Anthracycline-Free Protocol for Favorable-Risk Childhood ALL: A Noninferiority Comparison Between Malaysia-Singapore ALL 2003 and ALL 2010 Studies.

PURPOSE: To investigate whether, for children with favorable-risk B-cell precursor ALL (BCP-ALL), an anthracycline-free protocol is noninferior to a modified Berlin-Frankfurt-Muenster ALL-IC2002 protocol, which includes 120 mg/m2 of anthracyclines. PATIENTS AND METHODS: Three hundred sixty-nine children with favorable-risk BCP-ALL (age 1-9 years, no extramedullary disease, and no high-risk genetics) who cleared minimal residual disease (≤0.01%) at the end of remission induction were enrolled into Ma-Spore (MS) ALL trials. One hundred sixty-seven standard-risk (SR) patients (34% of Malaysia-Singapore ALL 2003 study [MS2003]) were treated with the MS2003-SR protocol and received 120 mg/m2 of anthracyclines during delayed intensification while 202 patients (42% of MS2010) received an anthracycline-free successor protocol. The primary outcome was a noninferiority margin of 1.15 in 6-year event-free survival (EFS) between the MS2003-SR and MS2010-SR cohorts. RESULTS: The 6-year EFS of MS2003-SR and MS2010-SR (anthracycline-free) cohorts was 95.2% ± 1.7% and 96.5% ± 1.5%, respectively (P = .46). The corresponding 6-year overall survival was 97.6% and 99.0% ± 0.7% (P = .81), respectively. The cumulative incidence of relapse was 3.6% and 2.6%, respectively (P = .42). After adjustment for race, sex, age, presenting WBC, day 8 prednisolone response, and favorable genetic subgroups, the hazard ratio for MS2010-SR EFS was 0.98 (95% CI, 0.84 to 1.14; P = .79), confirming noninferiority. Compared with MS2003-SR, MS2010-SR had significantly lower episodes of bacteremia (30% v 45.6%; P = .04) and intensive care unit admissions (1.5% v 9.5%; P = .004). CONCLUSION: In comparison with MS2003-SR, the anthracycline-free MS2010-SR protocol is not inferior and was less toxic as treatment for favorable-risk childhood BCP-ALL.

Associations of T-Cell Receptor Repertoire Diversity with L-Asparaginase Allergy in Childhood Acute Lymphoblastic Leukemia.

Asparaginase is a critical component of therapy for childhood acute lymphoblastic leukemia (ALL), but it is commonly associated with allergy, which results in morbidity and poorer outcomes. The underlying basis of this allergy is undoubtedly immune-mediated, but the exact components of T-cell immunity have yet to be characterized. We performed longitudinal TCR sequencing of 180 bone marrow samples from 67 children with B-ALL treated as part of the Ma-Spore-ALL-2010 trial, and we evaluated the associations of TCR profile with asparaginase hypersensitivity, with functional validation of asparaginase activity in a separate cohort of 113 children. We found that a more diverse and dynamically changing TCR repertoire was associated with increased risk of clinical hypersensitivity and decreased L-asp activity. Allergic patients had a higher proportion of infrequent clonotypes, as well as a significantly lower degree of shared clonotypes amongst the cohort. Allergic patients also had significantly higher longitudinal variability of clonotypes across timepoints, where a higher dissimilarity between diagnosis and week 5 represented an 8.1-fold increased risk of an allergic event. After an allergy had occurred, there was shaping and convergence of the TCR repertoire towards a common antigen. Understanding the immunological basis of T-cell responses in allergy lays the groundwork for developing predictive biomarkers or strategies to mediate this common toxicity in childhood ALL.

Life-threatening infections during treatment for acute lymphoblastic leukemia on the Malaysia-Singapore 2003 and 2010 clinical trials: A risk prediction model.

