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Effective treatment of pediatric acute leukemia is dependent on accurate genomic classification, typically derived from a combination of multiple time-consuming and costly techniques such as flow cytometry, fluorescence in situ hybridization (FISH), karyotype analysis, targeted PCR, and microarrays (Arber et al., 2016; Iacobucci & Mullighan, 2017; Narayanan & Weinberg, 2020). We investigated the feasibility of a comprehensive single-assay classification approach using long-read sequencing, with real-time genome target enrichment, to classify chromosomal abnormalities and structural variants characteristic of acute leukemia. We performed whole genome sequencing on DNA from diagnostic peripheral blood or bone marrow for 54 pediatric acute leukemia cases with diverse genomic subtypes. We demonstrated the characterization of known, clinically relevant karyotype abnormalities and structural variants concordant with standard-of-care clinical testing. Subtype-defining genomic alterations were identified in all cases following a maximum of forty-eight hours of sequencing. In 18 cases, we performed real-time analysis - concurrent with sequencing - and identified the driving alteration in as little as fifteen minutes (for karyotype) or up to six hours (for complex structural variants). Whole genome nanopore sequencing with adaptive sampling has the potential to provide detailed genomic classification of acute leukemia specimens with reduced cost and turnaround time compared to the current standard of care., Competing Interests: Competing Interests: JG and JRW have received compensation for travel to speak at Oxford Nanopore Technologies events. The remaining authors declare no conflicts of interest.

Approximately 80% of pediatric tumors occur in low- and middle-income countries (LMIC), where diagnostic tools essential for treatment decisions are often unavailable or incomplete. Development of cost-effective molecular diagnostics will help bridge the cancer diagnostic gap and ultimately improve pediatric cancer outcomes in LMIC settings. We investigated the feasibility of using nanopore whole transcriptome sequencing on formalin-fixed paraffin embedded (FFPE)-derived RNA and a composite machine learning model for pediatric solid tumor diagnosis. Transcriptome cDNA sequencing was performed on a heterogenous set of 221 FFPE and 32 fresh frozen pediatric solid tumor and lymphoma specimens on Oxford Nanopore Technologies' sequencing platforms. A composite machine learning model was then used to classify transcriptional profiles into clinically actionable tumor types and subtypes. In total, 95.6% and 89.7% of pediatric solid tumors and lymphoma specimens were correctly classified, respectively. 71.5% of pediatric solid tumors had prediction probabilities > 0.8 and were classified with 100% accuracy. Similarly, for lymphomas, 72.4% of samples that had prediction probabilities > 0.6 were classified with 97.6% accuracy. Additionally, FOXO1 fusion status was predicted accurately for 97.4% of rhabdomyosarcomas and MYCN amplification was predicted with 88% accuracy in neuroblastoma. Whole transcriptome sequencing from FFPE-derived pediatric solid tumor and lymphoma samples has the potential to provide clinical classification of both tissue lineage and core genomic classification. Further expansion, refinement, and validation of this approach is necessary to explore whether this technology could be part of the solution of addressing the diagnostic limitations in LMIC., (© 2024. The Author(s).)

