Type Of Pediatric Cancer Research Articles

ARID5B, IKZF1, GATA3, CEBPE, and CDKN2A germline polymorphisms and predisposition to childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most frequent type of pediatric cancer. Germline single nucleotide polymorphisms (SNPs), including ARID5B (rs10821936 T/C), IKZF1 (rs4132601 T/G), GATA3 (rs3824662 G/T), CEBPE (rs2239633 G/A), and CDKN2A (rs3731217 A/C) have been linked to pediatric ALL in different populations. Hitherto, no previous studies have tested the relationship between these SNPs and pediatric ALL in Gaza strip. Therefore, we investigated the association between these polymorphisms and the occurrence of childhood ALL in this part of Palestine. This case-control study recruited 100 healthy controls and 78 ALL patients. Allele-specific PCR (AS-PCR) technique was used for SNPs genotyping. Relevant statistical tests were used and the multifactor dimensionality reduction (MDR) approach was applied in the analysis of gene-gene interactions. Minor alleles of ARID5B rs10821936 T/C (p = 0.007) and IKZF1 rs4132601 T/G (p = 0.045) were significantly higher in ALL patients. The homozygous (TT) genotype of GATA3 rs3824662 G/T (p = 0.038), (CC) of ARID5B rs10821936 T/C (p = 0.008), and (AC and CC) genotypes of CDKN2A rs3731217 A/C (p < 0.0001) were significantly higher in ALL cases. On MDR analysis, the best model for ALL risk was the five-factor model combination of the examined SNPs (CVC = 10/10; TBA = 0.632; p < 0.0001). This work demonstrates the association of ARID5B rs10821936 T/C, IKZF1 rs4132601 T/G, GATA3 rs3824662 G/T, and CDKN2A rs3731217 A/C polymorphisms with increased risk of pediatric ALL among a patient cohort from Gaza Strip. Further studies with a larger sample size are needed in order to confirm these findings and test the value of these SNPs in prognosis and treatment sensitivity.

METB-01. EXTRACHROMOSOMAL DNA IS ASSOCIATED WITH ONCOGENE AMPLIFICATION AND POOR SURVIVAL ACROSS MULTIPLE PEDIATRIC CANCER TYPES

Abstract Circular extrachromosomal DNA (ecDNA) is an important driver of aggressive cancers. Its frequency and impact on the spectrum of pediatric cancers has yet to be fully described. To determine the ecDNA prevalence in various pediatric cancer types, we accessed whole genome sequencing (WGS) data in the Children’s Brain Tumor Network (CBTN) and St. Jude cancer cloud genomics platforms and utilized the bioinformatics tools Amplicon Architect (AA) and Amplicon Classifier (AC) to reconstruct ecDNA sequence structures. We then identified which genes were amplified on the ecDNA and the association of ecDNA with survival using Kaplan Meier curves. In total, we analyzed a retrospective cohort of over 1600 tumor biopsies from 1400 different patients, spanning 69 different tumor types. We identified ecDNA in 14 tumor types. The incidence of ecDNA within these 14 tumors was 16% with the highest percentage in osteosarcoma (47%), pineoblastoma (40%) and rhabdomyosarcoma (40%). The overall incidence of ecDNA across the patient cohort was 10%. In analyzing matched diagnostic/relapse and relapse/progression pairs, ecDNA was noted to evolve over time with progression of disease. Across the cohort, ecDNA was found to be significantly associated with poor survival. Our preliminary results demonstrate that ecDNA is prevalent in pediatric cancer, can contain known and potentially novel oncogenes and can be associated with poorer outcomes across many different pediatric cancer types.

