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Neuroblastomas harbor ALK aberrations clinically resistant to crizotinib yet sensitive pre-clinically to the third-generation ALK inhibitor lorlatinib. We conducted a first-in-child study evaluating lorlatinib with and without chemotherapy in children and adults with relapsed or refractory ALK-driven neuroblastoma. The trial is ongoing, and we report here on three cohorts that have met pre-specified primary endpoints: lorlatinib as a single agent in children (12 months to 123I-metaiodobenzylguanidine (MIBG) response. Lorlatinib was evaluated at 45–115 mg/m2/dose in children and 100–150 mg in adults. Common adverse events (AEs) were hypertriglyceridemia (90%), hypercholesterolemia (79%) and weight gain (87%). Neurobehavioral AEs occurred mainly in adults and resolved with dose hold/reduction. The RP2D of lorlatinib with and without chemotherapy in children was 115 mg/m2. The single-agent adult RP2D was 150 mg. The single-agent response rate (complete/partial/minor) for NCT03107988.

Neuroblastomas have neuroendocrine features and often show similar gene expression patterns to small cell lung cancer including high expression of delta-like ligand 3 (DLL3). Here we determine the efficacy of rovalpituzumab tesirine (Rova-T), an antibody–drug conjugated (ADC) with a pyrrolobenzodiazepine dimer toxin targeting DLL3, in preclinical models of human neuroblastoma.We evaluated DLL3 expression in RNA-sequencing datasets and performed IHC on neuroblastoma patient-derived xenograft (PDX), human neuroblastoma primary tumor and normal childhood tissue microarrays. We then evaluated the activity of Rova-T against 11 neuroblastoma PDX models using varying doses and schedules and compared antitumor activity with expression levels.DLL3 mRNA was differentially overexpressed in neuroblastoma at comparable levels to small cell lung cancer, as well as Wilms and rhabdoid tumors. DLL3 protein was robustly expressed across the neuroblastoma PDX array, but membranous staining was variable. The human neuroblastoma array, however, showed staining in only 44% of cases, whereas no significant staining was observed in the normal childhood tissue array. Rova-T showed a clear dose–response effect across the 11 models tested, with a single dose inducing a complete or partial response in 3 of 11 and stable disease in another 3 of 11 models. No overt signs of toxicity were observed, and there was no treatment-related mortality. Strong membranous staining was necessary, but not sufficient, for antitumor activity.Rova-T has activity in a subset of neuroblastoma preclinical models, but heterogeneous expression in these models and the near absence of expression seen in human tumors suggests that any DLL3-targeting clinical trial should be only performed with a robust companion diagnostic to evaluate DLL3 expression for patient selection.Significance:GD2-directed antibody therapy is standard of care for high-risk neuroblastoma; therapy is toxic, and relapses often occur. DLL3, an inhibitory Notch ligand, is overexpressed in several neuronal cancers. A DLL3-targeting ADC showed objective activity only in neuroblastoma models with high DLL3 expression. These data provide vigilance about clinical development of DLL3 immunotherapies for neuroblastoma.

Supplementary Figures 1-7 with legends included

Supplementary Data from Serial Profiling of Circulating Tumor DNA Identifies Dynamic Evolution of Clinically Actionable Genomic Alterations in High-Risk Neuroblastoma

