Chimeric antigen receptor T-cell therapy in patients with neurologic comorbidities.

Chimeric antigen receptor T cells (CAR-T) are an effective and potentially durable treatment for refractory and multiply relapsed B-cell acute lymphoblastic leukemia. Neurotoxicity is frequent after CAR-T cell therapy. Mechanisms driving neurotoxicity are incompletely understood, and symptoms can range from transient and mild to severe and life-threatening. Providers have exercised caution in providing CAR-T to patients with neurological comorbidities or extramedullary disease. Here, we report three patients with prior significant neurologic morbidity who safely tolerated CAR-T cell infusion after bridging therapy with conventional chemotherapy.

Nanopore sequencing of clonal IGH rearrangements in cell-free DNA as a biomarker for acute lymphoblastic leukemia.

Background: Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer, and patients with relapsed ALL have a poor prognosis. Detection of ALL blasts remaining at the end of treatment, or minimal residual disease (MRD), and spread of ALL into the central nervous system (CNS) have prognostic importance in ALL. Current methods to detect MRD and CNS disease in ALL rely on the presence of ALL blasts in patient samples. Cell-free DNA, or small fragments of DNA released by cancer cells into patient biofluids, has emerged as a robust and sensitive biomarker to assess cancer burden, although cfDNA analysis has not previously been applied to ALL. Methods: We present a simple and rapid workflow based on NanoporeMinION sequencing of PCR amplified B cell-specific rearrangement of the (IGH) locus in cfDNA from B-ALL patient samples. A cohort of 5 pediatric B-ALL patient samples was chosen for the study based on the MRD and CNS disease status. Results: Quantitation of IGH-variable sequences in cfDNA allowed us to detect clonal heterogeneity and track the response of individual B-ALL clones throughout treatment. cfDNA was detected in patient biofluids with clinical diagnoses of MRD and CNS disease, and leukemic clones could be detected even when diagnostic cell-count thresholds for MRD were not met. These data suggest that cfDNA assays may be useful in detecting the presence of ALL in the patient, even when blasts are not physically present in the biofluid sample. Conclusions: The Nanopore IGH detection workflow to monitor cell-free DNA is a simple, rapid, and inexpensive assay that may ultimately serve as a valuable complement to traditional clinical diagnostic approaches for ALL.

In situ mass spectrometry imaging reveals heterogeneous glycogen stores in human normal and cancerous tissues.

Glycogen dysregulation is a hallmark of aging, and aberrant glycogen drives metabolic reprogramming and pathogenesis in multiple diseases. However, glycogen heterogeneity in healthy and diseased tissues remains largely unknown. Herein, we describe a method to define spatial glycogen architecture in mouse and human tissues using matrix-assisted laser desorption/ionization mass spectrometry imaging. This assay provides robust and sensitive spatial glycogen quantification and architecture characterization in the brain, liver, kidney, testis, lung, bladder, and even the bone. Armed with this tool, we interrogated glycogen spatial distribution and architecture in different types of human cancers. We demonstrate that glycogen stores and architecture are heterogeneous among diseases. Additionally, we observe unique hyperphosphorylated glycogen accumulation in Ewing sarcoma, a pediatric bone cancer. Using preclinical models, we correct glycogen hyperphosphorylation in Ewing sarcoma through genetic and pharmacological interventions that ablate in vivo tumor growth, demonstrating the clinical therapeutic potential of targeting glycogen in Ewing sarcoma.

Informatics Methods and Infrastructure Needed to Study Factors Associated with High Incidence of Pediatric Brain and Central Nervous System Tumors in Kentucky.

Pediatric brain and central nervous system tumors (PBCNSTs) are the most common solid tumors and are the leading cause of disease-related death in US children. PBCNST incidence rates in Kentucky are significantly higher than in the United States as a whole, and are even higher among Kentucky's Appalachian children. To understand and eventually eliminate such disparities, population-based research is needed to gain a thorough understanding of the epidemiology and etiology of the disease. This multi-institutional population-based retrospective cohort study is designed to identify factors associated with the high incidence of PBCNST in Kentucky, leveraging the infrastructure provided by the Kentucky Cancer Registry, its Virtual Tissue Repository (VTR), and the National Institutes of Health Gabriella Miller Kids First Data Resource Center (DRC). Spatiotemporal scan statistics have been used to explore geographic patterns of risk measured by standardized incidence ratios (SIRs) with 95% confidence intervals. The VTR is being used to collect biospecimens for the population-based cohort of PBCNST tissues that are being sequenced by Center for Data Driven Discovery in Biomedicine (D3b) at the Children's Hospital of Philadelphia (CHOP) with support from the Kids First DRC. After adjusting for demographic factors, we assess their potential relationship to environmental factors. We have identified regions in north-central and eastern Appalachian Kentucky where children experienced a significant increased risk of developing PBCNST from 1995-2017 (SIR, 1.48; 95% CI, 1.34-1.62). The VTR has been successful in the collection of a population-based cohort of 215 PBCNST specimens. Timely establishment of legal agreements for data sharing and tissue acquisition proved to be challenging which has been somewhat mitigated by the adoption of national agreement templates. Coronavirus disease 2019 (COVID-19) severely limited the generation of sequencing results due to laboratory shutdowns. However, tissue specimens processed before the shutdown indicated that punches were inferior to scrolls for generating enough quality material for DNA and RNA extraction. Informatics infrastructures that were developed have demonstrated the feasibility of our approach to generate and retrieve molecular results. Our study shows that population-based studies using historical tissue specimens are feasible and practical, but require significant investments in technical infrastructures.

MYCN controls an alternative RNA splicing program in high-risk metastatic neuroblastoma.

The molecular mechanisms underlying the aggressive behavior of MYCN driven neuroblastoma (NBL) is under intense investigation; however, little is known about the impact of this family of transcription factors on the splicing program. Here we used high-throughput RNA sequencing to systematically study the expression of RNA isoforms in stage 4 MYCN-amplified NBL, an aggressive subtype of metastatic NBL. We show that MYCN-amplified NBL tumors display a distinct gene splicing pattern affecting multiple cancer hallmark functions. Six splicing factors displayed unique differential expression patterns in MYCN-amplified tumors and cell lines, and the binding motifs for some of these splicing factors are significantly enriched in differentially-spliced genes. Direct binding of MYCN to promoter regions of the splicing factors PTBP1 and HNRNPA1 detected by ChIP-seq demonstrates that MYCN controls the splicing pattern by direct regulation of the expression of these key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage4 NBL patients (p ≤ 0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, an isoform of pro-tumor-growth, result in repressed growth of NBL cells. Therefore, our study reveals a novel role of MYCN in controlling global splicing program through regulation of splicing factors in addition to its well-known role in the transcription program. These findings suggest a therapeutically potential to target the key splicing factors or gene isoforms in high-risk NBL with MYCN-amplification.