There were no pediatric oncology centers in southwest Ethiopia prior to 2016. This study aims to describe presenting diagnoses and initial outcomes at Jimma University Medical Center (JUMC), the first pediatric oncology unit (POU) in southwest Ethiopia, provide initial insights into regional pediatric cancer epidemiology, illustrate the rapid growth of pediatric cancer services, and highlight ongoing challenges. We used a retrospective descriptive approach to assess the epidemiologic pattern and initial treatment outcomes of pediatric cancer at JUMC POU from August 2016 through December 2022. During the study period, 749 children were diagnosed with cancer at JUMC. The mean age was 7.2 years (20 days-18 years). Acute lymphoblastic leukemia was the most common diagnosis (16.4%), followed by non-Hodgkin lymphoma (12.4%), Wilms tumor (11.1%), soft tissue sarcoma (8.8%), Hodgkin lymphoma (8.4%), and retinoblastoma (8.3%). Brain tumors accounted for only 2.7% of the diagnoses. Of the 703 patients that were not referred elsewhere, 42% of the patients abandoned treatment, 29% died, 17% completed treatment, and 7% remained on treatment at the time of this assessment. The findings emphasize the growth in the diagnosis and treatment of children with cancer in the southwest region of Ethiopia. The data suggests a different epidemiologic profile of childhood cancer cases diagnosed at the JUMC POU compared to high-income countries and neighbouring countries in Africa. Treatment abandonment remains a barrier to care. Ongoing areas of focus include establishment of a hospital-based cancer registry, reduction of treatment abandonment, improvement of diagnostic capacity, and increased access to advanced supportive care., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hordofa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The stark disparity in survival for children with cancer across the world has inspired a global call to expand chemotherapy access in low and middle income countries. Among the numerous barriers to success, a paucity of reliable information regarding chemotherapy pricing hinders the ability of governments and other key stakeholders to make informed budget decisions or negotiate lower medication prices. The aim of this study was to generate comparative price information on both individual chemotherapy agents and comprehensive treatment regimens for common childhood cancers using real-world data., Methods: Chemotherapy agents were selected based on their inclusion in the World Health Organization (WHO) Essential Medicines List for Children (EMLc) and their use in frontline regimens for the tracer cancer types prioritized by the WHO's Global Initiative for Childhood Cancer (GICC). Sources included IQVIA MIDAS data, obtained under license from IQVIA, and publicly available data from Management Sciences for Health (MSH). Data on chemotherapy prices and purchase volumes spanning 2012-2019 were aggregated according to WHO region and World Bank (WB) income classification. Cumulative chemotherapy prices for treatment regimens were compared across WB income classification., Findings: Data representing an estimated 1.1 billion doses of chemotherapy were obtained for 97 countries: 43 high income countries (HICs), 28 upper middle income countries (UMICs), and 26 low and lower middle income countries (LLMICs). Median drug prices in HICs were 0.9-20.4 times those of UMICs and 0.9-15.5 times those of LMICs. Regimen prices were generally higher for HICs, hematologic malignancies, non-adapted protocols, and higher risk stratification or stage, albeit with notable exceptions., Interpretation: This study represents the largest price analysis to date of chemotherapy agents used globally in childhood cancer therapy. The findings of this study form a basis for future cost-effectiveness analysis in pediatric cancer and should inform efforts of governments and stakeholders to negotiate drug prices and develop pooled purchasing strategies., Funding: NB received funding support from the American Lebanese Syrian Associated Charities and Cancer Center Support grant (CA21765) from the National Cancer Institute through the National Institutes of Health. TA received funding through the University of North Carolina Oncology K12 (K12CA120780) and the University Cancer Research Fund from the UNC Lineberger Comprehensive Cancer Center., Competing Interests: TA received support from NIH (K12CA120780 and R21CA259926), the 10.13039/100007602University Cancer Research Fund, Lineberger Comprehensive Cancer Center Fund from Lineberger Comprehensive Cancer Center, and 10.13039/100007737St. Jude Children's Research Hospital for grand rounds honoraria. NB received support from National Cancer Institute, American Lebanese Syrian Associated Charities (ALSAC), St. Jude Children’s Research Hospital NCI Comprehensive Cancer Center, and Memorial Sloan Kettering for grand rounds honoraria. SO received grant funding from Merck., (© 2023 The Author(s).)

Rare hematologic malignancies display evidence of both myeloid and lymphoid differentiation. Here, we describe such a novel bilineal event discovered in an adult woman with B-lymphoblastic leukemia (BLL). At the time of BLL diagnosis, the patient had a normal karyotype and a bulk sequencing panel identified pathogenic variants in BCOR, EZH2, RUNX1, and U2AF1, a genotype more typical of myeloid neoplasia. Additionally, the patient was noted to have 3-year history of cytopenias, and morphologic dyspoiesis was noted on post-treatment samples, raising the possibility of an antecedent hematologic disorder. To investigate the clonal architecture of her disease, we performed targeted sequencing on fractionated samples enriched for either B-lymphoblasts or circulating granulocytes. These studies revealed a truncal founder mutation in the spliceosome gene U2AF1 in both fractions, while distinct secondary mutations were present only in B-lymphoblasts (BCOR, NRAS) or myeloid cells (ASXL1, EZH2, RUNX1). These results indicate that both processes evolved from a common U2AF1-mutated precursor, which then acquired additional mutations during a process of divergent evolution and bilineal differentiation. Our findings highlight an atypical mechanism of BLL leukemogenesis and demonstrate the potential utility of fractionated sequencing in the characterization of acute leukemia., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Overexpression of Exportin-1 ( XPO1 ), a key regulator of nuclear-to-cytoplasmic transport, is associated with inferior patient outcomes across a range of adult malignancies. Targeting XPO1 with selinexor has demonstrated promising results in clinical trials, leading to FDA approval of its use for multiple relapsed/refractory cancers. However, XPO1 biology and selinexor sensitivity in childhood cancer is only recently being explored. In this review, we will focus on the differential biology of childhood and adult cancers as it relates to XPO1 and key cargo proteins. We will further explore the current state of pre-clinical and clinical development of XPO1 inhibitors in childhood cancers. Finally, we will outline potentially promising future therapeutic strategies for, as well as potential challenges to, integrating XPO1 inhibition to improve outcomes for children with cancer.

