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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.

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.

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. 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. 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

Although IKK-β has previously been shown as a negative regulator of IL-1β 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-1β 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-1β 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-1β 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-1β release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1β 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. Refereed/Peer-reviewed

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 C max‐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., We developed a system to efficiently and consistently generate patient‐derived cultures (PDCs) of neuroblastoma that recapitulated individual patient’s cytogenetic and immunohistochemical features, and phenotypic responses to standard‐of‐care chemotherapy. PDCs predominantly express the gene signature of the recently described neuroblastoma mesenchymal super‐enhancer state. This proof‐of concept provides translational evidence supporting the proposed role of the mesenchymal cell subpopulation in determining clinical treatment response and offers a novel platform for developing personalized ex vivo therapeutic decision‐making strategies for neuroblastoma.

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.

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.

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.A retrospective analysis of 2068 fever episodes during ALL therapy was used for model building and subsequent internal validation.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.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.

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.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.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.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.

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.

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

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

Accurate risk assignment in childhood acute lymphoblastic leukaemia is essential to avoid under- or over-treatment. We hypothesized that time-series gene expression profiles (GEPs) of bone marrow samples during remission-induction therapy can measure the response and be used for relapse prediction. We computed the time-series changes from diagnosis to Day 8 of remission-induction, termed Effective Response Metric (ERM-D8) and tested its ability to predict relapse against contemporary risk assignment methods, including National Cancer Institutes (NCI) criteria, genetics and minimal residual disease (MRD). ERM-D8 was trained on a set of 131 patients and validated on an independent set of 79 patients. In the independent blinded test set, unfavourable ERM-D8 patients had >3-fold increased risk of relapse compared to favourable ERM-D8 (5-year cumulative incidence of relapse 38·1% vs. 10·6%; P = 2·5 × 10-3 ). ERM-D8 remained predictive of relapse [P = 0·05; Hazard ratio 4·09, 95% confidence interval (CI) 1·03-16·23] after adjusting for NCI criteria, genetics, Day 8 peripheral response and Day 33 MRD. ERM-D8 improved risk stratification in favourable genetics subgroups (P = 0·01) and Day 33 MRD positive patients (P = 1·7 × 10-3 ). We conclude that our novel metric - ERM-D8 - based on time-series GEP after 8 days of remission-induction therapy can independently predict relapse even after adjusting for NCI risk, genetics, Day 8 peripheral blood response and MRD.

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.

BACKGROUND: Although cure rates of childhood acute lymphoblastic leukemia (ALL) have improved significantly with risk-adapted therapy, stark racial disparities persist in both the incidence and treatment outcomes of ALL. There is a paucity of data describing the genetic basis of these disparities, especially in relation to modern ALL molecular taxonomy and in the context of contemporary treatment regimens. AIMS: To determine the associations of genetic ancestry with ALL biology, and the relevance of genetic ancestry to survival outcomes of modern ALL therapy. Methods: This was a multi-national genomic study of 2,428 children with ALL on front-line trials from United States, Singapore, Malaysia, and Guatemala, representing diverse populations of European, African, Native American, East Asian, and South Asian descent. We performed RNA-sequencing to characterize ALL molecular subtype and genetic ancestry, and then evaluated associations of genetic ancestries with ALL biology and treatment outcomes. Results: Of 21 ALL subtypes, 11 showed significant associations with ancestry. The frequency of somatic DUX4 gene rearrangement was positively correlated with both East Asian and South Asian ancestries; and genomic alterations in ZNF384 and PAX5 increased with East Asian ancestry. By contrast, occurrence of CRLF2 rearrangements was linked to Native American ancestry. ETV6-RUNX1 fusion became less frequent as Native American ancestry increased, with the opposite observed for ETV6-RUNX1-like ALL. There was a marked preponderance of T-ALL in children of African descent. African ancestry was also positively correlated with the prevalence of TCF3-PBX1 and MEF2D fusions. Survival outcomes differed significantly by genetic ancestry, where African and Native American ancestries were both associated with poorer event-free survival (African: HR, 2.3; 95% CI, 1.4 – 3.8; P=0.001; Native American: HR, 2.5; 95% CI, 1·0 – 5.9; P=0.044) and overall survival (African: HR, 2·4; 95% CI, 1.2 – 4.7; P=0.012 for African; Native American: HR, 3.3; 95% CI, 1.1 – 10.0; P=0.033). Importantly, even after adjusting for biological subtypes and clinical features, Native American and African ancestries remained independently associated with poor prognosis. Conclusions: ALL biology and prognosis are highly associated with genetic ancestry, pointing to a genetic basis for racial disparities in ALL. Biology-driven treatment individualization is needed to eliminate racial gaps in outcomes. Citation Format: Shawn Lee, Federico Antillon, Deqing Pei, Wenjian Yang, Kathryn G Roberts, Zhenhua Li, Meenakshi Devidas, Wentao Yang, Cesar Najera, Hai Peng Lin, Ah Moy Tan, Hany Ariffin, Cheng Cheng, William E. Evans, Stephen P. Hunger, Sima Jeha, Charles G. Mullighan, Mignon L. Loh, Allen EJ Yeoh, Ching-Hon Pui, Jun J. Yang. The impact of genetic ancestry on the biology and prognosis of childhood acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-156.

