Expression of mGluR5 in Pediatric Hodgkin and Non-Hodgkin lymphoma-A Comparative Analysis of Immunohistochemical and Clinical Findings Regarding the Association between Tumor and Paraneoplastic Neurological Disease.

Autoantibodies targeting the neuronal antigen metabotropic glutamate receptor 5 (mGluR5) have been identified in patients with Ophelia syndrome, which describes a co-occurrence of paraneoplastic limbic encephalitis and Hodgkin lymphoma (HL). Little data exist regarding frequency and function of mGluR5 in HL and its potential role in causing seropositive paraneoplastic disease. We studied a representative cohort of pediatric HL and NHL patients (n = 57) using immunohistochemistry and fluorescence staining to investigate mGluR5 expression. All lymphoma tissues displayed positive mGluR5 staining, with focus on Hodgkin-Reed-Sternberg (H-RS) cells. We did not detect any mGluR5 staining in tumor-free lymph nodes, which is consistent with the absence of GRM5 transcripts in RNA-sequencing data from non-malignant B and T cells. The frequent presence in pediatric lymphoma falls in line with reports of mGluR5 expression and associated tumor progression in other malignancies. We tested for correlation with clinical features, focusing on disease progression and neurological symptoms. Low mGluR5 expression in H-RS cells correlated with young patient age (<15 years) and positive histology for EBV infection. Paraneoplastic or neurological symptoms were found exclusively in HL patients. While an impact of mGluR5 on HL severity remains possible, a prognostic value of mGluR5 expression levels requires further investigation.

L-Glyceraldehyde Inhibits Neuroblastoma Cell Growth via a Multi-Modal Mechanism on Metabolism and Signaling.

Glyceraldehyde (GA) is a three-carbon monosaccharide that can be present in cells as a by-product of fructose metabolism. Bruno Mendel and Otto Warburg showed that the application of GA to cancer cells inhibits glycolysis and their growth. However, the molecular mechanism by which this occurred was not clarified. We describe a novel multi-modal mechanism by which the L-isomer of GA (L-GA) inhibits neuroblastoma cell growth. L-GA induces significant changes in the metabolic profile, promotes oxidative stress and hinders nucleotide biosynthesis. GC-MS and 13C-labeling was employed to measure the flow of carbon through glycolytic intermediates under L-GA treatment. It was found that L-GA is a potent inhibitor of glycolysis due to its proposed targeting of NAD(H)-dependent reactions. This results in growth inhibition, apoptosis and a redox crisis in neuroblastoma cells. It was confirmed that the redox mechanisms were modulated via L-GA by proteomic analysis. Analysis of nucleotide pools in L-GA-treated cells depicted a previously unreported observation, in which nucleotide biosynthesis is significantly inhibited. The inhibitory action of L-GA was partially relieved with the co-application of the antioxidant N-acetyl-cysteine. We present novel evidence for a simple sugar that inhibits cancer cell proliferation via dysregulating its fragile homeostatic environment.

Defining the landscape of circular RNAs in neuroblastoma unveils a global suppressive function of MYCN.

Circular RNAs (circRNAs) are a regulatory RNA class. While cancer-driving functions have been identified for single circRNAs, how they modulate gene expression in cancer is not well understood. We investigate circRNA expression in the pediatric malignancy, neuroblastoma, through deep whole-transcriptome sequencing in 104 primary neuroblastomas covering all risk groups. We demonstrate that MYCN amplification, which defines a subset of high-risk cases, causes globally suppressed circRNA biogenesis directly dependent on the DHX9 RNA helicase. We detect similar mechanisms in shaping circRNA expression in the pediatric cancer medulloblastoma implying a general MYCN effect. Comparisons to other cancers identify 25 circRNAs that are specifically upregulated in neuroblastoma, including circARID1A. Transcribed from the ARID1A tumor suppressor gene, circARID1A promotes cell growth and survival, mediated by direct interaction with the KHSRP RNA-binding protein. Our study highlights the importance of MYCN regulating circRNAs in cancer and identifies molecular mechanisms, which explain their contribution to neuroblastoma pathogenesis.

Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells.

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond.

Treatment of Infants and Children With SARS-CoV-2 Monoclonal Antibodies: A European Case Series.

BACKGROUND: Although severe COVID-19 in children is rare, those with certain pre-existing health conditions are more prone to severe disease. Monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are potent antiviral agents that reduce adverse clinical outcomes in adults, but are commonly not approved for use in pediatric patients. METHODS: We retrospectively evaluated mAb treatment in children <12 years of age or <40kg with SARS-CoV-2 infection between January 1, 2021, and March 7, 2022, in 12 tertiary care centers in 3 European countries. RESULTS: We received data from 53 patients from Austria, Denmark and Germany. Median age was 5.4 years [0-13.8, interquartile range (IQR) = 6.2], and median body weight was 20 kg (3-50.1, IQR = 13). The most frequent SARS-CoV-2 variant in this study, if known, was Omicron, followed by Delta and Alpha. Pre-existing conditions included immunodeficiency, malignancy, hematologic disease, cardiac disease, chronic lung disease, chronic liver disease, kidney disease and diabetes. Forty-two patients received sotrovimab (79%), 9 casirivimab/imdevimab (17%) and 2 bamlanivimab (4%). All but 1 patient survived. Median duration of hospital stay was 3 days (0-56, IQR = 6). Seven patients required treatment in an intensive care unit, and 5 required high-flow nasal cannula treatment. Potential side effects included neutropenia (6/53, 11%), lymphopenia (3/53, 6%), nausea or vomiting (2/53, 4%), rise of alanine transaminase (1/53, 2%) and hypotonia (1/53, 2%). CONCLUSIONS: MAb treatment was well tolerated by children in this cohort.

A Deep Dive into the Circulating ctDNA Cosmos to Vanquish Neuroblastoma.

SUMMARY: Single biopsies fail to reflect intratumor heterogeneity and tumor evolution. In this issue of Cancer Discovery, Bosse and colleagues show an important role for circulating cell-free tumor DNA sequencing to detect the genomic evolution of neuroblastoma under ALK inhibitor therapy and identify novel (sub)clonal pathogenic variants involved in disease progression under conventional therapy. See related article by Bosse et al., p. 2800 (5).

Efficacy and Feasibility of Proton Beam Therapy in Relapsed High-Risk Neuroblastoma-Experiences from the Prospective KiProReg Registry.

BACKGROUND: Despite an intensive multimodal treatment approach, approximately 50% of high-risk (HR) neuroblastoma (NB) patients experience progression. Despite the advances in targeted therapy, high-dose chemotherapy, and other systemic treatment options, radiation therapy (RT) to sites of relapsed disease can be an option to reduce tumor burden and improve chance for disease control. METHODS: Patients who received salvage irradiation with proton beam therapy (PBT) for local or metastatic relapse of HR NB within the prospective registry trials KiProReg and ProReg were eligible for this retrospective analysis. Data on patient characteristics, multimodality therapy, adverse events, and oncologic endpoints were evaluated. Adverse events were assessed before, during, and after PBT according to common terminology criteria for adverse events (CTCAE) V4.0. RESULTS: Between September 2013 and September 2020, twenty (11 male; 9 female) consecutive patients experiencing local (N = 9) or distant recurrence (N = 25) were identified for this analysis. Distant recurrences included osteomedullary (N = 11) or CNS lesions (N = 14). Salvage therapy consisted of re-induction chemo- or chemo-immuno-therapy (N = 19), surgery (N = 6), high-dose chemotherapy and stem cell transplantation (N = 13), radiation (N = 20), and concurrent systemic therapy. Systemic therapy concurrent to RT was given to six patients and included temozolomide (N = 4), carboplatine (N = 1), or anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKI) (N = 1). A median dose of 36 Gy was applied to the 34 recurrent sites. Local RT was applied to 15 patients, while five patients, received craniospinal irradiation for CNS relapse. After a median follow-up (FU) of 20 months (4-66), the estimated rate for local control, distant metastatic free survival, and overall survival at 3 years was 68.0%, 37.9%, and 61.6%, respectively. During RT, ten patients (50%) presented with a higher-grade acute hematologic adverse event. Late higher-grade sequelae included transient myelitis with transverse section (N = 2) and secondary malignancy outside of the RT field (N = 1). CONCLUSION: Our study demonstrates the efficacy and safety of RT/PBT for recurrent HR NB in a multimodality second-line approach. To better define the role of RT for these patients, prospective studies would be desirable.

