-144) months.Conclusions: The prevalence of Early T precursor phenotype within T ALL was 10.5% in our setting. The most frequent progenitor marker was CD34 and CD33 among myeloid markers. Of note, translocations involving 12p13 region were found in 5 patients. Sixty percent of patients remain disease free, although longer follow-up is needed in order to define prognosis of this group in our cohort of patients. The lineage switch case supports the notion of the myeloid differentiation potential of these blasts. DisclosuresNo relevant conflicts of interest to declare.

Cerebral Venous Thrombosis As a Complication of Treatment for Acute Lymphoblastic Leukaemia in Children and Young Adults

▪Cerebral venous thrombosis (CVT) is a significant complication of therapy in children and young adults with acute lymphoblastic leukaemia (ALL). Previous reports indicate a considerable risk of mortality and morbidity in relation to CVT. In addition, there is potential for suboptimal treatment of ALL due to avoidance of Asparaginase and the need for reversal of anticoagulant therapy for invasive procedures such as intrathecal chemotherapy. Anticoagulation may be complicated by haemorrhage.This retrospective study aimed to identify the clinical features, therapy and outcome of CVT occurring during treatment of ALL in individuals, aged 1 to 24 years, who participated in the Medical Research Council UK ALL 2003 (MRC UKALL 2003) randomised controlled trial.Cases of CVT were identified from adverse event reporting. All reports were screened for central nervous system thrombosis and grade 4 serious adverse events in the category of coagulation/thrombosis. Reporting centres were asked for additional clinical information.3126 children and young adults were enrolled in the study between 2003 and 2011, with median age 5 years, including 1776 males (56.8%). The 3 initial treatment regimens: A, B and C: corresponded to standard-risk, intermediate-risk and high-risk, respectively. 55% were treated with regimen A, 32% regimen B and 13% regimen C. 48 individuals (1.5%) developed CVT and data were returned for 42. Median age was 10 (range: 1 to 18) years and 25 cases were male (59.5%). 71.4% of cases (30/42) occurred during induction therapy. 18 cases were in individuals treated with regimen A, 22 regimen B and 2 regimen C. Median time between the most recent Asparaginase dose and CVT was 12.5 days (range: 1-111 days). 29 cases (68.4%) had received 2 doses of Asparaginase prior to thrombosis, 5 cases >2 doses and 8 cases 1 dose.Presenting symptoms were: seizures, 22 cases (52.3%); neurological impairment, 20 (47.6%); headache, 19 (45.2%); reduced conscious level, 8 (19.0%); nausea/vomiting, 8 (19.0%). Identified thrombotic risk factors included: hospitalisation, 22 (52.3%); active infection, 5; recent immobility, 5; dehydration, 6; combined oral contraceptive pill, 2. None were obese, had a high white cell count at time of CVT diagnosis or a history of recent major surgery. 13 (31.0%) had >1 thrombotic risk factor and 16 (38.1%) had no additional clinical risk factor identified. 10 had a thrombophilia screen performed, one with a repeatedly low protein S level (male, 1 year of age). 7 cases (16.7%) had cerebral infarction on imaging and 11 (26.2%) were complicated by intracranial haemorrhage (ICH).38 cases (90.5%) received anticoagulant therapy, 37 of which received low molecular weight heparin (LMWH) and one unfractionated heparin. One case had catheter-directed thrombolysis. 4 were not anticoagulated, 3 due to ICH. 2 received Antithrombin replacement therapy. Anticoagulation therapy was continued for a median of 3 months. 65.0% of cases due more Asparaginase were re-exposed (26/40), 19 of which received thromboprophylaxis during re-exposure. 2 remain on long-term therapeutic anticoagulation. In 23 cases (54.8%) CVT resulted in a delay or change to planned treatment.Neurological morbidity was reported in 4 cases (9.5%): seizure disorder, 2; hemiplegia, 1; residual hemiplegia and hemianopia with severe developmental delay, 1. 36 cases (85.7%) remain in first remission. Survival in those with CVT was 90.1%, comparable to actuarial 5-year survival of the entire cohort (91.5%). There were 4 deaths, three due to leukaemia (two in first relapse) and one due to pneumonia as a complication of stem cell transplant. There were no deaths due to CVT or bleeding on anticoagulant therapy.This study highlights the clinical features of CVT in children and young adults treated for ALL in the MRC UKALL 2003 trial, in particular the timing in relation to Asparaginase treatment, presenting features and neurological outcome. These data support LMWH as safe treatment for CVT in this clinical setting. We have demonstrated the tendency of clinicians to adjust treatment regimen and/or avoid further Asparaginase exposure in response to CVT. However, overall survival at 5 years did not differ for the cohort with CVT. Future studies should aim to further evaluate clinical predictors, alongside potential coagulation and genetic markers, in order to identify a high-risk group that may benefit from measures to reduce CVT during ALL therapy. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Microbiome: Metagenomic Comparsion of Childhood Acute Lymphoblastic and Acute Myeloid Leukamias