Background: Haploidentical hematopoietic cell transplantation (HCT) provides an alternative option for patients without HLA-matched donors. Graft-versus-host disease (GVHD), engraftment failure, and infectious complications are main causes of non-relapse mortality (NRM). We hypothesized that selective depletion of TCRαβ+ and CD45RA+ naïve T cells will permit hematopoietic engraftment while effectively reducing GVHD and providing donor immune reconstitution through adoptive transfer of donor's mature NK cells, γδ T cells and CD45RO+ memory T cells. Methods: Mobilized PBSC products were divided into two fractions in 9:1 ratio and depleted using CliniMACS device after labelling with TCRαβ and CD45RA reagents (Miltenyi Biotec, Bergish-Gladbach, Germany) respectively. All except 6 patients received the standard conditioning regimen of fludarabine 160mg/m 2 divided daily over 4 days, thiotepa 10mg/kg divided twice daily for 1 day, and melphalan 70 - 140mg/m 2 for 1 day, in combination with either total lymphoid irradiation 6Gy (n=53) or 7.5Gy (n=12) over 3 equal fractions, or total body irradiation of 2Gy (n=17), or thymoglobulin (n=2). Short term GVHD prophylaxis was given for 30 days to 1 patient using MMF, 73 using tacrolimus, and 2 using sirolimus. Results: Between January 2017 and July 2021, we transplanted 85 patients, including 78 adults (median age, 48 years; range 20 - 70) and 7 children (median age, 13 years, range 7 - 17), with high risk AML (n=44), ALL (n=19), MDS (n=9), plasma cell neoplasm (n=4), mast cell leukemia (n=1), acute undifferentiated leukemia (n=1), CMMoL (n=2), CML (n=1) and lymphoma (n=2). Patients were infused with TCRαβ and CD45RA depleted grafts containing median of 6.19 x 10 6 (range 3.54 - 20.78) CD34+ cells/kg, 0.00 x 10 4 (range 0 - 0.97) CD45RA+CD3+ cells/kg, and 1.10 x 10 6 (range 0.15 - 11.67) CD45RO+CD3+ cells/kg. TCRαβ depleted graft fraction contained a median of 0.42 x 10 4 (range 0 - 11.30) TCRαβ+ cells/kg, and 7.61 x 10 6 (range 0.62 - 31.84) TCRγδ+ cells/kg. Only 1 patient experienced primary graft failure with no secondary graft failures. All others had engraftment of ANC > 500 cells/µL at median of 12 days (range 8 - 22) and PLT > 20,000 cells/µL at median of 11 days (range 7 - 17). 6 patients with high donor-specific HLA antibodies (DSA) titres engrafted successfully after desensitisation with plasma exchange, rituximab, and immunoglobulin. 29 patients (34%) developed acute GVHD of grade II - IV (Gd II, n=20; Gd III, n=5; Gd IV, n=4), with a cumulative incidence (CI) of grade II-IV and grade III-IV of 31% (95% CI 21-42%) and 11% (95% CI 5-19%) respectively, at 100 days. Chronic GVHD was seen in only 4 patients at a 2-year CI of 6% (95% CI 2-13%). 1-year CI of non-relapse mortality (NRM) and relapse were 22.7% (95% CI 13.9 - 32.9%) and 15.7% (95% CI 8.3 - 25.3%) respectively. 4 of the 17 NRM were attributed to aGVHD. Viral reactivation included CMV (n=32), HHV-6 (n=22), EBV (n=15), and adenovirus (n=8). 15 patients (17.6%) died of infection within 180 days, including 6 patients with fatal bacteraemia (bacteria, n=4; candidemia, n=2) and 1 patient from disseminated adenovirus infection. At a median follow up of 448 days (range 16- 1648) in surviving patients, 2-year overall (OS), event-free (EFS), and GVHD-free/relapse-free (GRFS) survival were 64.2 %, 54.0 %, and 49.0%, respectively (Figure 1). In multivariate analysis, only HCT-comorbidity index (HCT-CI) showed significant impact on OS (HR 3.38; 95% CI 1.42 - 8.02; p=0.0059), EFS (HR 2.62; 95% CI 1.18 - 5.76; p=0.0017), and NRM (HR 4.92; 95% CI 1.79 - 13.58; p=0.0021). Disease risk index (DRI) showed a trend in higher risk of relapse (HR 2.83; 95% CI 0.96 - 8.33; p=0.059). 2-year OS, EFS, and GRFS for the subset of 58 patients (adults, n=52; children, n=6) with HCT-CI score of 0 were 76.6 %, 63.4%, and 57.5 %, respectively (Figure 2). Conclusions: Our preliminary results suggest that RIC haplo-HCT with TCRαβ and CD45RA+ depleted grafts allow successful allograft in high-risk patients lacking suitable matched donors, including patients with high levels of DSA. Acute GVHD was generally abortive, leading to low incidence of chronic GVHD. Best outcomes are seen in patients with favourable HCT-CI. Further efforts are needed to reduce the risk of infection-related death in patients with high risk HCT-CI, and relapse in patients with high risk DRI, through optimization of anti-microbial prophylaxis or prophylactic infusion of memory-cell DLI. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Purpose/Objective(s) Total lymphatic irradiation (TLI) is a procedure in which a patient's major lymph nodes and spleen are treated with low doses of radiation for recipient immunosuppression as part of multi-agent conditioning for cardiac, lung or hematopoietic stem cell transplantation, to facilitate engraftment and to decrease the risk of rejection. TLI is considered an attractive option instead of total body irradiation (TBI) to mitigate side effects of radiation exposure to normal tissues. There are no established guidelines on technique and dose fractionation. Herein, we share our experience. Materials/Methods Retrospective review of children who received TLI for hematopoietic stem cell transplants at the hospital was done. Results Twenty-two patients who had TLI from 2004 to 2020, with a median age of 7.5 years (range 0.9-19.0) were included. Twelve had benign conditions and 10 had malignant conditions. Eighteen had haploidentical grafts, 2 had matched sibling and 2 had unrelated donors. Four patients had previously failed engraftment prior to TLI. Conditioning included multi-agent chemotherapy for all including rabbit-ATG for 12 patients. All patients had computer-tomographic scans simulation except for 2 patients who were treated in the earlier years when only orthogonal XR was available. Dose fractionation was single prior to 2017 (5-7Gy/1#) and in recent times more fractionated (6-7.5Gy/3#). The treatment targets were the spleen and the following nodal stations: cervical, axillary, mediastinal, hilar, paraaortic, pelvic, inguinal and femoral. Normal organs including the medial thyroid, heart, lungs, and abdominal organs were shielded with multi-leaf collimators using three-dimensional conformal technique (Figure 1, 2). Of 12 patients with benign conditions, all are alive at last follow up. Of 10 patients with malignant conditions, 3 are dead from disease. Five out of 12 patients who had haploidentical grafts, 0/2 matched sibling graft and 1/2 unrelated grafts experienced rejection. The median follow up is 14.0 months (IQR 6-41.5). None developed acute pneumonitis. One patient developed thyroid cancer 14 years after TLI. Conclusion Herein we describe our TLI experience and technique. More studies should be undertaken to evaluate the safety and efficacy of TLI compared to TBI. Going forward, a consensus on technique and dose should be developed.