AIM: Life-threatening infections significantly impact the care of children undergoing therapy for acute lymphoblastic leukemia (ALL) who are at risk of severe sepsis due to both host and treatment factors. Our aim was to develop a life-threatening infection risk prediction model that would allow remote rapid triage of patients to reduce time to first dose of antibiotics and sepsis-related mortality. METHODS: A retrospective analysis of 2068 fever episodes during ALL therapy was used for model building and subsequent internal validation. RESULTS: Three hundred and seventy-seven patients were treated for ALL in two institutions with comparable critical and supportive care resources. A total of 55 patients accounted for 71 admissions to the critical care unit for sepsis that led to eight septic deaths during a 16-year study period. A retrospective analysis of risk factors for sepsis enabled us to build a model focused on 13 variables that discriminated admissions requiring critical care well: area under the receiver operating characteristic curve of .82; 95% CI .76-.87, p<.001, and Brier score of .033. Significant univariate predictors included neutropenia, presence of symptoms of abdominal pain, diarrhea, fever during induction or steroid-based phases, and the lack of any localizing source of infection at time of presentation. CONCLUSION: We have developed a risk prediction model that can reliably identify ALL patients undergoing treatment who are at a higher risk of life-threatening sepsis. Clinical applicability can potentially be extended to low-middle income settings, and its utility should be further studied in real-world settings.

Association of Genetic Ancestry With the Molecular Subtypes and Prognosis of Childhood Acute Lymphoblastic Leukemia.

IMPORTANCE: Racial and ethnic disparities persist in the incidence and treatment outcomes of childhood acute lymphoblastic leukemia (ALL). However, there is a paucity of data describing the genetic basis of these disparities, especially in association with modern ALL molecular taxonomy and in the context of contemporary treatment regimens. OBJECTIVE: To evaluate the association of genetic ancestry with childhood ALL molecular subtypes and outcomes of modern ALL therapy. DESIGN, SETTING, AND PARTICIPANTS: This multinational, multicenter genetic association study was conducted from March 1, 2000, to November 20, 2020, among 2428 children and adolescents with ALL enrolled in frontline trials from the United States, South East Asia (Singapore and Malaysia), and Latin America (Guatemala), representing diverse populations of European, African, Native American, East Asian, and South Asian descent. Statistical analysis was conducted from February 3, 2020, to April 19, 2021. MAIN OUTCOMES AND MEASURES: Molecular subtypes of ALL and genetic ancestry were comprehensively characterized by performing RNA sequencing. Associations of genetic ancestries with ALL molecular subtypes and treatment outcomes were then evaluated. RESULTS: Among the participants in the study, 1340 of 2318 (57.8%) were male, and the mean (SD) age was 7.8 (5.3) years. Of 21 ALL subtypes identified, 8 were associated with ancestry. East Asian ancestry was positively associated with the frequency of somatic DUX4 (odds ratio [OR], 1.30 [95% CI, 1.16-1.45]; P < .001) and ZNF384 (OR, 1.40 [95% CI, 1.18-1.66]; P < .001) gene rearrangements and negatively associated with BCR-ABL1-like ALL (OR, 0.79 [95% CI, 0.66-0.92]; P = .002) and T-cell ALL (OR, 0.80 [95% CI, 0.71-0.90]; P < .001). By contrast, occurrence of CRLF2 rearrangements was associated with Native American ancestry (OR, 1.48 [95% CI, 1.29-1.69]; P < .001). When the percentage of Native American ancestry increased, ETV6-RUNX1 fusion became less frequent (OR, 0.80 [95% CI, 0.70-0.91]; P < .001), with the opposite trend observed for ETV6-RUNX1-like ALL. There was a marked preponderance of T-cell ALL in children of African descent compared with those with a high percentage of Native American ancestry (African: OR, 1.22 [95% CI, 1.07-1.37]; P = .003; Native American: OR, 0.53 [95% CI, 0.40-0.67]; P < .001). African ancestry was also positively associated with the prevalence of TCF3-PBX1 (OR, 1.49 [95% CI, 1.25-1.76]; P < .001) and negatively associated with DUX4 rearrangements (OR, 0.70 [95% CI, 0.48-0.93]; P = .01) and hyperdiploidy (OR, 0.77 [95% CI, 0.68-0.86]; P < .001). African and Native American ancestries as continuous variables were both associated with poorer event-free survival (for every 25% increase in ancestry: hazard ratio [HR], 1.2; 95% CI, 1.1-1.4; P = .001 for African ancestry; HR, 1.3; 95% CI, 1.0-1.6; P = .04 for Native American ancestry) and overall survival (for every 25% increase in ancestry: HR, 1.2; 95% CI, 1.1-1.5; P = .01 for African ancestry; HR, 1.4; 95% CI, 1.0-1.8; P = .03 for Native American ancestry). Even after adjusting for biological subtypes and clinical features, Native American and African ancestries remained associated with poor prognosis. CONCLUSIONS AND RELEVANCE: This study suggests that ALL molecular subtypes and prognosis are associated with genetic ancestry, potentially pointing to a genetic basis for some of the racial and ethnic disparities in ALL. Therefore, molecular subtype-driven treatment individualization is needed to help address racial and ethnic gaps in outcomes.