Abstract 2557: Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma

Abstract Retinoblastoma is the most common intraocular cancer of childhood, with approximately 8,000 new cases each year worldwide. While survival rates are relatively high in developed countries, death can occur in up to half of children in some regions. In addition, blindness is common in patients who survive, and improved therapies are clearly needed. While loss of RB gene function is essential for the initiation of almost all retinoblastoma, additional molecular changes driving their growth and dissemination are poorly understood. Improved understanding of the molecular pathways driving retinoblastoma growth and dissemination may allow us to develop more effective and less toxic therapies. Alterations leading to BCL-6 corepressor (BCOR) loss of function are the second most common mutations in retinoblastoma after changes affecting the RB locus, and are found in more aggressive tumors. BCOR mutations are also associated with metastasis and poor prognosis in other types of pediatric cancer, suggesting that targeting it will represent a new therapeutic approach for aggressive retinoblastoma. Here, we found that BCOR knockdown or knockout in two retinoblastoma cell lines with high baseline levels of protein caused significantly increased invasion/migration, proliferation and clonogenicity. In contrast, overexpression of BCOR in retinoblastoma cells with low/no baseline expression showed opposite effects. In orthotopic xenograft models, BCOR knockout dramatically increased the size and spread of retinoblastoma xenografts as compared to parental controls. In order to determine the molecular pathways modulated by BCOR, we used RNA sequencing and found that loss of function induces IGF1 transcription, resulting in activation of IGF1R and the ERK signaling pathway. Pharmacological inhibition of IGF1/IGF1R signaling using Lincitinib or AEW541 inhibited proliferation and migration in BCOR knockout retinoblastoma cells in vitro, and Lincitinib dramatically slowed xenograft growth in vivo, suggesting that induction of IGF1 represents an important downstream effector following BCOR loss. Our studies support a functional role for BCOR mutations in aggressive retinoblastoma, and suggest that this is mediated at least in part by increased IGF1R signaling, which represents a new therapeutic target for these malignant childhood tumors of the eye. Citation Format: Su-Chan Lee, Lujain Alaali, Charles G. Eberhart. Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2557.

Abstract 243: Long-read sequencing of pediatric cancer genomes identifies multiple clinically relevant variants

Abstract Genetic characterization of pediatric cancer samples clinically for diagnostic, prognostic, or therapeutic information currently relies on an assortment of methods that complement and partially overlap one another, such as fluorescence in situ hybridization (FISH), karyotyping, microarray, and sequencing. This need for multiple tests results in longer analysis time, increased total costs, and higher consumption of sometimes limited tumor material. Long-read sequencing (lrSeq) using HiFi technology, i.e., circular consensus sequencing of reads 12-16kb in length, is able to provide sequence level changes with highly accurate base quality (&amp;gt;99%), as well as information on copy number variants, structural variants, and methylation status, all fully phased. Here, we assess the ability of lrSeq to detect clinically relevant genetic alterations (CRGAs) in pediatric cancer samples. We selected 10 patient cases of solid and liquid tumors with known CRGAs from prior clinical testing. These included 3 cases of T-cell acute lymphoblastic leukemia (T-ALL): one with t(9;17) involving NOTCH1, one with t(7;7) involving TRB, and one with biallelic CDKN2A deletion; 2 high-grade glioma (HGG) cases: one with t(2;9) causing a WDCP-NTRK2 fusion, and one with a TP53 mutation; 2 cases of acute myeloid leukemia (AML): one with biallelic mutations in CEPBA and one with a KMT2A-rearrangement (KMT2A-r; t(v;11q23.3)); 1 case of B-cell ALL with KMT2A-r, t(v;11q23.3); 1 neuroblastoma (NBL) case with regional amplifications of chromosome 12 including MDM2; and 1 case of anaplastic large cell lymphoma (ALCL) with an NPM1-ALK fusion (t(2;5)). DNA from these samples was sequenced on the PacBio Sequel II System to a target depth of 25x. HiFi reads were processed using the PacBio Human WGS Workflow. Methylation prediction was made from Primrose and 5mC modification probability of CpG sites were computed by pb-CpG-tools. We then assessed phased CpG methylation in known imprinted genes.We found that lrSeq detected translocations in 6 out of 6 cases, providing precise break ends and often clarifying unclear cytogenetic findings (such as partner genes). HiFi sequencing also detected 3 of 3 sequence alterations in CEBPA and TP53; biallelic deletion of CDKN2A; and known regions with loss of heterozygosity (LOH) in 2 out of 2 cases. However, it had difficulty detecting all amplified regions of chromosome 12 in the case of NBL, such as the one containing MDM2. Finally, we were able to detect methylation differences in a phased manner by parental haplotype in known imprinting regions across the genome. The difference in 5mC modification probability between the two alleles of the known imprinted genes was 81.5. Our results demonstrate that lrSeq can detect multiple CRGAs across several different types of genetic variants and pediatric cancers, bringing us closer to the promise of an all-in-one genetic test for cancer characterization. Citation Format: Byunggil Yoo, Warren Cheung, Irina Pushel, Lisa Lansdon, Chengpeng Bi, Erin Guest, Tomi Pastinen, Midhat Farooqi. Long-read sequencing of pediatric cancer genomes identifies multiple clinically relevant variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 243.