Context: Although cure rates of childhood acute lymphoblastic leukemia (ALL) have improved with risk-adapted therapy, stark racial disparities persist in both the incidence and treatment outcomes of ALL. There is a paucity of data describing the genetic basis of these disparities, especially in relation to modern ALL molecular taxonomy and in the context of contemporary treatment regimens. Objective: Determine the associations of genetic ancestry with ALL biology and the relevance of genetic ancestry to survival outcomes of modern ALL therapy. Design: This was a multi-national genomic study of 2,428 children with ALL in front-line trials from the United States, Singapore, Malaysia, and Guatemala, representing diverse populations of European, African, Native American, East Asian, and South Asian descent. We performed RNA sequencing to characterize ALL molecular subtype and genetic ancestry and then evaluated associations of genetic ancestries with ALL biology and treatment outcomes. Results: Of 21 ALL subtypes, 11 showed significant associations with ancestry. The frequency of somatic DUX4 gene rearrangement was positively correlated with both East Asian and South Asian ancestries, and alterations in ZNF384 and PAX5 increased with East Asian ancestry. By contrast, occurrence of CRLF2 rearrangements was linked to Native American ancestry. ETV6-RUNX1 fusion became less frequent as Native American ancestry increased, with the opposite observed for ETV6-RUNX1-like. There was a marked preponderance of T-ALL in children of African descent. African ancestry was also positively correlated with the prevalence of TCF3-PBX1 and MEF2D fusions. Survival outcomes differed significantly by genetic ancestry, where African and Native American ancestries were both associated with poorer event-free survival (African: HR, 2.3; 95% CI, 1.4–3.8; P=0.001; Native American: HR, 2.5; 95% CI, 1.0–5.9; P=0.044) and overall survival (African: HR, 2·4; 95% CI, 1.2-4.7; P=0.012 for African; Native American: HR, 3.3; 95% CI, 1.1-10.0; P=0.033). Importantly, even after adjusting for biological subtypes and clinical features, Native American and African ancestries remained independently associated with poor prognosis. Conclusions: ALL biology and prognosis are highly associated with genetic ancestry, pointing to a genetic basis for racial disparities in ALL. Biology-driven treatment individualization is needed to eliminate racial gaps in outcomes.

Publisher's Note: There is an [Inside Blood Commentary][1] on this article in this issue. To the editor: Our understanding of acute lymphoblastic leukemia (ALL) has expanded tremendously in the past few years because of large-scale genomic studies.[1][2],[2][3] ALL is characterized by a relatively

Background: Haploidentical hematopoietic cell transplantation (HCT) provides an alternative option for patients without HLA-matched donor. Graft-versus-host disease (GVHD), engraftment failure, and infectious complications continue to be the main causes of non-relapse mortality (NRM). We hypothesized that selective depletion of TCRαβ+ and CD45RA+ naïve T-cells subset will permit hematopoietic engraftment, while effectively reducing GVHD, and provide improved donor immune reconstitution through adoptive transfer of donor's mature NK-cells, γδ T-cells, and CD45RO+ memory T-cells. Methods: Mobilized PBSC products were divided into two fractions in 9:1 ratio and depleted using CliniMACS device after labeling with TCRαβ and CD45RA reagents (Miltenyi Biotec, Bergish-Gladbach, Germany), respectively. The conditioning regimen consisted of fludarabine 160mg/m2 divided daily over 4 days, thiotepa 10mg/kg divided twice daily for 1 day, and melphalan 70-140mg/m2 for 1 day, in combination with total lymphoid irradiation 6Gy (n=35) or 7.5Gy (n=12) over 3 equal fractions, total body irradiation of 2Gy (n=13) or antithymocyte globuline (n=2). Short term GVHD prophylaxis for 30 days was given to 1 patient using MMF, 50 patients using tacrolimus, and 2 patients using sirolimus. Results: We transplanted 62 patients, including 55 adults (median age, 47 years; range 20-69) and 7 children (median age, 13 years, range 7-17) with high risk AML (n=34), ALL (n=15), MDS (n=7), plasma cell neoplasm (n=2), mast cell leukemia (n=1), acute undifferentiated leukemia (n=1), and NK/T-cell lymphoma (n=1). The patients were infused with TCRαβ and CD45RA depleted graft containing a median of 6.79 x 106 (range 3.54-20.78) CD34+ cells/kg, 0.00 x 104 (range 0-0.97) CD45RA+CD3+ cells/kg, and 1.09 x 106 (range 0.15-11.67) CD45RO+CD3+ cells/kg. The TCRαβ depleted graft fraction contained a median of 0.20 x 104 (range 0-11.30) TCRαβ+ cells/kg, and 8.52 x 106 (range 0.62-30.00) TCRγδ+ cells/kg. Only 1 patient experienced primary graft failure. All others had engraftment of ANC > 500 cells/µL at a median of 10 days (range 8-22) and platelet > 20,000 cells/µL at a median of 12 days (range 8-22). There was no secondary graft failure. Six patients with high titers of donor-specific HLA antibodies (DSA) engrafted successfully after effective desensitisation with plasma exchange, rituximab, and immunoglobulin. Twenty-two patients (35%) developed acute GVHD of grade II - IV (Gd II, n=15; Gd III, n=5; Gd IV, n=2). Three patients experienced chronic GVHD, giving 2-year cumulative incidence of 7 %. Day 180 cumulative incidence of NRM and relapse were 24.8% (95% CI 14.3-36.8%) and 14.9% (95% CI 6.3-27.0%), respectively. Four of the 16 NRM were attributed to aGVHD. Viral reactivation included CMV (n=25), HHV-6 (n=14), EBV (n=11), and adenovirus (n=7). Fourteen patients died of infection, including 5 patients who had fatal blood stream infection (bacteria, n=3; candidemia, n=2) and 1 patient from disseminated adenovirus infection within 180 days. With a median follow-up of 298 days (range 21-1298) in surviving patients, the 2-year overall (OS), event-free (EFS), and GVHD-free/relapse-free (GRFS) survival were 58% (95% CI 37.6-73.4%), 43% (95% CI 27-57%), and 39% (95% CI 24-54%), respectively (Figure 1). In multivariable analysis, only HCT-comorbidity index (HCT-CI) showed significant impact on OS (HR 6.24; 95% CI 2.05-19.05; p=0.0013), EFS (HR 4.80; 95% CI 1.73-13.35; p=0.0027), and RRM (HR 6.49; 95% CI 1.44-29.25; p=0.015), whereas disease risk index (DRI) impacts risk of relapse (HR 4.50; 95% CI 1.39-14.52; p=0.012). The 2-year OS, EFS, and GRFS for the subset of 30 patients (adults, n=25; children, n=5) with HCT-CI score of 0 and low/intermediate risk DRI were 68%, 68%, and 60%, respectively (Figure 2). Conclusions: Our preliminary results suggest that RIC haplo-HCT with TCRαβ and CD45RA+ depleted grafts allowed successful allograft in high-risk patients lacking a suitable matched donor, including patients with high level of DSA. Acute GVHD was generally abortive, leading to low incidence of chronic GVHD. The best outcome is seen in patients with favorable HCT-CI and DRI. Further efforts are needed to lower the risk of death due to blood stream infection, and relapse in patients with high risk DRI, such as optimisation of anti-microbial prophylaxis or repeated doses of memory-cell donor lymphocyte infusion (DLI). Disclosures No relevant conflicts of interest to declare.