Distinct clinical characteristics of DUX4- and PAX5-altered childhood B-lymphoblastic leukemia.

Among the recently described subtypes in childhood B-lymphoblastic leukemia (B-ALL) were DUX4- and PAX5-altered (PAX5alt). By using whole transcriptome RNA sequencing in 377 children with B-ALL from the Malaysia-Singapore ALL 2003 (MS2003) and Malaysia-Singapore ALL 2010 (MS2010) studies, we found that, after hyperdiploid and ETV6-RUNX1, the third and fourth most common subtypes were DUX4 (n = 51; 14%) and PAX5alt (n = 36; 10%). DUX4 also formed the largest genetic subtype among patients with poor day-33 minimal residual disease (MRD; n = 12 of 44). But despite the poor MRD, outcome of DUX4 B-ALL was excellent (5-year cumulative risk of relapse [CIR], 8.9%; 95% confidence interval [CI], 2.8%-19.5% and 5-year overall survival, 97.8%; 95% CI, 85.3%-99.7%). In MS2003, 21% of patients with DUX4 B-ALL had poor peripheral blood response to prednisolone at day 8, higher than other subtypes (8%; P = .03). In MS2010, with vincristine at day 1, no day-8 poor peripheral blood response was observed in the DUX4 subtype (P = .03). The PAX5alt group had an intermediate risk of relapse (5-year CIR, 18.1%) but when IKZF1 was not deleted, outcome was excellent with no relapse among 23 patients. Compared with MS2003, outcome of PAX5alt B-ALL with IKZF1 codeletion was improved by treatment intensification in MS2010 (5-year CIR, 80.0% vs 0%; P = .05). In conclusion, despite its poor initial response, DUX4 B-ALL had a favorable overall outcome, and the prognosis of PAX5alt was strongly dependent on IKZF1 codeletion.

Practical Considerations for Using RNA Sequencing in Management of B-Lymphoblastic Leukemia: Malaysia-Singapore Acute Lymphoblastic Leukemia 2020 Implementation Strategy.

Despite the immense genetic heterogeneity of B-lymphoblastic leukemia [or precursor B-cell acute lymphoblastic leukemia (B-ALL)], RNA sequencing (RNA-Seq) could comprehensively interrogate its genetic drivers, assigning a specific molecular subtype in >90% of patients. However, study groups have only started to use RNA-Seq. For broader clinical use, technical, quality control, and appropriate performance validation are needed. We describe the development and validation of an RNA-Seq workflow for subtype classification, TPMT/NUDT15/TP53 variant discovery, and immunoglobulin heavy chain (IGH) disease clone identification for Malaysia-Singapore acute lymphoblastic leukemia (ALL) 2020. We validated this workflow in 377 patients in our preceding Malaysia-Singapore ALL 2003/Malaysia-Singapore ALL 2010 studies and proposed the quality control measures for RNA quality, library size, sequencing, and data analysis using the International Organization for Standardization 15189 quality and competence standard for medical laboratories. Compared with conventional methods, we achieved >95% accuracy in oncogene fusion identification, digital karyotyping, and TPMT and NUDT15 variant discovery. We found seven pathogenic TP53 mutations, confirmed with Sanger sequencing, which conferred a poorer outcome. Applying this workflow prospectively to the first 21 patients in Malaysia-Singapore ALL 2020, we identified the genetic drivers and IGH disease clones in >90% of patients with concordant TPMT, NUDT15, and TP53 variants using PCR-based methods. The median turnaround time was 12 days, which was clinically actionable. In conclusion, RNA-Seq workflow could be used clinically in management of B-cell ALL patients.