Blinatumomab, a bispecific antibody that directs CD3+ T cells to CD19+ tumor cells, shows variable efficacy in B-progenitor acute lymphoblastic leukemia (B-ALL). To determine tumor-intrinsic and -extrinsic determinants of response, we studied 44 adults with relapsed or refractory B-ALL (including 2 minimal residual disease positive) treated with blinatumomab using bulk tumor and single-cell sequencing. The overall response rate in patients with hematological disease was 55%, with a high response rate in those with CRLF2-rearranged Philadelphia chromosome-like ALL (12 [75%] of 16). Pretreatment samples of responders exhibited a tumor-intrinsic transcriptomic signature of heightened immune response. Multiple mechanisms resulted in loss of CD19 expression, including CD19 mutations, CD19-mutant allele-specific expression, low CD19 RNA expression, and mutations in CD19 signaling complex member CD81. Patients with low hypodiploid ALL were prone to CD19- relapse resulting from aneuploidy-mediated loss of the nonmutated CD19 allele. Increased expression of a CD19 isoform with intraexonic splicing of exon 2, CD19 ex2part, at baseline or during therapy was associated with treatment failure. These analyses demonstrate both tumor-intrinsic and -extrinsic factors influence blinatumomab response. We show that CD19 mutations are commonly detected in CD19- relapse during blinatumomab treatment. Identification of the CD19 ex2part splice variant represents a new biomarker predictive of blinatumomab therapy failure., (© 2021 by The American Society of Hematology.)

Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as SIN3A , USP7 , and CHD8 , we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients., (© 2019 by The American Society of Hematology.)

Mixed phenotype acute leukaemia (MPAL) is a high-risk subtype of leukaemia with myeloid and lymphoid features, limited genetic characterization, and a lack of consensus regarding appropriate therapy. Here we show that the two principal subtypes of MPAL, T/myeloid (T/M) and B/myeloid (B/M), are genetically distinct. Rearrangement of ZNF384 is common in B/M MPAL, and biallelic WT1 alterations are common in T/M MPAL, which shares genomic features with early T-cell precursor acute lymphoblastic leukaemia. We show that the intratumoral immunophenotypic heterogeneity characteristic of MPAL is independent of somatic genetic variation, that founding lesions arise in primitive haematopoietic progenitors, and that individual phenotypic subpopulations can reconstitute the immunophenotypic diversity in vivo. These findings indicate that the cell of origin and founding lesions, rather than an accumulation of distinct genomic alterations, prime tumour cells for lineage promiscuity. Moreover, these findings position MPAL in the spectrum of immature leukaemias and provide a genetically informed framework for future clinical trials of potential treatments for MPAL.

Despite attempts to improve the definitions of ambiguous lineage leukemia (ALAL) during the last 2 decades, general therapy recommendations are missing. Herein, we report a large cohort of children with ALAL and propose a treatment strategy. A retrospective multinational study (International Berlin-Frankfurt-Münster Study of Leukemias of Ambiguous Lineage [iBFM-AMBI2012]) of 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (EFS) of patients with acute lymphoblastic leukemia (ALL)-type primary therapy (80% ± 4%) was superior to that of children who received acute myeloid leukemia (AML)-type or combined-type treatment (36% ± 7.2% and 50% ± 12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5-year EFS of CD19 + leukemia was 83% ± 5.3% on ALL-type primary treatment compared with 0% ± 0% and 28% ± 14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using World Health Organization and/or European Group for Immunophenotyping of Leukemia definitions) and bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19 + ALAL. AML-type treatment is preferred in a minority of ALAL cases with CD19 - and no other lymphoid features. No overall benefit of transplantation was documented, and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change in treatment type, this is to be considered only in cases with ≥5% blasts after remission induction. The results provide a basis for a prospective trial., (© 2018 by The American Society of Hematology.)

Hoxa2 gene is a primary player in regulation of craniofacial programs of head development in vertebrates. Here we investigate the evolution of a Hoxa2 neural crest enhancer identified originally in mouse by comparing and contrasting the fugu hoxa2a and hoxa2b genes with their orthologous teleost and mammalian sequences. Using sequence analyses in combination with transgenic regulatory assays in zebrafish and mouse embryos we demonstrate subfunctionalization of regulatory activity for expression in hindbrain segments and neural crest cells between these two fugu co-orthologs. hoxa2a regulatory sequences have retained the ability to mediate expression in neural crest cells while those of hoxa2b include cis-elements that direct expression in rhombomeres. Functional dissection of the neural crest regulatory potential of the fugu hoxa2a and hoxa2b genes identify the previously unknown cis-element NC5, which is implicated in generating the differential activity of the enhancers from these genes. The NC5 region plays a similar role in the ability of this enhancer to mediate reporter expression in mice, suggesting it is a conserved component involved in control of neural crest expression of Hoxa2 in vertebrate craniofacial development., (Copyright © 2015 Elsevier Inc. All rights reserved.)