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.

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.

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

Most B cell precursor acute lymphoblastic leukemia (BCP ALL) can be classified into known major genetic subtypes, while a substantial proportion of BCP ALL remains poorly characterized in relation to its underlying genomic abnormalities. We therefore initiated a large-scale international study to reanalyze and delineate the transcriptome landscape of 1,223 BCP ALL cases using RNA sequencing. Fourteen BCP ALL gene expression subgroups (G1 to G14) were identified. Apart from extending eight previously described subgroups (G1 to G8 associated with MEF2D fusions, TCF3–PBX1 fusions, ETV6–RUNX1–positive/ETV6–RUNX1–like, DUX4 fusions, ZNF384 fusions, BCR–ABL1/Ph–like, high hyperdiploidy, and KMT2A fusions), we defined six additional gene expression subgroups: G9 was associated with both PAX5 and CRLF2 fusions; G10 and G11 with mutations in PAX5 (p.P80R) and IKZF1 (p.N159Y), respectively; G12 with IGH–CEBPE fusion and mutations in ZEB2 (p.H1038R); and G13 and G14 with TCF3/4–HLF and NUTM1 fusions, respectively. In pediatric BCP ALL, subgroups G2 to G5 and G7 (51 to 65/67 chromosomes) were associated with low-risk, G7 (with ≤50 chromosomes) and G9 were intermediate-risk, whereas G1, G6, and G8 were defined as high-risk subgroups. In adult BCP ALL, G1, G2, G6, and G8 were associated with high risk, while G4, G5, and G7 had relatively favorable outcomes. This large-scale transcriptome sequence analysis of BCP ALL revealed distinct molecular subgroups that reflect discrete pathways of BCP ALL, informing disease classification and prognostic stratification. The combined results strongly advocate that RNA sequencing be introduced into the clinical diagnostic workup of BCP ALL.

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., Cancer: Screening checklists catch Asian children at risk of hereditary tumours Existing checklists designed to screen children for a genetic predisposition to cancer are sensitive enough to detect at-risk individuals in an Asian population. Joanne Ngeow from the National Cancer Centre Singapore and colleagues reviewed the medical records of 102 paediatric patients diagnosed with cancer and profiled for inherited mutations in 100 cancer-associated genes. They crosschecked the records against two published screening instruments used by clinical geneticists to identify patients with a hereditary cancer risk. Although only ten patients harboured known cancer predisposition mutations, the checklists flagged 54 individuals. However, all ten pathogenic gene carriers screened positive on the diagnostic aids. The checklists thus seem to err on the side of over-identification and don’t miss many true cases of hereditary cancer in this Asian population.

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 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.

Purpose Although IKZF1 deletion ( IKZF1del) confers a higher risk of relapse in childhood B-cell acute lymphoblastic leukemia (B-ALL), it is uncertain whether treatment intensification will reverse this risk and improve outcomes. The Malaysia-Singapore ALL 2010 study (MS2010) prospectively upgraded the risk assignment of patients with IKZF1del to the next highest level and added imatinib to the treatment of all patients with BCR- ABL1 fusion. Patients and Methods In total, 823 patients with B-ALL treated in the Malyasia-Singapore ALL 2003 study (MS2003; n = 507) and MS2010 (n = 316) were screened for IKZF1del using the multiplex ligation-dependent probe amplification assay. The impact of IKZF1del on the 5-year cumulative incidence of relapse (CIR) was compared between the two studies. Results Patient characteristics were similar in both cohorts, including IKZF1del frequencies (59 of 410 [14.4%] v 50 of 275 [18.2%]; P = .2). In MS2003, where IKZF1del was not used in risk assignment, IKZF1del conferred a significantly higher 5-year CIR (30.4% v 8.1%; P = 8.7 × 10−7), particularly in the intermediate-risk group who lacked high-risk features (25.0% v 7.5%; P = .01). For patients with BCR-ABL1–negative disease, IKZF1del conferred a higher 5-year CIR (20.5% v 8.0%; P = .01). In MS2010, the 5-year CIR of patients with IKZF1del significantly decreased to 13.5% ( P = .05) and no longer showed a significant difference in patients with BCR-ABL1-negative disease (11.4% v 4.4%; P = .09). The 5-year overall survival for patients with IKZF1del improved from 69.6% in MS2003 to 91.6% in MS2010 ( P = .007). Conclusion Intensifying therapy for childhood B-ALL with IKZF1del significantly reduced the risk of relapse and improved overall survival. Incorporating IKZF1del screening significantly improved treatment outcomes in contemporary ALL therapy.

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 specimens10 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.