Pediatric Cancer Types, Diagnosis, and Treatment

The current study's objective was to conduct a review study on pediatric malignancies from several angles, including kinds, diagnosis, and treatment. In order to accomplish this, the primary data sources, including Google, GoogleScholar, Sciencedirect, PubMed, and others, were examined. The findings of this study demonstrated that, despite being uncommon, childhood malignancies are lethal once they manifest. His study's findings also demonstrated the presence of therapy choices and diagnostic technologies that increase survival rates to above 80% of cases. Hence, pediatric cancer affects youngsters under the age of 18. Each form of pediatric cancer has a unique set of symptoms and available therapies. Juvenile cancer can be challenging to diagnose and is typically detected by physical examinations, blood tests, imaging scans, and biopsies. Pediatric cancer treatment is determined by the type, stage, age, and condition of the child. The prognosis for pediatric cancer has significantly improved thanks to recent developments in diagnosis and treatment.

In vivo loss of tumorigenicity in a patient-derived orthotopic xenograft mouse model of ependymoma.

Ependymomas (EPN) are the third most common malignant brain cancer in children. Treatment strategies for pediatric EPN have remained unchanged over recent decades, with 10-year survival rates stagnating at just 67% for children aged 0-14 years. Moreover, a proportion of patients who survive treatment often suffer long-term neurological side effects as a result of therapy. It is evident that there is a need for safer, more effective treatments for pediatric EPN patients. There are ten distinct subgroups of EPN, each with their own molecular and prognostic features. To identify and facilitate the testing of new treatments for EPN, in vivo laboratory models representative of the diverse molecular subtypes are required. Here, we describe the establishment of a patient-derived orthotopic xenograft (PDOX) model of posterior fossa A (PFA) EPN, derived from a metastatic cranial lesion. Patient and PDOX tumors were analyzed using immunohistochemistry, DNA methylation profiling, whole genome sequencing (WGS) and RNA sequencing. Both patient and PDOX tumors classified as PFA EPN by methylation profiling, and shared similar histological features consistent with this molecular subgroup. RNA sequencing revealed that gene expression patterns were maintained across the primary and metastatic tumors, as well as the PDOX. Copy number profiling revealed gains of chromosomes 7, 8 and 19, and loss of chromosomes 2q and 6q in the PDOX and matched patient tumor. No clinically significant single nucleotide variants were identified, consistent with the low mutation rates observed in PFA EPN. Overexpression of EZHIP RNA and protein, a common feature of PFA EPN, was also observed. Despite the aggressive nature of the tumor in the patient, this PDOX was unable to be maintained past two passages in vivo. Others who have successfully developed PDOX models report some of the lowest success rates for EPN compared to other pediatric brain cancer types attempted, with loss of tumorigenicity not uncommon, highlighting the challenges of propagating these tumors in the laboratory. Here, we discuss our collective experiences with PFA EPN PDOX model generation and propose potential approaches to improve future success in establishing preclinical EPN models.

The opportunities and challenges for nutritional intervention in childhood cancers.

Diet dictates nutrient availability in the tumor microenvironment, thus affecting tumor metabolic activity and growth. Intrinsically, tumors develop unique metabolic features and are sensitive to environmental nutrient concentrations. Tumor-driven nutrient dependencies provide opportunities to control tumor growth by nutritional restriction or supplementation. This review summarized the existing data on nutrition and pediatric cancers after systematically searching articles up to 2023 from four databases (PubMed, Web of Science, Scopus, and Ovid MEDLINE). Epidemiological studies linked malnutrition with advanced disease stages and poor clinical outcomes in pediatric cancer patients. Experimental studies identified several nutrient dependencies (i.e., amino acids, lipids, vitamins, etc.) in major pediatric cancer types. Dietary modifications such as calorie restriction, ketogenic diet, and nutrient restriction/supplementation supported pediatric cancer treatment, but studies remain limited. Future research should expand epidemiological studies through data sharing and multi-institutional collaborations and continue to discover critical and novel nutrient dependencies to find optimal nutritional approaches for pediatric cancer patients.