Neuroblastomas have neuroendocrine features and often show similar gene expression patterns to small cell lung cancer including high expression of delta-like ligand 3 (DLL3). Here we determine the efficacy of rovalpituzumab tesirine (Rova-T), an antibody–drug conjugated (ADC) with a pyrrolobenzodiazepine dimer toxin targeting DLL3, in preclinical models of human neuroblastoma. We evaluated DLL3 expression in RNA-sequencing datasets and performed IHC on neuroblastoma patient-derived xenograft (PDX), human neuroblastoma primary tumor and normal childhood tissue microarrays. We then evaluated the activity of Rova-T against 11 neuroblastoma PDX models using varying doses and schedules and compared antitumor activity with expression levels. DLL3 mRNA was differentially overexpressed in neuroblastoma at comparable levels to small cell lung cancer, as well as Wilms and rhabdoid tumors. DLL3 protein was robustly expressed across the neuroblastoma PDX array, but membranous staining was variable. The human neuroblastoma array, however, showed staining in only 44% of cases, whereas no significant staining was observed in the normal childhood tissue array. Rova-T showed a clear dose–response effect across the 11 models tested, with a single dose inducing a complete or partial response in 3 of 11 and stable disease in another 3 of 11 models. No overt signs of toxicity were observed, and there was no treatment-related mortality. Strong membranous staining was necessary, but not sufficient, for antitumor activity. Rova-T has activity in a subset of neuroblastoma preclinical models, but heterogeneous expression in these models and the near absence of expression seen in human tumors suggests that any DLL3-targeting clinical trial should be only performed with a robust companion diagnostic to evaluate DLL3 expression for patient selection. Significance: GD2-directed antibody therapy is standard of care for high-risk neuroblastoma; therapy is toxic, and relapses often occur. DLL3, an inhibitory Notch ligand, is overexpressed in several neuronal cancers. A DLL3-targeting ADC showed objective activity only in neuroblastoma models with high DLL3 expression. These data provide vigilance about clinical development of DLL3 immunotherapies for neuroblastoma.

Neuroblastoma evolution, heterogeneity, and resistance remain inadequately defined, suggesting a role for circulating tumor DNA (ctDNA) sequencing. To define the utility of ctDNA profiling in neuroblastoma, 167 blood samples from 48 high-risk patients were evaluated for ctDNA using comprehensive genomic profiling. At least one pathogenic genomic alteration was identified in 56% of samples and 73% of evaluable patients, including clinically actionable ALK and RAS–MAPK pathway variants. Fifteen patients received ALK inhibition (ALKi), and ctDNA data revealed dynamic genomic evolution under ALKi therapeutic pressure. Serial ctDNA profiling detected disease evolution in 15 of 16 patients with a recurrently identified variant—in some cases confirming disease progression prior to standard surveillance methods. Finally, ctDNA-defined ERRFI1 loss-of-function variants were validated in neuroblastoma cellular models, with the mutant proteins exhibiting loss of wild-type ERRFI1's tumor-suppressive functions. Taken together, ctDNA is prevalent in children with high-risk neuroblastoma and should be followed throughout neuroblastoma treatment. Significance: ctDNA is prevalent in children with neuroblastoma. Serial ctDNA profiling in patients with neuroblastoma improves the detection of potentially clinically actionable and functionally relevant variants in cancer driver genes and delineates dynamic tumor evolution and disease progression beyond that of standard tumor sequencing and clinical surveillance practices. See related commentary by Deubzer et al., p. 2727. This article is highlighted in the In This Issue feature, p. 2711

Current approaches to detect and characterize mosaic chromosomal aneuploidy are limited by sensitivity, efficiency, cost, or the need to culture cells. We describe the mosaic aneuploidy detection by massively parallel sequencing (MAD-seq) capture assay and the MADSEQ analytical approach that allow low (MADSEQ analytical approach can be applied to exome or whole-genome sequencing data, revealing previously unrecognized aneuploidy or copy number neutral loss of heterozygosity in samples studied by the 1000 Genomes Project, cell lines from public repositories, and one of the Illumina Platinum Genomes samples. We have made the meMAD-seq capture design and MADSEQ analytical software open for unrestricted use, with the goal that they can be applied in clinical samples to allow new insights into the unrecognized prevalence of mosaic chromosomal aneuploidy in humans and its phenotypic associations.