Introduction: Treatment of acute lymphoblastic leukaemia (ALL) using intensive chemotherapy has resulted in high cure rates but also substantial morbidity. Infective complications represent a significant proportion of treatment-related toxicity. The objective of this study was to describe the microbiological aetiology and clinical outcome of episodes of chemotherapy-induced febrile neutropaenia in a cohort of children treated for ALL at our institution. Materials and Methods: Patients with ALL were treated with either the HKSGALL93 or the Malaysia-Singapore (Ma-Spore) 2003 chemotherapy protocols. The records of 197 patients who completed the intensive phase of treatment, defined as the period of treatment from induction, central nervous system (CNS)-directed therapy to reinduction from June 2000 to January 2010 were retrospectively reviewed. Results: There were a total of 587 episodes of febrile neutropaenia in 197 patients, translating to an overall rate of 2.98 episodes per patient. A causative pathogen was isolated in 22.7% of episodes. An equal proportion of Gram-positive bacteria (36.4%) and Gram-negative bacteria (36.4%) were most frequently isolated followed by viral pathogens (17.4%), fungal pathogens (8.4%) and other bacteria (1.2%). Fungal organisms accounted for a higher proportion of clinically severe episodes of febrile neutropaenia requiring admission to the high-dependency or intensive care unit (23.1%). The overall mortality rate from all episodes was 1.5%. Conclusion: Febrile neutropaenia continues to be of concern in ALL patients undergoing intensive chemotherapy. The majority of episodes will not have an identifiable causative organism. Gram-positive bacteria and Gram-negative bacteria were the most common causative pathogens identified. With appropriate antimicrobial therapy and supportive management, the overall risk of mortality from febrile neutropaenia is extremely low. Key words: Infective complications, Bacterial infections, Mortality, Toxicity

A 13-year-old boy underwent allogeneic hematopoietic stem cell transplantation (HSCT) for underlying acute lymphoblastic leukemia and achieved neutrophil engraftment 28 days after HSCT. He developed ichthyosis 6 weeks after HSCT and then keratotic follicular papules, palmoplantar keratoderma, and a seborrheic dermatitis-like eruption 18 weeks after HSCT. From skin biopsies he was diagnosed with eczematoid graft-versus host disease (GVHD), which showed spongiosis with scattered necrotic keratinocytes. He responded to oral and topical steroids and an increase in cyclosporine dose. Although uncommon, eczematoid GVHD must be considered in children who have undergone HSCT and then develop an atypical eczematous eruption, especially in the absence of a history of atopy.

Children with hemophagocytic lymphohistiocytosis (HLH) are at an increased risk of critical illness. In this study, we described the clinical characteristics of critically ill children with HLH and identify factors associated with poor clinical outcomes. Children who were diagnosed with HLH with emergent admission to Children's Intensive Care Unit (CICU) between January 1, 2000 and October 31, 2015 were included. The primary outcome was CICU mortality. Over the 15-year period, there were 14 critically ill patients with HLH with 23 CICU admissions. Median age at HLH diagnosis was 8.2 years (interquartile range [IQR], 2.9 to 11.3). Overall CICU mortality was 8 of 23 CICU admissions (34.8%). Factors that were associated with CICU mortality in critically ill children with HLH identified in this study include: a worse median pediatric index of mortality 2 score (4.7% in survivors [IQR, 2.9% to 11.6%] vs. 2.4% [IQR, 1.2% to 4.3%]; P=0.031); higher median peak serum lactate level (mmol/L) within 24 hours of admission (5.6 [IQR, 2.7 to 17.4] vs. 1.6 [IQR, 1.2 to 2.8]; P=0.032); the need for mechanical ventilation (100% vs. 46.7%; P=0.019); inotropic support (87.5% vs. 20.0%; P=0.006); renal replacement therapy (50% vs. 0%; P=0.008); and blood product transfusion episodes (24.5 [IQR, 14.3 to 46.8] vs. 3.0 [IQR, 1.0 to 9.0]; P=0.002). Further studies are required to validate the factors that are associated with poor outcomes in critically ill children with HLH.

The authors describe a series of 15 intracranial germ cell tumors (IGCTs) excluding mature teratomas; 3 cases in children younger than 3 years of age who were treated at 3 different international institutions over the course of 20 years, and 12 from a PubMed search. These tumors, with possible in utero origins, often occur in atypical locations. The clinical behavior differed significantly from these tumors' counterparts in older children. In this young age group germinoma is highly aggressive, whereas nongerminomatous germ cell tumors may be cured without radiotherapy. Ongoing genomic studies reveal insights to attain an understanding of the biology of these tumors. New treatment strategies are needed to improve outcomes for IGCTs in this age group, particularly for germinomas.