Incidence of subsequent malignancies after total body irradiation-based allogeneic HSCT in children with ALL - long-term follow-up from the prospective ALL-SCT 2003 trial.

Total body irradiation (TBI)-based conditioning is associated with superior leukemia-free survival in children with ALL undergoing HSCT. However, the risk for subsequent malignant neoplasms (SMN) remains a significant concern. We analyzed 705 pediatric patients enrolled in the prospective ALL-SCT-BFM-2003 trial and its subsequent registry. Patients >2 years received conditioning with TBI 12 Gy/etoposide (n = 558) and children ≤2 years of age or with contraindications for TBI received busulfan/cyclophosphamide/etoposide (n = 110). The 5- and 10-year cumulative incidence of SMN was 0.02 ± 0.01 and 0.13 ± 0.03, respectively. In total, 39 SMN (34 solid tumors, 5 MDS/AML) were diagnosed in 33 patients at a median of 5.8 years (1.7-13.4), exclusively in the TBI group. Of 33 affected patients, 21 (64%) are alive at a median follow-up of 5.1 years (0-9.9) after diagnosis of their first SMN. In univariate analysis, neither age at HSCT, donor type, acute GVHD, chronic GVHD, nor CMV constituted a significant risk factor for SMN. The only significant risk factor was TBI versus non-TBI based conditioning. This analysis confirms and quantifies the increased risk of SMN in children with ALL after conditioning with TBI. Future strategies to avoid TBI will need careful tailoring within prospective, controlled studies to prevent unfavorable outcomes.

Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells.

BACKGROUND: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. METHODS: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. RESULTS: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. CONCLUSIONS: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

Circulating Cell-Free DNA Assessment in Biofluids from Children with Neuroblastoma Demonstrates Feasibility and Potential for Minimally Invasive Molecular Diagnostics.

Liquid biopsy strategies in pediatric patients are challenging due to low body weight. This study investigated cfDNA size distribution and concentration in blood, bone marrow, cerebrospinal fluid, and urine from 84 patients with neuroblastoma classified as low (n = 28), intermediate (n = 6), or high risk (n = 50) to provide key data for liquid biopsy biobanking strategies. The average volume of blood and bone marrow plasma provided ranged between 1 and 2 mL. Analysis of 637 DNA electropherograms obtained by Agilent TapeStation measurement revealed five different major profiles and characteristic DNA size distribution patterns for each of the biofluids. The proportion of samples containing primarily cfDNA was, at 85.5%, the highest for blood plasma. The median cfDNA concentration amounted to 6.28 ng/mL (blood plasma), 58.2 ng/mL (bone marrow plasma), 0.08 ng/mL (cerebrospinal fluid), and 0.49 ng/mL (urine) in samples. Meta-analysis of the dataset demonstrated that multiple cfDNA-based assays employing the same biofluid sample optimally require sampling volumes of 1 mL for blood and bone marrow plasma, 2 mL for cerebrospinal fluid, and as large as possible for urine samples. A favorable response to treatment was associated with a rapid decrease in blood-based cfDNA concentration in patients with high-risk neuroblastoma. Blood-based cfDNA concentration was not sufficient as a single parameter to indicate high-risk disease recurrence. We provide proof of concept that monitoring neuroblastoma-specific markers in very small blood volumes from infants is feasible.