Introduction: A growing body of data has shown that higher cumulative anesthesia exposure is associated with neurocognitive impairment in children with chronic disease, including those with hematologic malignancies [Banerjee JAMA Oncology 2019]. To limit general anesthesia (GA) exposure alternative means of sedation and acceptance of alternatives by patients, families and providers need to be explored for children with acute lymphoblastic leukemia or lymphoma (ALL/LL) undergoing repeated lumbar punctures (LP) to deliver intrathecal (IT) chemotherapy. Methods: Patients age 1 to 25 years with ALL/LL treated at the Children's Hospital of Philadelphia main outpatient clinic (CHOP Main) and two CHOP satellite oncology offices (KOP, VOR) were analyzed for baseline rates of sedation for lumbar punctures. GA was defined as the use of intravenous (IV) propofol or an inhalational flurane. Moderate sedation was defined as IV fentanyl (maximum 2 mcg/kg dosing) for analgesia and midazolam (maximum 0.1 mg/kg) for anxiolysis. IV or oral midazolam only was considered mild sedation. We created a comprehensive quality improvement (QI) pathway to implement the use of mild/moderate sedation as a new Division of Oncology standard for LP-only procedures. Our QI goal was a 10% reduction in use of GA with concomitant measurement of outpatient clinic length of stay (LOS) as an additional metric. LOS was measured as time from triage to port de-access. Results: Our interdisciplinary QI team leveraged the CHOP QI framework utilizing tools that included a driver diagram (Figure 1) and impact/effort matrix to develop and prioritize interventions associated with achieving the QI goal. The Plan-Do-Study Act methodology was used to optimize the intervention. This required extensive collaboration with pediatric oncologists, nurse practitioners, nursing, child life, pharmacy, and QI staff to encourage methodology adoption and to optimize clinic workflow for maximal provider/patient participation and procedural efficiency. In the immediate period preceding the QI intervention (October 2019 to February 2020), baseline rates of GA use for across all 3 clinic sites were 73.1%, 15% (plus 34.6% deep sedation), and 24.7% at CHOP Main, KOP, and VNJ, respectively in 310 LP only procedures for patients with ALL/LL. After a delay in implementation due to COVID, the rate of GA usage at CHOP Main from May to July 2020 was reduced to 55.6% for 81 LPs (p=0.001) (Figure 2), while GA rates at the satellite clinics were unchanged. Decreased GA use at CHOP main was replaced by a significant increase in mild/moderate sedation (26.9% to 44.5%; p=0.001). Of the 18 newly diagnosed patients participating in the intervention to date, 16 receive their LPs with no or mild/moderate sedation. The median age of this cohort is 7 years of age with 6 patients ≤ 5 and 12 patients ≤ 10 years. The other two patients have medical syndromes deemed not conducive to replacing GA with moderate sedation. No child has experienced complications from moderate sedation or returned to GA due to sedation intolerance. Overall clinic LOS decreased significantly for all patients getting LPs (GA and sedation) (mean 4.88 hours vs 4.08 hours; p=0.033). An unintended benefit of the QI initiative is that the average LOS for patients receiving moderate sedation decreased from 6.67 hours to 4.15 hours (p=0.017) with the new streamlined clinic workflow. Conclusion: Baseline rates of sedation varied by oncology clinic site and appeared to align with ease of access to GA. GA is available in the oncology main clinic 3 days/week, and 73% of ALL/LL patients were using GA for LPs prior to the intervention. Since March 2020, nearly all patients with newly diagnosed ALL/LL are now undergoing LPs with no or mild/moderate sedation. Our data show that with appropriate education and preparation, LP with mild/moderate sedation is a feasible alternative to GA even for very young patients and reduces clinic LOS. We have expanded this QI intervention to include children with other newly-diagnosed cancers who require LPs and/or IT chemotherapy. Future directions include converting patients in ALL/LL maintenance and those undergoing other minimally invasive procedures from GA to moderate sedation. Disclosures Tasian: Incyte Corporation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Rheingold:Pfizer: Research Funding.