Gastrointestinal and oncologic outcomes of pediatric gastrointestinal lymphoma following upfront resection or biopsy of bowel masses.

PURPOSE: Complete upfront resection of pediatric gastrointestinal lymphomas is recommended over biopsy whenever feasible, but either approach may have adverse sequelae. We sought to compare gastrointestinal and oncological outcomes of pediatric gastrointestinal lymphomas who underwent attempted upfront resection or biopsy of the presenting bowel mass. METHODS: We retrospectively reviewed charts of children with gastrointestinal lymphomas treated on LMB89 and LMB96 protocols from 2000 to 2019 who underwent upfront gastrointestinal surgery, and compared resection and biopsy groups. RESULTS: Of 33 children with abdominal lymphomas, 20 had upfront gastrointestinal surgery-10 each had resection or biopsy. Patients with attempted upfront resections had fewer postoperative gastrointestinal complications compared to biopsies (10% vs. 60%, p = 0.057), but longer time to chemotherapy initiation (median 11.5 vs. 4.5 days, p < 0.001). Three resection patients were surgically down-staged. Second surgeries were required in 30% and 40% of resected and biopsied patients, respectively, at median 4.6 months. Survival was similar in both groups, but better in patients on LMB96 protocol and stage II/III disease. CONCLUSIONS: Children with upfront attempted resection had low rates of surgical down-staging, greater delay in chemotherapy initiation, but fewer gastrointestinal complications and subsequent surgeries than biopsies. Survival was similar regardless of upfront surgery, likely reflecting beneficial effects of newer protocols.

Clinical and health-related quality of life outcomes of transfusion-dependent thalassaemia patients in Singapore.

INTRODUCTION: Transfusion-dependent thalassaemia is associated with complications related to iron overload from frequent red cell transfusions which affect quality of life. We collected data on the clinical outcomes, complications, socioeconomic status and health-related quality of life (HRQoL) of transfusion-dependent thalassaemia patients in Singapore, and analysed the associations between clinical and socioeconomic factors with development of transfusion-related complications and HRQoL scores. MATERIALS AND METHODS: This was a cross-sectional study of transfusion-dependent thalassaemia patients treated at four major public hospitals in Singapore. Clinical information was obtained from retrospective reviews of medical records. Socioeconomic data and patient-reported compliance to iron chelators were obtained from prospective interviews of patients or caregivers using a questionnaire. A validated, disease-specific HRQoL instrument, the TranQOL, was administered to patients and caregivers during a routine clinic or transfusion visit. RESULTS: Liver iron loading was the most common transfusion-related complication and occurred in 79% of patients. Cardiac iron loading was noted in 28.3% and endocrine complications were present in 34.2%. Liver iron loading was significantly associated with higher mean ferritin level. Cardiac iron loading was significantly associated with increasing age, higher mean ferritin level and type of iron chelator. Endocrine complications were associated with increasing age, higher mean ferritin level, type of iron chelator and poorer patient-reported compliance to iron chelators. The lowest TranQOL scores were reported by caregiver parents of patients aged less than 18 years. Lower TranQOL scores were significantly associated with increasing age, especially in the 31-50 age cohort, and with reception of social assistance. CONCLUSION: The main morbidities noted in transfusion-dependent thalassaemia patients in Singapore are from complications associated with iron loading. The cohort of older thalassaemia patients aged 31-50 experienced significantly higher rates of cardiac iron loading, endocrine complications and lower TranQOL scores compared to younger age cohorts.

Excellent Survival Outcomes of Pediatric Patients With Acute Myeloid Leukemia Treated With the MASPORE 2006 Protocol.