Targeted Analysis of Cell-free Circulating Tumor DNA is Suitable for Early Relapse and Actionable Target Detection in Patients with Neuroblastoma.

PURPOSE: Treating refractory or relapsed neuroblastoma remains challenging. Monitoring body fluids for tumor-derived molecular information indicating minimal residual disease supports more frequent diagnostic surveillance and may have the power to detect resistant subclones before they give rise to relapses. If actionable targets are identified from liquid biopsies, targeted treatment options can be considered earlier. EXPERIMENTAL DESIGN: Droplet digital PCR assays assessing MYCN and ALK copy numbers and allelic frequencies of ALK p.F1174L and ALK p.R1275Q mutations were applied to longitudinally collected liquid biopsies and matched tumor tissue samples from 31 patients with high-risk neuroblastoma. Total cell-free DNA (cfDNA) levels and marker detection were compared with data from routine clinical diagnostics. RESULTS: Total cfDNA concentrations in blood plasma from patients with high-risk neuroblastoma were higher than in healthy controls and consistently correlated with neuron-specific enolase levels and lactate dehydrogenase activity but not with 123I-meta-iodobenzylguanidine scores at relapse diagnosis. Targeted cfDNA diagnostics proved superior for early relapse detection to all current diagnostics in 2 patients. Marker analysis in cfDNA indicated intratumor heterogeneity for cell clones harboring MYCN amplifications and druggable ALK alterations that were not detectable in matched tumor tissue samples in 17 patients from our cohort. Proof of concept is provided for molecular target detection in cerebrospinal fluid from patients with isolated central nervous system relapses. CONCLUSIONS: Tumor-specific alterations can be identified and monitored during disease course in liquid biopsies from pediatric patients with high-risk neuroblastoma. This approach to cfDNA surveillance warrants further prospective validation and exploitation for diagnostic purposes and to guide therapeutic decisions.

Hemostatic Management in an Infant With Neuroblastoma and Severe Hemophilia B With Extended Half-life Recombinant Factor IX Fusion Protein.

In the rare co-occurrence of childhood cancer and severe hemophilia, hemostatic management is of paramount therapeutic importance. We present the case of an 11-month-old boy with severe congenital hemophilia B, who was diagnosed with metastatic high-risk neuroblastoma. He consequently developed paraneoplastic coagulopathy with life-threatening tumor hemorrhage and intracranial hemorrhage, showing central nervous system relapse. Management consisted of factor IX replacement with extended half-life factor IX fusion protein, adjusted to bleeding risk. Additional interventions included factor XIII, fibrinogen, fresh frozen plasma, tranexamic acid, and platelet transfusions. The half-life of factor IX products was markedly reduced requiring close factor IX monitoring and adequate replacement. This intensified treatment allowed chemotherapy, autologous stem cell transplantation, and GD2 antibody immune therapy without bleeding or thrombosis.

Spatial and temporal intratumour heterogeneity has potential consequences for single biopsy-based neuroblastoma treatment decisions.

Intratumour heterogeneity is a major cause of treatment failure in cancer. We present in-depth analyses combining transcriptomic and genomic profiling with ultra-deep targeted sequencing of multiregional biopsies in 10 patients with neuroblastoma, a devastating childhood tumour. We observe high spatial and temporal heterogeneity in somatic mutations and somatic copy-number alterations which are reflected on the transcriptomic level. Mutations in some druggable target genes including ALK and FGFR1 are heterogeneous at diagnosis and/or relapse, raising the issue whether current target prioritization and molecular risk stratification procedures in single biopsies are sufficiently reliable for therapy decisions. The genetic heterogeneity in gene mutations and chromosome aberrations observed in deep analyses from patient courses suggest clonal evolution before treatment and under treatment pressure, and support early emergence of metastatic clones and ongoing chromosomal instability during disease evolution. We report continuous clonal evolution on mutational and copy number levels in neuroblastoma, and detail its implications for therapy selection, risk stratification and therapy resistance.