OBJECTIVES/GOALS: Neuroblastoma (NB) is an embryonal cancer of the sympathetic nervous system that affects mostly infants and young children. The complex genetic background present across NB patients results in diverse clinical response and difficulty in individualizing therapy. Currently, NB patients undergo a regimen of genotoxic chemotherapeutics, radiation therapy, and new immunotherapy that, while effective, has significant side effects, including excruciating pain. One promising avenue for targeted therapy in neuroblastoma focuses on anaplastic lymphoma kinase (ALK), a cell surface neural receptor tyrosine kinase. We previously identified activating point mutations within the tyrosine kinase domain of ALK as the primary cause of hereditary NB, and we and others subsequently showed that these same alterations are the most common somatic single-nucleotide mutations in the sporadic forms of the disease. Crizotinib, a first-generation small molecule ATP-competitive inhibitor of the ALK tyrosine kinase, showed limited anti-tumor activity in patients with relapsed NB harboring ALK F1174 and F1245 mutations. We have demonstrated that lorlatinib, a novel ATP-competitive ALK inhibitor, overcomes this de novo resistance in preclinical models of ALK-driven NB. Recent clinical trials with lorlatinib in patients with non-small cell lung cancer harboring an ALK fusion, and in patients with NB harboring ALK mutations show the emergence of multiple or compound ALK mutations as a mechanism of resistance. We postulate that these compound mutations disrupt the interaction between and ALK and cause resistance. In this study, we employ a computational approach to model mutated ALK in complex with lorlatinib as well as ATP to understand whether the new mutations alter the affinity or mode of lorlatinib/ATP binding to ALK, and thus cause suboptimal ALK inhibition. METHODS/STUDY POPULATION: We employ methods in computational structural biology and drug design, primarily based on molecular modeling, molecular dynamics (MD), and molecular docking. Based on existing crystal structures of wildtype ALK, we model the mutations and perform MD simulations in order to characterize the activation state of the protein as well as perform ensemble docking calculations to assess the binding affinities and modes in ALK-lorlatinib and ALK-ATP complexes. RESULTS/ANTICIPATED RESULTS: We expect that the compound mutations cause resistance to lorlatinib either by lowering protein affinity for the drug or increasing the affinity for ATP. Alternatively, the compound mutations may disrupt the protein activation state, in which case ALK may no longer be active, and another protein/pathway could be driving the resistance. DISCUSSION/SIGNIFICANCE OF IMPACT: The results of this study will enable the understanding of the mechanism of resistance to lorlatinib and facilitate the design of new ALK inhibitors, or help develop more optimal and mechanism-guided therapies aimed to overcome the resistance.

Mechanisms of acquired resistance to ALK inhibition therapy in neuroblastoma have not yet been elucidated. In a recent issue of Nature, Debruyne et al. demonstrate that resistant MYCN-amplified ALK-mutated neuroblastoma cells overexpress BORIS, resulting in wide-ranging changes in chromatin interaction and transcriptional reprogramming.

SUMMARY Accelerating cures for children with cancer remains an immediate challenge as a result of extensive oncogenic heterogeneity between and within histologies, distinct molecular mechanisms evolving between diagnosis and relapsed disease, and limited therapeutic options. To systematically prioritize and rationally test novel agents in preclinical murine models, researchers within the Pediatric Preclinical Testing Consortium are continuously developing patient-derived xenografts (PDXs)—many of which are refractory to current standard-of-care treatments—from high-risk childhood cancers. Here, we genomically characterize 261 PDX models from 37 unique pediatric cancers; demonstrate faithful recapitulation of histologies and subtypes; and refine our understanding of relapsed disease. In addition, we use expression signatures to classify tumors for TP53 and NF1 pathway inactivation. We anticipate that these data will serve as a resource for pediatric oncology drug development and will guide rational clinical trial design for children with cancer., Graphical Abstract, In Brief Rokita et. al provide an extensively annotated genomic dataset of somatic oncogenic regulation across 37 distinct pediatric malignancies. The 261 patient-derived xenograft models are available to the scientific community, and the genomic annotations will enable rational preclinical agent prioritization and acceleration of therapeutic targets for early-phase pediatric oncology clinical trials.