Background At diagnosis, recurrent genomic aberrations - aneuploidy and oncogene fusion that drive B-lymphoblastic leukemia - best predict treatment response. Currently, karyotyping and RT-PCR can discern only 50% of the genomic aberrations driving childhood B-ALL. RNA sequencing (RNA-Seq) interrogates the whole transcriptome through deep sequencing. Using RNA-Seq to study expressed SNP variants and expression level of each chromosome, we can identify their copy number; this enables digital karyotyping. For each patient, this digital karyotyping together with oncogene fusion sequencing and transcriptome profiling allow full molecular interrogation of the genomic aberrations that drive leukemia. We molecular subtyped and risk assigned 377 children with B-lymphoblastic leukemia using RNA-Seq. We showed that in childhood B-lymphoblastic leukemia, a single platform RNA-seq significantly improved risk assignment. Methods Whole transcriptome RNA-Seq was performed on the diagnostic bone marrow or peripheral blood samples of 377 children enrolled on the Ma-Spore ALL 2003 and Ma-Spore ALL 2010 studies. Oncogene fusions, variant calling, hierarchical clustering and gross chromosomal copy number calling were performed using RNA-Seq. These are combined for molecular assignment. As compared with conventional risk stratification using a combination of cytogenetics, DNA index and oncogene fusion panel, risk assignment including newly discovered molecular subtypes are made using RNA-Seq. Results Patients are classified into 20 molecularly distinct subtypes (Table 1) in 5 categories: 1. Oncogene fusion category with 10 subtypes. 2. Gene expression profiles with 5 subtypes. 3. Aneuploidy defines 3 subtypes, namely high hyperdiploidy, hypodiploidy and near haploid. 4. Biallelic mutation of PAX5 characterized by PAX5 P80R. 5. B-others. The 20 molecular subtypes are then risk assigned to Low, Intermediate or High risk groups (Table 1). Specifically, 1. Low Risk (LR) - 66% (250/377) of the cohort 2. Intermediate Risk (IR) - 15% (57 of 377) of the cohort 3. High risk (HR) - 12% (44/377) of the cohort 4. B-others - 7% (26/377) of the cohort In the patients with available survival data, compared to 39% by conventional methods, RNA-Seq improves risk assignment by assigning 93% of the total cohort into a distinct risk group (Figure 1). The number of LR patients by RNA-Seq nearly doubled (RNA-Seq n=238 vs conventional n=125), with comparable cumulative incidence of relapse (CIR; 6.2% vs 5.2%) and overall survival (OS; 98.2% vs 99.1%). The number of HR patients also increased >2-fold (RNA-Seq n=41 vs conventional n=17), with a slightly lower CIR (41.2% vs 52.9%) and a similar OS (65.4% vs 64.7%). The RNA-Seq defined risk groups have more significantly distinct outcome (P-value comparing CIR: 1×10-8 vs1×10-7; P-value comparing OS: 2×10-12 vs3×10-7; Figure 1). In conventional risk assignment, 67% of the relapses and 68% of the deaths are in B-Others (61% of patients). Using RNA-Seq, HR (11% of patients) accounts for the most relapses (38%) and deaths (59%) while B-Others (7% of patients) account for 7% of relapses and 5% of deaths. In multivariate analysis using competing risk regression on CIR together with age, gender, WBC and Day 33 MRD, only risk stratification by RNA-Seq and Day 33 MRD remained prognostically significant (RNA-Seq subtype P=6.4×10-4 or 5.9×10-3 for HR or IR vs SR; MRD P=1.3×10-5 or 0.020 for HR or IR vs SR). While in Cox proportional hazards regression on OS, Day 33 MRD lost its significance; RNA-Seq risk stratification is the most significant factor that predicts OS (RNA-Seq subtype P=3.4×10-4 or 0.27 for HR or IR vs SR; MRD P=0.19 or 0.24 for HR or IR vs SR). Conclusion When compared to conventional karyotyping and RT-PCR, in B-lymphoblastic leukemia, RNA-Seq significantly improved molecular diagnosis and risk assignment; it assigns 93% of patients into a distinct molecular and risk group. In Cox proportional hazards regression on OS, Day 33 MRD lost its prognostic significance, and RNA-Seq risk stratification assignment is the only significant predictive factor of overall survival. Disclosures No relevant conflicts of interest to declare.

Background: Haploidentical hematopoietic cell transplantation (HCT) provides an alternative option for patients without HLA-matched donor. Graft-versus-host disease (GVHD), engraftment failure, and infectious complications continue to be the main causes of non-relapse mortality (NRM). We hypothesized that selective depletion of TCRαβ+ and CD45RA+ naive T-cells subset will permit hematopoietic engraftment, while effectively reducing GVHD, and provide improved donor immune reconstitution through adoptive transfer of donor’s mature NK-cells, γδ T-cells, and CD45RO+ memory T-cells. Methods: Mobilized PBSC products were divided into two fractions in 9:1 ratio and depleted using CliniMACS device after labeling with TCRαβ and CD45RA reagents (Miltenyi Biotec, Bergish-Gladbach, Germany), respectively. The conditioning regimen consisted of fludarabine 160mg/m2 divided daily over 4 days, thiotepa 10mg/kg divided twice daily for 1 day, and melphalan 70-140mg/m2 for 1 day, in combination with total lymphoid irradiation 6Gy (n=35) or 7.5Gy (n=12) over 3 equal fractions, total body irradiation of 2Gy (n=13) or antithymocyte globuline (n=2). Short term GVHD prophylaxis for 30 days was given to 1 patient using MMF, 50 patients using tacrolimus, and 2 patients using sirolimus. Results: We transplanted 62 patients, including 55 adults (median age, 47 years; range 20-69) and 7 children (median age, 13 years, range 7-17) with high risk AML (n=34), ALL (n=15), MDS (n=7), plasma cell neoplasm (n=2), mast cell leukemia (n=1), acute undifferentiated leukemia (n=1), and NK/T-cell lymphoma (n=1). The patients were infused with TCRαβ and CD45RA depleted graft containing a median of 6.79 x 106 (range 3.54-20.78) CD34+ cells/kg, 0.00 x 104 (range 0-0.97) CD45RA+CD3+ cells/kg, and 1.09 x 106 (range 0.15-11.67) CD45RO+CD3+ cells/kg. The TCRαβ depleted graft fraction contained a median of 0.20 x 104 (range 0-11.30) TCRαβ+ cells/kg, and 8.52 x 106 (range 0.62-30.00) TCRγδ+ cells/kg. Only 1 patient experienced primary graft failure. All others had engraftment of ANC > 500 cells/µL at a median of 10 days (range 8-22) and platelet > 20,000 cells/µL at a median of 12 days (range 8-22). There was no secondary graft failure. Six patients with high titers of donor-specific HLA antibodies (DSA) engrafted successfully after effective desensitisation with plasma exchange, rituximab, and immunoglobulin. Twenty-two patients (35%) developed acute GVHD of grade II - IV (Gd II, n=15; Gd III, n=5; Gd IV, n=2). Three patients experienced chronic GVHD, giving 2-year cumulative incidence of 7 %. Day 180 cumulative incidence of NRM and relapse were 24.8% (95% CI 14.3-36.8%) and 14.9% (95% CI 6.3-27.0%), respectively. Four of the 16 NRM were attributed to aGVHD. Viral reactivation included CMV (n=25), HHV-6 (n=14), EBV (n=11), and adenovirus (n=7). Fourteen patients died of infection, including 5 patients who had fatal blood stream infection (bacteria, n=3; candidemia, n=2) and 1 patient from disseminated adenovirus infection within 180 days. With a median follow-up of 298 days (range 21-1298) in surviving patients, the 2-year overall (OS), event-free (EFS), and GVHD-free/relapse-free (GRFS) survival were 58% (95% CI 37.6-73.4%), 43% (95% CI 27-57%), and 39% (95% CI 24-54%), respectively (Figure 1). In multivariable analysis, only HCT-comorbidity index (HCT-CI) showed significant impact on OS (HR 6.24; 95% CI 2.05-19.05; p=0.0013), EFS (HR 4.80; 95% CI 1.73-13.35; p=0.0027), and RRM (HR 6.49; 95% CI 1.44-29.25; p=0.015), whereas disease risk index (DRI) impacts risk of relapse (HR 4.50; 95% CI 1.39-14.52; p=0.012). The 2-year OS, EFS, and GRFS for the subset of 30 patients (adults, n=25; children, n=5) with HCT-CI score of 0 and low/intermediate risk DRI were 68%, 68%, and 60%, respectively (Figure 2). Conclusions: Our preliminary results suggest that RIC haplo-HCT with TCRαβ and CD45RA+ depleted grafts allowed successful allograft in high-risk patients lacking a suitable matched donor, including patients with high level of DSA. Acute GVHD was generally abortive, leading to low incidence of chronic GVHD. The best outcome is seen in patients with favorable HCT-CI and DRI. Further efforts are needed to lower the risk of death due to blood stream infection, and relapse in patients with high risk DRI, such as optimisation of anti-microbial prophylaxis or repeated doses of memory-cell donor lymphocyte infusion (DLI). Download : Download high-res image (119KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.