Molecular mechanisms of pediatric intractable leukemia

Recent advances in intensive treatment strategies have resulted in an overall treatment rate of approximately 70% for pediatric cancers. The most notable improvement in clinical outcomes has been observed for acute lymphocytic leukemia, with the cure rate increasing from approximately 10% 50 years ago to 90% at present. However, relapsed and refractory cases of childhood leukemia continue to have a poor prognosis, and there is no standard treatment strategy for many cases. Moreover, even in cases with favorable outcomes, growth retardation and endocrine disorders are major complications. To improve the cure rate of pediatric cancers, we have focused on the molecular mechanisms of leukemia, the most common type of pediatric cancer. In this symposium, recent findings on T-cell acute lymphocytic leukemia will be outlined.

Phosphorylation-Mediated Activation of β-Catenin-TCF4-CEGRs/ALCDs Pathway Is an Essential Event in Development of Aggressive Hepatoblastoma.

Hepatoblastoma (HBL), a deadly malignancy in children, is the most common type of pediatric liver cancer. We recently demonstrated that β-catenin, phosphorylated at S675 (ph-S675-β-catenin), causes pathological alterations in fibrolamellar hepatocellular carcinoma (FLC), by activating oncogenes and fibrotic genes via human genomic regions, known as cancer-enhancing genomic regions or aggressive liver cancer domains (CEGRs/ALCDs). The aim of this study was to determine the role of the ph-S675-β-catenin-TCF4-CEGRs/ALCDs pathway in HBL. The ph-S675-β-catenin-TCF4-CEGRs/ALCDs pathway was examined in a large cohort of HBL specimens, in HBL cell lines HepG2 and Huh6, and in patient-derived xenografts (PDXs). β-catenin is phosphorylated at S675 in a large portion of tested HBL patients. In these patients, ph-S675-β-catenin forms complexes with TCF4 and opens CEGRs/ALCDs-dependent oncogenes for transcription, leading to a massive overexpression of the oncogenes. The inhibition of the β-catenin-TCF4-CEGRs/ALCDs axis inhibits the proliferation of cancer cells and tumor growth in HBL cell lines and HBL-PDXs. The ph-S675-β-catenin is abundant in mitotic cells. We found that markers of HBL Glypican 3 (GPC3) and Alpha Fetoprotein (AFP) are increased in HBL patients by β-catenin-TCF4-p300 complexes. The phosphorylation-mediated activation of the β-catenin-TCF4-p300-CEGRs/ALCDs pathway increases oncogene expression in patients with aggressive liver cancer and promotes the development of hepatoblastoma.

Deep learning for MYC binding site recognition.

Motivation: The definition of the genome distribution of the Myc transcription factor is extremely important since it may help predict its transcriptional activity particularly in the context of cancer. Myc is among the most powerful oncogenes involved in the occurrence and development of more than 80% of different types of pediatric and adult cancers. Myc regulates thousands of genes which can be in part different, depending on the type of tissues and tumours. Myc distribution along the genome has been determined experimentally through chromatin immunoprecipitation This approach, although powerful, is very time consuming and cannot be routinely applied to tumours of individual patients. Thus, it becomes of paramount importance to develop in silico tools that can effectively and rapidly predict its distribution on a given cell genome. New advanced computational tools (DeeperBind) can then be successfully employed to determine the function of Myc in a specific tumour, and may help to devise new directions and approaches to experiments first and personalized and more effective therapeutic treatments for a single patient later on. Results: The use of DeeperBind with DeepRAM on Colab platform (Google) can effectively predict the binding sites for the MYC factor with an accuracy above 0.96 AUC, when trained with multiple cell lines. The analysis of the filters in DeeperBind trained models shows, besides the consensus sequence CACGTG classically associated to the MYC factor, also the other consensus sequences G/C box or TGGGA, respectively bound by the SP1 and MIZ-1 transcription factors, which are known to mediate the MYC repressive response. Overall, our findings suggest a stronger synergy between the machine learning tools as DeeperBind and biological experiments, which may reduce the time consuming experiments by providing a direction to guide them.