10504 Background: Lorlatinib, a potent ALK inhibitor, exerts unprecedented activity against neuroblastoma (NB) derived xenografts harboring common crizotinib-resistant ALK mutations, leading to a first in child phase I study. Methods: R/R NB patients (pts) > 12 months, with ALK mutations/amplification and prior ALK inhibitor (ALKi) treatment were eligible. Lorlatinib was administered in 28-day courses (C). For pts < 18 years, 5 dose levels (DL) (45, 60, 75, 95, 115 mg/m2/day) were assessed. DL5 (115 mg/m2/day) expansion is enrolling (cohort A1). For patients > 18 years, two DL (DL3a the adult RP2D of 100 mg/day and DL4a at 150mg/day) were assessed (cohort A2). Primary endpoint was dose-limiting toxicity (DLT) during C1 and neurocognitive toxicity through C2. Blood samples for circulating tumor DNA (ctDNA) were matched to radiologic restaging. Results: From 9/2017 to 1/2019, 33 eligible patients enrolled (13 with prior ALKi therapy), with median age (range) 5.5 years (2-17) on A1, 21.5 years (15-50) on A2. In A1, 3 pts each enrolled onto DL1-3, with no DLT’s. 5/10 pts enrolled on DL4, with no DLT’s. 1/3 on DL5 had a DLT of grade 3 diarrhea, with expansion ongoing. In cohort A2, 5 patients enrolled at 100 mg/day with no DLT’s; 6 enrolled at 150 mg/day, with one DLT (grade 4 reversible psychosis). Most common treatment-related adverse events were weight gain (90%, grade 1-3), hyperlipidemia (90%, grade 1-3), concentration/memory impairment (23%, grade 1-2), peripheral neuropathy (13%; grade 1-2, A2 only), and peripheral edema (10%; grade 1, A2 only). Lorlatinib steady state exposure at DL3 and DL4 was in the range of exposures seen in adult lung cancer patients at the 100 mg and 200 mg DLs. In A1, 1/18 had partial response (PR), 3/18 had minor responses (MR), and 4/18 had stable disease (SD). Of pts with MR, 2/3 had PR of soft tissue and 1/3 had complete response (CR) by MIBG. In A2 pts, 1/10 had CR, 3/10 PR, and 3/10 MR; Notably, 2/3 with PR had CR by MIBG. Of the pts with MR, one had PR and one CR by MIBG. Responses occurred across dose levels, ALK mutations, and in ALKi pre-treated pts with median courses of 2 (1-24) on A1 and 10.5 (2-28) on A2. Serial ctDNA results showed mutant ALK variant allele frequency trajectories that correlated with clinical response and emergence of novel ALK mutations in cis that corresponded with disease progression. Conclusions: Inhibition of ALK-driven NB with lorlatinib occurs with manageable toxicity and objective anti-tumor activity. Prospective ctDNA allows for monitoring of disease and evolution of resistance. Clinical trial information: NCT03107988.

Background The causes of intellectual disability (ID) are diverse and de novo mutations are increasingly recognised to account for a significant proportion of ID. Methods and results In this study, we performed whole exome sequencing on a large cohort of patients with ID or neurodevelopmental delay and identified four novel de novo predicted deleterious missense variants in HECW2 in six probands with ID/developmental delay and hypotonia. Other common features include seizures, strabismus, nystagmus, cortical visual impairment and dysmorphic facial features. HECW2 is an ubiquitin ligase that stabilises p73, a crucial mediator of neurodevelopment and neurogenesis. Conclusion This study implicates pathogenic genetic variants in HECW2 as potential causes of neurodevelopmental disorders in humans.

Current approaches to detect and characterize mosaic chromosomal aneuploidy are limited by sensitivity, efficiency, cost or the need to culture cells. We describe a combination of a new sequencing-based assay and a novel analytical approach that allows low levels of mosaicism for chromosomal aneuploidy to be detected, assigned to a meiotic or mitotic origin, and quantified as a proportion of the cells in the sample. We show results from a multi-ethnic assay design that is suitable for populations of diverse racial and ethnic origins, and how the MADSEQ analytical approach applied to exome sequencing data reveals unrecognized aneuploidy in 1000 Genomes samples and cell lines from public repositories. We have made the assay design and analytical software open for unrestricted use, with the goal that it can be applied in clinical samples to allow new insights into the unrecognized prevalence of mosaic chromosomal aneuploidy and its phenotypic associations.

The potential for non-genetic susceptibility to mediate part of the risk of autism spectrum disorder (ASD) has prompted a number of studies to date, all showing evidence for epigenetic differences characterizing the individuals with ASD. The modest differences in DNA methylation observed have indicated an underlying cellular mosaicism for epigenetic dysregulation. The studies to date have not comprehensively addressed potential confounding issues like cell subcomposition differences, transcriptional and DNA sequence variability. Additionally, it is possible that mutations in protein-coding genes encoding transcriptional and chromatin regulatory proteins lead to the epigenetic changes in a subset of individuals with ASD. More definitive studies are now needed to allow higher confidence insights into epigenetic events occurring in early embryogenesis in individuals with ASD.