PURPOSE: To determine the prognostic factors in pediatric patients with acute myeloid leukemia (AML) and to assess whether their outcomes have improved over time. PATIENTS AND METHODS: Sixty-two patients with AML excluding acute promyelocytic leukemia were retrospectively analyzed. Patients in the earlier cohort (n = 36) were treated on the Medical Research Council (MRC) AML12 protocol, whereas those in the recent cohort (n = 26) were treated on the Malaysia-Singapore AML protocol (MASPORE 2006), which differed in terms of risk group stratification, cumulative anthracycline dose, and timing of hematopoietic stem-cell transplantation for high-risk patients. RESULTS: Significant improvements in 10-year overall survival and event-free survival were observed in patients treated with the recent MASPORE 2006 protocol compared to the earlier MRC AML12 protocol (overall survival: 88.0% ± 6.5% vs 50.1% ± 8.6%, P = .002; event-free survival: 72.1% ± 9.0 vs 50.1% ± 8.6%, P = .045). In univariate analysis, patients in the recent cohort had significantly lower intensive care unit admission rate (11.5% vs 47.2%, P = .005) and numerically lower relapse rate (26.9% vs 50.0%, P = .068) compared to the earlier cohort. Multivariate analysis showed that treatment protocol was the only independent predictive factor for overall survival (hazard ratio = 0.21; 95% confidence interval, 0.06-0.73, P = .014). CONCLUSION: Outcomes of pediatric AML patients have improved over time. The more recent MASPORE 2006 protocol led to significant improvement in long-term survival rates and reduction in intensive care unit admission rate.

Bleeder With a Clot: Thrombosis Following Treatment of Bleeding in a Child With Severe Hemophilia B.

A 16-year-old boy with severe hemophilia B and minimal bleeding manifestations in his early childhood presented with gastrointestinal bleeding at 11 years of age. Following administration of prothrombin complex concentrate, he developed peripheral venous thrombosis and cerebral sinovenous thrombosis, posing a management dilemma. His cerebral sinovenous thrombosis resolved spontaneously, proving watchful waiting to be a useful strategy. He developed spontaneous intracranial bleed at 14 years of age for which he was treated with factor IX concentrate and commenced on prophylaxis. We discuss the factors contributing to genotype-phenotype dissonance in severe hemophilia and considerations before commencing prophylaxis in such cases.

Intracellular vincristine levels in lymphoblasts affect treatment outcome in childhood B-lymphoblastic leukaemia: Ma-Spore ALL 2010 study.

AIMS: Vincristine (VCR) is a key drug in the successful multidrug chemotherapy for childhood acute lymphoblastic leukaemia (ALL). However, it remains unclear how VCR pharmacokinetics affects its antileukaemic efficacy. The objective of this study is to explore the VCR pharmacokinetic parameters and intracellular VCR levels in an up-front window of Ma-Spore ALL 2010 (MS2010) study. METHODS: We randomised 429 children with newly diagnosed ALL to 15-minute vs 3-hour infusion for the first dose of VCR to study if prolonging the first dose of VCR infusion improved response. In a subgroup of 115 B-ALL and 20 T-ALL patients, we performed VCR plasma (n = 135 patients) and intracellular (n = 66 patients) pharmacokinetic studies. The correlations between pharmacokinetic parameters and intracellular VCR levels with early treatment response, final outcome and ABCB1 genotypes were analysed. RESULTS: There was no significant difference between 15-minute and 3-hour infusion schedules in median Day 8 peripheral or bone marrow blast response. Plasma VCR pharmacokinetic parameters did not predict outcome. However, in B-ALL, Day 33 minimal residual disease (MRD) negative patients and patients in continuous complete remission had significantly higher median intracellular VCR24h levels (P = .03 and P = .04, respectively). The median VCR24h intracellular levels were similar among the common genetic subtypes of ALL (P = .4). Patients homozygous for wild-type ABCB1 2677GG had significantly higher median intracellular VCR24h (P = .04) than 2677TT. CONCLUSION: We showed that in childhood B-ALL, the intracellular VCR24h levels in lymphoblasts affected treatment outcomes. The intracellular VCR24h level was independent of leukaemia subtype but dependent on host ABCB1 G2677T genotype.

Neuroblastoma patient-derived cultures are enriched for a mesenchymal gene signature and reflect individual drug response.