Background As a single platform, RNA sequencing (RNA-Seq) can accurately identify majority of oncogenic driver fusion genes and aneuploidy. This provides refined molecular subtyping and risk stratification; RNA-Seq is increasing used upfront for genetic subtype assignment in on-going contemporary ALL trials. The IGH gene is highly expressed in our diagnostic ALL samples using RNA-Seq. We investigated the usefulness of RNA-Seq in identifying IGH disease sequences for MRD monitoring in 258 childhood B-lymphoblastic leukemia (B-LL) samples. Methods RNA-seq was performed on a total of 259 diagnostic bone marrow or peripheral blood samples from children with B-LL treated in the Ma-Spore ALL 2003 and ALL 2010 studies. The reads were aligned to germline IGH gene V, D and J segments, and reads covered IGH VHDJH or DJH junctional regions are clustered into clones. Overall, the distribution of read count of the IGH background clones follows Zipf's law, i.e. the read count of the IGH background clones is negative linearly correlated with the number of IGH clones with that read count in the log-log scale (Figure 1A). IGH disease clones are outliers with exceedingly high read count. For each sample, a linear model is fitted between log transformed read count (using only read count ≤10) and log transformed number of IGH clones. The expected number of IGH clones (E-value) of a read count can be predicted by the fitted linear model. A clone with E-value Results In 90.3% of the patients, 497 IGH disease clones (median 2, range 0-7 clones/patient) are identified. The number of IGH disease clones significantly correlated with genetic subtype (P=1.9×10-8; Table 1). Specifically, high hyperdiploid patients have the most IGH disease clones (median 3) with 67% (22/33) have >2 IGH disease clones, due to the gain of chromosome 14. In contrast, DUX4 high expressers have the least (median 1) IGH disease clones with 19% (8/43) have no IGH disease clone, due to the rearrangements involving one of the IGH locus. Of the 348 IGH disease clones identified by Sanger sequencing, 90.8% are also identified by RNA-Seq; and in the 199 Sanger sequences that yielded sensitive markers, 191 (96.0%) are also defined as IGH disease clones by RNA-Seq. In addition, RNA-Seq identified 43% more IGH disease clones. In 69 patients lacking sensitive IGH targets by Sanger sequencing, the disease clones identified by RNA-Seq were used collectively for MRD monitoring by targeted deep sequencing of IGH (IGH-Seq) and showed high correlation with conventional RQ-PCR MRD using non-IGH targets (R=0.93; P=1.3x10-14;Figure 1C). In addition, IGH-Seq showed improved sensitivity with quantifiable MRD in 20% more samples compared to RQ-PCR (78% vs 58%; Figure 1C). In the samples that are positive below sensitivity or negative by RQ-PCR, IGH-Seq obtained quantifiable MRD for 60% (9/15) or 25% (5/20) of the samples (Figure 1C). Conclusion In conclusion, IGH disease clone sequences are highly expressed and can be distinctly identified from whole transcriptome RNA-Seq in >90% of children with B-LL. These can be used for molecular MRD monitoring using RQ-PCR or NGS-based IGH-Seq. In addition to identifying the constellation of oncogene fusions and chromosomal aneuploidies that drive B-LL and RNA-Seq can identify IGH MRD sequences for molecular MRD monitoring. Disclosures No relevant conflicts of interest to declare.