The protein MeCP2 mediates epigenetic regulation by binding methyl-CpG (mCpG) sites on chromatin. MeCP2 consists of six domains of which one, the methyl binding domain (MBD), binds mCpG sites in duplex DNA. We show that solution conditions with physiological or greater salt concentrations or the presence of nonspecific competitor DNA is necessary for the MBD to discriminate mCpG from CpG with high specificity. The specificity for mCpG over CpG is >100-fold under these solution conditions. In contrast, the MBD does not discriminate hydroxymethyl-CpG from CpG. The MBD is unusual among site-specific DNA binding proteins in that (i) specificity is not conferred by the enhanced affinity for the specific site but rather by suppression of its affinity for generic DNA, (ii) its specific binding to mCpG is highly electrostatic, and (iii) it takes up as well as displaces monovalent cations upon DNA binding. The MBD displays an unusually high affinity for single-stranded DNA independent of modification or sequence. In addition, the MBD forms a discrete dimer on DNA via a noncooperative binding pathway. Because the affinity of the second monomer is 1 order of magnitude greater than that of nonspecific binding, the MBD dimer is a unique molecular complex. The significance of these results in the context of neuronal function and development and MeCP2-related developmental disorders such as Rett syndrome is discussed.

DNA mutational events are increasingly being identified in autism spectrum disorder (ASD), but the potential additional role of dysregulation of the epigenome in the pathogenesis of the condition remains unclear. The epigenome is of interest as a possible mediator of environmental effects during development, encoding a cellular memory reflected by altered function of progeny cells. Advanced maternal age (AMA) is associated with an increased risk of having a child with ASD for reasons that are not understood. To explore whether AMA involves covert aneuploidy or epigenetic dysregulation leading to ASD in the offspring, we tested a homogeneous ectodermal cell type from 47 individuals with ASD compared with 48 typically developing (TD) controls born to mothers of ≥35 years, using a quantitative genome-wide DNA methylation assay. We show that DNA methylation patterns are dysregulated in ectodermal cells in these individuals, having accounted for confounding effects due to subject age, sex and ancestral haplotype. We did not find mosaic aneuploidy or copy number variability to occur at differentially-methylated regions in these subjects. Of note, the loci with distinctive DNA methylation were found at genes expressed in the brain and encoding protein products significantly enriched for interactions with those produced by known ASD-causing genes, representing a perturbation by epigenomic dysregulation of the same networks compromised by DNA mutational mechanisms. The results indicate the presence of a mosaic subpopulation of epigenetically-dysregulated, ectodermally-derived cells in subjects with ASD. The epigenetic dysregulation observed in these ASD subjects born to older mothers may be associated with aging parental gametes, environmental influences during embryogenesis or could be the consequence of mutations of the chromatin regulatory genes increasingly implicated in ASD. The results indicate that epigenetic dysregulatory mechanisms may complement and interact with DNA mutations in the pathogenesis of the disorder., Author Summary Older mothers have a higher than expected risk of having a child with an autism spectrum disorder (ASD). The reason for this increased risk is unknown. The eggs of older mothers are more prone to abnormalities of chromosome numbers, suggesting this as one possible mechanism of the increased ASD risk. Age is also associated with a loss of control of epigenetic regulatory patterns that govern gene expression, indicating a second potential mechanism. To test both possibilities, we sampled cells from the same developmental origin as the brain, and performed genome-wide tests looking for unusual chromosome numbers and DNA methylation patterns. The studies were performed on individuals with ASD and typically developing controls, all born to mothers at least 35 years of age at the time of birth. We found the cells from individuals with ASD to have changes in DNA methylation at a number of loci, especially near genes encoding proteins known to interact with those already implicated in ASD. We conclude that epigenetic dysregulation occurring in gametes or early embryonic life may be one of the contributors to the development of ASD.