Purpose: To describe and compare the pre-treatment bone marrow metagenomes of childhood acute lymphoblastic leukemia (ALL) and childhood acute myeloid leukemia (AML).Background: Leukemia is the most common cancer in children, comprised mainly of ALL (~80%) followed by AML (~17%). The role of infections and their timing in the etiology of pre B-cell ALL’s (but not AML’s) is supported by a considerable body of epidemiologic evidence developed in the past 20 years (Gilham, Peto et al. 2005, Ma, Urayama et al. 2009). There are two main infection related hypotheses. Greaves postulates that delayed infection, akin to the hygiene hypothesis, increases risk of leukemia (Greaves and Alexander 1993, Greaves 1997). The second hypothesis set forth by Kinlen is not mutually exclusive and postulates the role of a transforming yet common virus where introduction of the infectious agent to previously naive communities trigger leukemia (Kinlen 1995). While many viral agents have been investigated (reviewed in (O’Connor and Boneva 2007)) no definitive infectious agent has been identified but, until recently, broad surveys of the microbiome were not possible. Recent developments in next generation sequencing and bioinformatics now allow for unbiased investigation into the totality of organisms that inhabit the human body.Methods: Pre-treatment bone marrow specimens were collected in pre B-cell ALL and AML cases participating in the California Childhood Leukemia Study. AML’s with similar age distribution to the ALL’s were utilized as controls, to represent the infection history of an immune suppressed child. We interrogated the viral metagenomes of 60 ALL’s and 30 AML’s, pooled and reduced with ribosomal capture, then sequenced with high coverage next generation RNA sequencing on an Illumina HiSeq 2500. The resulting sequences were analyzed using a custom bioinformatics pipeline, DARK, to remove human sequences, to identify viral transcripts, and to categorize all transcripts present in the pre-treatment bone marrow. Bacterial metagenomes of the same subjects were analyzed using 16S rRNA amplicon sequencing and the QIIME pipeline.Results: We analyzed over 500 million 100bp reads and categorized human, non-human and unknown sequences. We found striking differences between the viral metagenomes of childhood ALL and AML, with ALL subjects presenting with a greater viral infection load than AML’s. Furthermore, differences in the presence of transcripts aligning to both exogenous viruses and endogenous retroviruses were observed with some viruses occurring exclusively in ALL’s. We found no difference between the bacterial metagenomes of childhood ALL and AML suggesting no differential contamination occurred during collection and analysis.Discussion: This study provides the first measurement of all identifiable infectious agents in both childhood ALL an AML. Our data suggest that bacteria do not play a role in the etiology of ALL. The greater number and frequency of viral infectious agents were observed in ALL versus AML contribute to an etiologic hypothesis of ALL involving viral infections yet require replication and additional follow up. Moreover the clinical relevance of these infections and their relation to outcome should be investigated.ReferencesGilham, C., J. Peto, J. Simpson, E. Roman, T. O. Eden, M. F. Greaves, F. E. Alexander and U. Investigators (2005). "Day care in infancy and risk of childhood acute lymphoblastic leukaemia: findings from UK case-control study." BMJ 330(7503): 1294.Greaves, M. F. (1997). "Aetiology of acute leukaemia." Lancet 349(9048): 344-349.Greaves, M. F. and F. E. Alexander (1993). "An infectious etiology for common acute lymphoblastic leukemia in childhood?" Leukemia 7(3): 349-360.Kinlen, L. J. (1995). "Epidemiological evidence for an infective basis in childhood leukaemia." Br J Cancer 71(1): 1-5.Ma, X., K. Urayama, J. Chang, J. L. Wiemels and P. A. Buffler (2009). "Infection and pediatric acute lymphoblastic leukemia." Blood Cells Mol Dis 42(2): 117-120.O’Connor, S. M. and R. S. Boneva (2007). "Infectious etiologies of childhood leukemia: plausibility and challenges to proof." Environ Health Perspect 115(1): 146-150. DisclosuresNo relevant conflicts of interest to declare.

Thiopurine dose intensity and treatment outcome in childhood lymphoblastic leukaemia: the influence of thiopurine methyltransferase pharmacogenetics.