Ex vivo evaluation of personalized models can facilitate individualized treatment selection for patients, and advance the discovery of novel therapeutic options. However, for embryonal malignancies, representative primary cultures have been difficult to establish. We developed patient-derived cell cultures (PDCs) from chemo-naïve and post-treatment neuroblastoma tumors in a consistent and efficient manner, and characterized their in vitro growth dynamics, histomorphology, gene expression, and functional chemo-response. From 34 neuroblastoma tumors, 22 engrafted in vitro to generate 31 individual PDC lines, with higher engraftment seen with metastatic tumors. PDCs displayed characteristic immunohistochemical staining patterns of PHOX2B, TH, and GD2 synthase. Concordance of MYCN amplification, 1p and 11q deletion between PDCs and patient tumors was 83.3%, 72.7%, and 80.0% respectively. PDCs displayed a predominantly mesenchymal-type gene expression signature and showed upregulation of pro-angiogenic factors that were similarly enriched in culture medium and paired patient serum samples. When tested with standard-of-care cytotoxics at human Cmax -equivalent concentrations, MYCN-amplified and non-MYCN-amplified PDCs showed a differential response to cyclophosphamide and topotecan, which mirrored the corresponding patients' responses, and correlated with gene signatures of chemosensitivity. In this translational proof-of-concept study, early-phase neuroblastoma PDCs enriched for the mesenchymal cell subpopulation recapitulated the individual molecular and phenotypic profile of patient tumors, and highlighted their potential as a platform for individualized ex vivo drug-response testing.

Dominant-negative NFKBIA mutation promotes IL-1ß production causing hepatic disease with severe immunodeficiency.

Although IKK-ß has previously been shown as a negative regulator of IL-1ß secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1ß expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1ß secretion was elevated in the patient's stimulated leukocytes, in her induced pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hypersecretion of IL-1ß correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1ß release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1ß secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.

DISSEMINATED BACILLUS-CALMETTE-GUÉRIN INFECTIONS AND PRIMARY IMMUNODEFICIENCY DISORDERS IN SINGAPORE: A SINGLE CENTER 15-YEAR RETROSPECTIVE REVIEW.

BACKGROUND: Disseminated Bacillus Calmette-Guérin (BCG) disease (BCGosis) is a classical feature of children with primary immunodeficiency disorders (PIDs). METHODS: A 15-year retrospective review was conducted in KK Women's and Children's Hospital in Singapore, from January 2003 to October 2017. RESULTS: Ten patients were identified, the majority male (60.0%). The median age at presentation of symptoms of BCG infections was 3.8 (0.8 - 7.4) months. All the patients had likely underlying PIDS - four with Severe Combined Immunodeficiency (SCID), three with Mendelian Susceptibility to Mycobacterial Diseases (MSMD), one with Anhidrotic Ectodermal Dysplasia with Primary Immunodeficiency (EDA-ID), one with combined immunodeficiency (CID), and one with STAT-1 gain-of-function mutation. Definitive BCGosis was confirmed in all patients by the identification of Mycobacterium bovis subsp BCG from microbiological cultures. The susceptibility profiles of Mycobacterium bovis subsp BCG are as follows: Rifampicin (88.9%), Isoniazid (44.47%), Ethambutol (100.0%), Streptomycin (100.0%), Kanamycin (100.0%), Ethionamide (25.0%), and Ofloxacin (100.0%). Four patients (40.0%) received a three-drug regimen. Five patients (50.0%) underwent hematopoietic stem cell transplant (HSCT), of which three (60%) have recovered. Overall mortality was 50.0%. CONCLUSION: Disseminated BCG disease (BCGosis) should prompt immunology evaluation to determine the diagnosis of the immune defect. A three-drug regimen is adequate for treatment if the patient undergoes early HSCT.

Identifying IGH disease clones for MRD monitoring in childhood B-cell acute lymphoblastic leukemia using RNA-Seq.