Neuroblastoma Risk Assessment and Treatment Stratification with Hybrid Capture-Based Panel Sequencing.

For many years, the risk-based therapy stratification of children with neuroblastoma has relied on clinical and molecular covariates. In recent years, genome analysis has revealed further alterations defining risk, tumor biology, and therapeutic targets. The implementation of a robust and scalable method for analyzing traditional and new molecular markers in routine diagnostics is an urgent clinical need. Here, we investigated targeted panel sequencing as a diagnostic approach to analyze all relevant genomic neuroblastoma risk markers in one assay. Our "neuroblastoma hybrid capture sequencing panel" (NB-HCSP) assay employs a technology for the high-coverage sequencing (>1000×) of 55 selected genes and neuroblastoma-relevant genomic regions, which allows for the detection of single nucleotide changes, structural rearrangements, and copy number alterations. We validated our assay by analyzing 15 neuroblastoma cell lines and a cohort of 20 neuroblastomas, for which reference routine diagnostic data and genome sequencing data were available. We observed a high concordance for risk markers identified by the NB-HSCP assay, clinical routine diagnostics, and genome sequencing. Subsequently, we demonstrated clinical applicability of the NB-HCSP assay by analyzing routine clinical samples. We conclude that the NB-HCSP assay may be implemented into routine diagnostics as a single assay that covers all essential covariates for initial neuroblastoma classification, extended risk stratification, and targeted therapy selection.

A Reproducible Bioprinted 3D Tumor Model Serves as a Preselection Tool for CAR T Cell Therapy Optimization.

Chimeric antigen receptor (CAR) T cell performance against solid tumors in mouse models and clinical trials is often less effective than predicted by CAR construct selection in two-dimensional (2D) cocultures. Three-dimensional (3D) solid tumor architecture is likely to be crucial for CAR T cell efficacy. We used a three-dimensional (3D) bioprinting approach for large-scale generation of highly reproducible 3D human tumor models for the test case, neuroblastoma, and compared these to 2D cocultures for evaluation of CAR T cells targeting the L1 cell adhesion molecule, L1CAM. CAR T cells infiltrated the model, and both CAR T and tumor cells were viable for long-term experiments and could be isolated as single-cell suspensions for whole-cell assays quantifying CAR T cell activation, effector function and tumor cell cytotoxicity. L1CAM-specific CAR T cell activation by neuroblastoma cells was stronger in the 3D model than in 2D cocultures, but neuroblastoma cell lysis was lower. The bioprinted 3D neuroblastoma model is highly reproducible and allows detection and quantification of CAR T cell tumor infiltration, representing a superior in vitro analysis tool for preclinical CAR T cell characterization likely to better select CAR T cells for in vivo performance than 2D cocultures.

Germline Mutations Including the Rare Pathogenic Variant c.3206delC in the ATM Gene Cause Ataxia Teleangiectasia-Associated Primary Central Nervous System Lymphoma.

We here report the case of a 2-year-old patient with a primary central nervous system lymphoma of B-cell origin. Due to their past medical history of repeated respiratory tract infections and the marked chemotherapy-associated toxicity and infectious comorbidity, we suspected that the patient also suffered from an inherited immune deficiency disorder. Despite the lack of classical pathognomonic symptoms for ataxia teleangiectasia and missing evidence for a cancer predisposition syndrome in the family, genetic testing identified biallelic germline mutations, including the rare pathogenic variant c.3206delC (p.Pro1069Leufs*2), in the ataxia telangiectasia-mutated (ATM) gene. The case highlights the importance of searching for immune deficiency disorders associated with primary central nervous system lymphoma before treatment initiation and the urgent need to develop novel treatment strategies for cancer patients with underlying immunodeficiency syndromes.

Inhibiting PHGDH with NCT-503 reroutes glucose-derived carbons into the TCA cycle, independently of its on-target effect.

The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.