SummaryThe impact of thiopurine methyltransferase (TPMT) genotype on thiopurine dose intensity, myelosuppression and treatment outcome was investigated in the United Kingdom childhood acute lymphoblastic leukaemia (ALL) trial ALL97. TPMT heterozygotes had significantly more frequent cytopenias and therefore required dose adjustments below target levels significantly more often than TPMT wild‐type patients although the average dose range was similar for both genotypes. Event‐free survival (EFS) for patients heterozygous for the more common TPMT*1/*3A variant allele (n = 99, 5‐year EFS 88%) was better than for both wild‐type TPMT*1/*1 (n = 1206, EFS 80%, P = 0·05) and TPMT*1/*3C patients (n = 17, EFS 53%, P = 0·002); outcomes supported by a multivariate Cox regression analysis. Poor compliance without subsequent clinician intervention was associated with a worse EFS (P = 0·02) and such non‐compliance may have contributed to the poorer outcome for TPMT*1/*3C patients. Patients prescribed escalated doses had a worse EFS (P = 0·04), but there was no difference in EFS by dose intensity or duration of cytopenias. In contrast to reports from some USA and Nordic trials, TPMT heterozygosity was not associated with a higher rate of second cancers. In conclusion, TPMT*1/*3A heterozygotes had a better EFS than TPMT wild‐type patients. Thiopurine induced cytopenias were not detrimental to treatment outcome.

The genomic landscape of acute lymphoblastic leukemia in children and young adults.

Our understanding of the genetic basis of childhood acute lymphoblastic leukemia (ALL) has been greatly advanced by genomic profiling and sequencing studies. These efforts have characterized the genetic basis of recently described and poorly understood subtypes of ALL, including early T-cell precursor ALL, Philadelphia chromosome-like (Ph-like) ALL, and ALL with intrachromosomal amplification of chromosome 21, and have identified several rational therapeutic targets in high-risk ALL, notably ABL1-class and JAK-STAT inhibitors in Ph-like ALL. Deep sequencing studies are also refining our understanding of the genetic basis of clonal heterogeneity and relapse. These studies have elucidated the nature of clonal evolution during disease progression and identified genetic changes that confer resistance to specific therapeutic agents, including CREBBP and NT5C2. Genomic profiling has also identified common and rare inherited genetic variants that influence the risk of developing leukemia. These efforts are now being extended to ALL in adolescents and adults with the goal of fully defining the genetic landscape of ALL to further improve treatment outcomes in high-risk populations.

T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial

Chimeric antigen receptor (CAR) modified T cells targeting CD19 have shown activity in case series of patients with acute and chronic lymphocytic leukaemia and B-cell lymphomas, but feasibility, toxicity, and response rates of consecutively enrolled patients treated with a consistent regimen and assessed on an intention-to-treat basis have not been reported. We aimed to define feasibility, toxicity, maximum tolerated dose, response rate, and biological correlates of response in children and young adults with refractory B-cell malignancies treated with CD19-CAR T cells. This phase 1, dose-escalation trial consecutively enrolled children and young adults (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukaemia or non-Hodgkin lymphoma. Autologous T cells were engineered via an 11-day manufacturing process to express a CD19-CAR incorporating an anti-CD19 single-chain variable fragment plus TCR zeta and CD28 signalling domains. All patients received fludarabine and cyclophosphamide before a single infusion of CD19-CAR T cells. Using a standard 3 + 3 design to establish the maximum tolerated dose, patients received either 1 × 10(6) CAR-transduced T cells per kg (dose 1), 3 × 10(6) CAR-transduced T cells per kg (dose 2), or the entire CAR T-cell product if sufficient numbers of cells to meet the assigned dose were not generated. After the dose-escalation phase, an expansion cohort was treated at the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT01593696. Between July 2, 2012, and June 20, 2014, 21 patients (including eight who had previously undergone allogeneic haematopoietic stem-cell transplantation) were enrolled and infused with CD19-CAR T cells. 19 received the prescribed dose of CD19-CAR T cells, whereas the assigned dose concentration could not be generated for two patients (90% feasible). All patients enrolled were assessed for response. The maximum tolerated dose was defined as 1 × 10(6) CD19-CAR T cells per kg. All toxicities were fully reversible, with the most severe being grade 4 cytokine release syndrome that occurred in three (14%) of 21 patients (95% CI 3·0-36·3). The most common non-haematological grade 3 adverse events were fever (nine [43%] of 21 patients), hypokalaemia (nine [43%] of 21 patients), fever and neutropenia (eight [38%] of 21 patients), and cytokine release syndrome (three [14%) of 21 patients). CD19-CAR T cell therapy is feasible, safe, and mediates potent anti-leukaemic activity in children and young adults with chemotherapy-resistant B-precursor acute lymphoblastic leukaemia. All toxicities were reversible and prolonged B-cell aplasia did not occur. National Institutes of Health Intramural funds and St Baldrick's Foundation.