Identifying patient-specific clonal IGH/TCR junctional sequences is critical for minimal residual disease (MRD) monitoring. Conventionally these junctional sequences are identified using laborious Sanger sequencing of excised heteroduplex bands. We found that the IGH is highly expressed in our diagnostic B-cell acute lymphoblastic leukemia (B-ALL) samples using RNA-Seq. Therefore, we used RNA-Seq to identify IGH disease clone sequences in 258 childhood B-ALL samples for MRD monitoring. The amount of background IGH rearrangements uncovered by RNA-Seq followed the Zipf's law with IGH disease clones easily identified as outliers. Four hundred and ninety-seven IGH disease clones (median 2, range 0-7 clones/patient) are identified in 90.3% of patients. High hyperdiploid patients have the most IGH disease clones (median 3) while DUX4 subtype has the least (median 1) due to the rearrangements involving the IGH locus. In all, 90.8% of IGH disease clones found by Sanger sequencing are also identified by RNA-Seq. In addition, RNA-Seq identified 43% more IGH disease clones. In 69 patients lacking sensitive IGH targets, targeted NGS IGH MRD showed high correlation (R = 0.93; P = 1.3 × 10-14), better relapse prediction than conventional RQ-PCR MRD using non-IGH targets. In conclusion, RNA-Seq can identify patient-specific clonal IGH junctional sequences for MRD monitoring, adding to its usefulness for molecular diagnosis in childhood B-ALL.

A simplified approach for anaesthetic management of diagnostic procedures in children with anterior mediastinal mass.

INTRODUCTION: Children with an anterior mediastinal mass (AMM) need general anaesthesia (GA) or deep sedation for diagnostic procedures more often than adult patients. Anaesthetic management to prevent such complications includes maintenance of spontaneous ventilation (SV) and prebiopsy corticosteroids/radiotherapy. METHODS: We reviewed the medical records of children with AMM who were brought to the operating theatre for diagnostic procedures (prior to chemotherapy) between 2001 and 2013. Our aim was to describe the clinical features, radiological findings and anaesthetic management, as well as determine any association with complications. RESULTS: 25 patients (age range 10 months-14 years) were identified during the study period. Corticosteroid therapy was started before the biopsy for one patient. All 25 patients had GA/sedation. A senior paediatric anaesthesiologist was involved in all procedures. Among 13 high-risk patients, SV was maintained in 11 (84.6%) patients, ketamine was used as the main anaesthetic in 8 (61.5%) patients, 6 (46.2%) patients were in a sitting position and no airway adjunct was used for 7 (53.8%) patients. There were 3 (12.0%) minor complications. CONCLUSION: Based on our results, we propose a simplified workflow, wherein airway compression of any degree is considered high risk. For patients with high-risk features, multidisciplinary input should be sought to decide whether the child would be fit for a procedure under GA/sedation or considered unfit for any procedure. Recommendations include the use of less invasive methods, involving experienced anaesthesiologists to plan the anaesthetic technique and maintaining SV.

Primary Adrenal Angiomatoid Fibrous Histiocytoma With Novel EWSR1-ATF1 Gene Fusion Exon-Exon Breakpoint.

We describe the clinical, pathological, and molecular features of a primary adrenal angiomatoid fibrous histiocytoma (AFH) in an 11-year-old girl presenting with pyrexia of unknown origin. We performed next-generation sequencing-based anchored multiplex polymerase chain reaction (Archer® FusionPlex® sarcoma assay), which revealed an EWSR1-ATF1 gene fusion with novel breakpoints in exon 11 of EWSR1 and exon 3 of ATF1. The pyrexia resolved fully after surgical resection, and the patient was disease-free on follow-up at 1 year and 6 months. This case exemplifies the value of molecular testing of pediatric neoplasms presenting at unusual sites for diagnosis and identification of novel gene fusion breakpoints.

Retroperitoneal tumors and congenital variations in vascular anatomy of retroperitoneal great vessels.