As ~90% children with Acute Lymphoblastic Leukemia (ALL) are now cured, the next challenge of contemporary ALL therapy is curing with less toxicity. To reduce toxicity, the Malaysia-Singapore ALL 2003 (MS2003) study lowered the intensity of induction in Standard(SR)/Intermediate Risk(IR) patients by removing Daunorubicin. However, toxicities causing prolonged delays during Consolidation and Delayed-Intensification(DI) remained major problems. To address these delays, the MS2010 study: (1) Replaced half of all cytarabine blocks with 2 doses of Vincristine(VCR) per block (2) Implemented Doxorubicin-free DI Protocol V (3) Added 1 dose of L-Asparaginase(L-Asp) to each DI block. Here, we report the significant improvements in MS2010 compared to MS2003 in terms of treatment-related toxicities. Methods MS2003 (Yeoh AEJ et al. J Clin Oncol 2012) started with a 3 drug induction for non-HR patients in a BFM ALL-IC 2002 backbone where SR consisted of 2x Protocol III and IR consisted of 3x Protocol III. In MS2010 (Yeoh AEJ et al. J Clin Oncol 2018), SR consisted of 2x Protocol V and IR consisted of 2x Protocol III + 1x Protocol V (Table 1). We performed a per-protocol analysis of 315 children with de novo SR/IR ALL, treated on MS2003 (n=183) and MS2010 (n=132) in 2 centers in Singapore, with a median follow up of 10.7 and 6.4years respectively. We analyzed clinical toxicity data, focusing on hospitalizations for fever (regardless of neutrophil count). Mean differences were adjusted for baseline characteristics including sex, race, age and Down syndrome. Results Overall decreased hospitalization stay and phase delays. In MS2010, the total duration of hospitalization for fever are fewer and shorter; this is most apparent for DI phases. For SR group, total length of stay during DI decreased from 13.7 to 6.1 days with adjusted(adj.) δ=7.0 days (95% CI 3.5-10.5, p In MS2010, overall delays in treatment are significantly reduced -- SR: From 65.9 to 16.4days, adj.δ=48.8 days(95% CI 39.8-57.8, p MS2010 Protocol III had 5 doses of L-Asp compared to 4 doses of L-Asp in MS2003 Protocol III. Although L-Asp is generally considered less myelosuppressive, there was a significant increase in the number of hospitalizations during MS2010 Protocol III blocks. This translated to longer hospitalizations per patient in MS2010 during those specific phases (III#1: adj.δ=-1.6 days 95% CI -2.8 to -0.3 p=0.017; III#2: adj.δ=-1.9 days 95% CI -3.2 to -0.6 p=0.006). These increases in hospitalizations in MS2010 Protocol IIIa were most likely due to an additional single dose of L-Asp. Equivalent survival outcomes Based on a per-protocol analysis, 5 year Event-Free survival(EFS) outcomes between the cohorts were equivalent. In the SR cohort, 5yr EFS was 93.6% (MS2003) vs. 95.5% (MS2010) p=0.685. In the IR cohorts, 5yr EFS improved from 89.4% (MS2003) to 95.6% (MS2010) p=0.151, although statistical significance was not reached. Overall, significant improvements: reduction in hospitalisation stays and reduction in treatment delays were observed in MS2010. This was achieved through a combination of (1) Replacing half of all cytarabine blocks with 2 doses of VCR per block; and (2) New DI Protocol V with no Anthracyclines. Despite the AIEOP-BFM 2000 experience of increased toxicity with Protocol III, our modifications to consolidation Ib and Protocol III have led to less phase delays, tolerable toxicities with comparable outcomes. Disclosures No relevant conflicts of interest to declare.

Hepatoblastoma is a highly malignant embryonal liver tumor that occurs almost exclusively in infants and toddlers. Trisomy 18 is the second most common autosomal trisomy after trisomy 21 and is generally considered a lethal disorder. Ten cases of hepatoblastoma in children with trisomy 18 have been published to date. Here, we report on two female patients with trisomy 18 and pretreatment extent of disease (PRETEXT) stage 1 hepatoblastoma, which support the presence of a nonrandom association between hepatoblastoma and trisomy 18. Both patients underwent primary surgical resection without any neoadjuvant or adjuvant chemotherapy. The histologies returned as pure fetal epithelial type, and combined fetal and embryonal epithelial type. There was no evidence of recurrence on serial abdominal ultrasound and serum alpha-fetoprotein levels on follow-up. Conclusion: Primary surgical resection is a treatment approach that can be considered in children with trisomy 18 and PRETEXT stage 1 tumor. However, in view of the overall prognosis for trisomy 18, the decision on the optimal treatment is a delicate one and has to be individualized in the context of the best interests of the child.

Purpose: To analyze the clinical manifestations and treatment outcomes for patients with retinoblastoma in Singapore from 1997 to 2010. Methods: Medical records of 51 patients (67 eyes) diagnosed as having retinoblastoma were analyzed. Data on laterality, genetics, presentation, disease severity, treatment, and prognosis were collected. Results: The mean age of presentation was 25.7 ± 19.9 months. Sixteen (31.4%) of the patients had bilateral disease, of whom 2 had an associated pineal tumor. Leukocoria was the most common sign. Two had metastasis at diagnosis. Only 3 patients (5.9%) had a family history. Using the International Intraocular Retinoblastoma Classification, 6.0% were Group A, 6.0% were Group B, 3.0% were Group C, 38.8% were Group D, and 49.2% were Group E. Chemotherapy and focal therapy were administered for 4 of 35 (12.9%) patients with unilateral retinoblastoma (50% had successful globe preservation) and 13 of 16 (81.3%) patients with bilateral retinoblastoma (42.3% had successful globe preservation). Overall, globe preservation was achieved in 100% of Groups A, B, and C, and 23.1% of Group D cases. The 5-year survival rate overall, for unilateral retinoblastoma, and for bilateral retinoblastoma was 91%, 97%, and 76% respectively. Conclusion: The overall 5-year survival rate is comparable to international data in most developed countries. However, most patients presented with advanced disease, making the rate of globe preservation lower than in some developed countries. Better education of the public and healthcare professionals may increase awareness and enable early detection of the disease. [J Pediatr Ophthalmol Strabismus 2013;50:106–112.]

Chromosomal translocations are a genomic hallmark of many hematologic malignancies. Often as initiating events, these structural abnormalities result in fusion proteins involving transcription factors important for hematopoietic differentiation and/or signaling molecules regulating cell proliferation and cell cycle. In contrast, epigenetic regulator genes are more frequently targeted by somatic sequence mutations, possibly as secondary events to further potentiate leukemogenesis. Through comprehensive whole-transcriptome sequencing of 231 children with acute lymphoblastic leukemia (ALL), we identified 58 putative functional and predominant fusion genes in 54.1% of patients (n = 125), 31 of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature predominantly driven by chromosomal rearrangements of the ZNF384 gene with histone acetyltransferases EP300 and CREBBP. ZNF384-rearranged ALL showed significant up-regulation of CLCF1 and BTLA expression, and ZNF384 fusion proteins consistently showed higher activity to promote transcription of these target genes relative to wild-type ZNF384 in vitro. Ectopic expression of EP300-ZNF384 and CREBBP-ZNF384 fusion altered differentiation of mouse hematopoietic stem and progenitor cells and also potentiated oncogenic transformation in vitro. EP300- and CREBBP-ZNF384 fusions resulted in loss of histone lysine acetyltransferase activity in a dominant-negative fashion, with concomitant global reduction of histone acetylation and increased sensitivity of leukemia cells to histone deacetylase inhibitors. In conclusion, our results indicate that gene fusion is a common class of genomic abnormalities in childhood ALL and that recurrent translocations involving EP300 and CREBBP may cause epigenetic deregulation with potential for therapeutic targeting.