BACKGROUND/OBJECTIVES: Variations in vascular anatomy (VIVAs) of the retroperitoneal great vessels are uncommon but can potentially complicate surgical procedures and negatively affect treatment outcomes, yet their incidence and clinical impact are poorly studied. We sought to assess the incidence and clinical impact of VIVAs of retroperitoneal great vessels in patients with retroperitoneal tumors. METHODS: We retrospectively analyzed imaging, surgical, treatment and survival data of all pediatric patients with retroperitoneal tumors who underwent resection between January 2007 and October 2016, comparing preoperative scans with corresponding intraoperative observations, and subsequent surgical outcomes. RESULTS: Among 66 children with renal, adrenal and paravertebral tumors, 6 (9%) had retroperitoneal VIVAs. Retroperitoneal VIVAs were present only with right-sided tumors and significantly associated with more frequent intraoperative complications (P = 0.013). While the presence of retroperitoneal VIVAs was not directly associated with survival outcomes, relapse was more frequent in patients with VIVAs (33%) than those without (18.3%, P = 0.378), and relapse was also associated with lower overall and event-free survival (P < 0.001). CONCLUSIONS: VIVAs of retroperitoneal great vessels occurred in 9% of our patients with retroperitoneal tumors. Retroperitoneal VIVAs were associated with higher rates of intraoperative complications and disease relapse but was not directly related to survival outcomes. TYPE OF STUDY: Retrospective review study. LEVEL OF EVIDENCE: Level III Retrospective comparative study.

Clinical, pathological and loss of heterozygosity differences in Wilms tumors between Asian and non-Asian children.

Wilms tumor demonstrates significant interethnic epidemiological, histological and outcome differences, and is rare and poorly studied among Asians. We compared the clinicopathological, and loss of heterozygosity (LOH) profile and survival outcomes of Asian and non-Asian patients with Wilms tumor. Clinical charts and histological slides from patients with malignant renal tumors over a period of 20 years were retrospectively reviewed. We adapted a genotyping assay to determine 1p36 and 16q21-22 LOH in formalin-fixed paraffin-embedded (FFPE) specimens, and compared these characteristics between Asian and non-Asian patients. Fifty-three (79.1%) Asian and 14 (20.9%) non-Asian patients had Wilms tumors. Compared to non-Asians, Asians were younger (mean 4.6 and 4.0 years, respectively), had more equal gender distribution (female: male = 1.8 and 1.0, respectively), fewer tumors with unfavorable histology (25.0% and 4.1%, respectively, p = 0.05), and less advanced disease at presentation, yet similar nodal metastases rates (16.7% and 18.4%, respectively). No Asian patients had bilateral tumors. Our adapted genotyping assay accurately determined LOH in FFPE specimens <10 years post-fixation. Among 30 Asian patients, 1p and 16q LOH were each detected in 5 (16.7%) patients, respectively-similar to rates reported in other ethnicities. Yet after similar treatment with National Wilms Tumor Study regimens, 15-year event-free and overall survival for Asian patients was 95.7% and 96.3% respectively. In summary, despite similar nodal metastasis and LOH rates, Asian patients had fewer unfavorable histology tumors, lower-stage disease, and better survival outcomes. The bases for these differences and implications on treatment strategy for these patients warrant further study.

Clinical relevance of screening checklists for detecting cancer predisposition syndromes in Asian childhood tumours.

Assessment of cancer predisposition syndromes (CPS) in childhood tumours is challenging to paediatric oncologists due to inconsistent recognizable clinical phenotypes and family histories, especially in cohorts with unknown prevalence of germline mutations. Screening checklists were developed to facilitate CPS detection in paediatric patients; however, their clinical value have yet been validated. Our study aims to assess the utility of clinical screening checklists validated by genetic sequencing in an Asian cohort of childhood tumours. We evaluated 102 patients under age 18 years recruited over a period of 31 months. Patient records were reviewed against two published checklists and germline mutations in 100 cancer-associated genes were profiled through a combination of whole-exome sequencing and multiplex ligation-dependent probe amplification on blood-derived genomic DNA. Pathogenic germline mutations were identified in ten (10%) patients across six known cancer predisposition genes: TP53, DICER1, NF1, FH, SDHD and VHL. Fifty-four (53%) patients screened positive on both checklists, including all ten pathogenic germline carriers. TP53 was most frequently mutated, affecting five children with adrenocortical carcinoma, sarcomas and diffuse astrocytoma. Disparity in prevalence of germline mutations across tumour types suggested variable genetic susceptibility and implied potential contribution of novel susceptibility genes. Only five (50%) children with pathogenic germline mutations had a family history of cancer. We conclude that CPS screening checklists are adequately sensitive to detect at-risk children and are relevant for clinical application. In addition, our study showed that 10% of Asian paediatric solid tumours have a heritable component, consistent with other populations.