EPCO-45. EXTRACHROMOSOMAL DNA PROMOTES ONCOGENE AMPLIFICATION ACROSS MULTIPLE PEDIATRIC CANCER TYPES

Abstract Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive cancers. Its frequency and impact on pediatric cancers has yet to be fully described. To determine the ecDNA prevalence in various pediatric cancer types, we accessed whole genome sequencing (WGS) data in the Children’s Brain Tumor Network (CBTN) and St. Jude cancer cloud genomics platforms and utilized the bioinformatics tools Amplicon Architect (AA) and Amplicon Classifier (AC) to reconstruct ecDNA sequence structures. We then identified which genes were amplified on the ecDNA and how ecDNA correlated with survival, using Kaplan Meier curves. Different cancer types were noted to have varying prevalence of ecDNA with the highest detection in pediatric high-grade gliomas (30% of cases). In analyzing matched diagnostic/relapse and relapse/progression pairs, ecDNA was noted to evolve over time with progression of disease. Our preliminary results demonstrate that ecDNA is prevalent in pediatric cancer, can contain known and potential novel oncogenes and can be associated with poorer outcomes across many different pediatric cancer types.

EXTH-81. ITGA5 IS A NOVEL IMMUNOTHERAPEUTIC TARGET AGAINST TREATMENT REFRACTORY MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common type of malignant pediatric brain cancer. Current standard of care (SOC) involves maximal safe resection and neuraxis radiotherapy and chemotherapy in individuals older than 3 years. To date, these cytotoxic SOC combined with craniospinal irradiation led to devastating neurocognitive and developmental deficits impacting quality of life for pediatric patients. The biological heterogeneity of MB is highlighted by the existence of four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4). Group 3 and Group 4 have the poorest patient outcomes because of their aggressive, metastatic nature, and so often remain treatment refractory to SOC. Group 3 has a poor prognosis due to its high incidence of leptomeningeal spread and an overall survival rate of less than 50%. The cytotoxic nature and lack of response in specific subtypes to SOC underscores the urgent need for developing and translating novel treatment options including immunotherapies. In our earlier work, we have developed a therapy-adapted patient derived xenograft (PDX) model of the Group 3 MB as the tumor cells undergoes therapy in vitro and in vivo. N-glycocapture surfaceome profiling of the MB cells through this PDX model identified Integrin α5 (ITGA5) as one of the most differentially expressed targets found at recurrence when compared to engraftment and untreated timepoints. Through shRNA knockdown and small molecule inhibition, we identify ITGA5 expression marks a MB cell subpopulation with increased self-renewal ability both in vitro and in vivo. Access to recurrent MB (rMB) post-therapy allowed us to investigate the changes in the surfaceome of MB cells using proteomics profiling to identify promising rMB-specific targets for rational development of novel immunotherapies.

EPCO-37. IDENTIFICATION OF KEY EPIGENETIC REGULATORS FOR MEDULLOBLASTOMA INVASION AND METASTASIS

Abstract Medulloblastoma (MB) is most common type of pediatric brain cancer. It starts from the hind brain or cerebellum. It is actually an embryonic malignant cancer of the cerebellum is mainly reported over 20% of all central nervous system (CNS) neoplasms in children. MB frequently affected the 3.8-6.9 per million children population in North America and Europe. MB embracing four subgroups: Sonic Hedgehog, Wingless, Group 3, and Group 4. However, the molecular and cellular factors and their mechanism in compelling the maintenance of malignancy are have not well understood. Invasion of tumor cells across the extracellular matrix to the other neighboring tissues is main key event critical for metastasis of the medulloblastoma metastasis. Several factors affect the medulloblastoma genesis, progression, invasion and metastasis. But epigenetic regulators are the crucial regulators MB progression, invasion and metastasis. In order to identify the key regulators for medulloblastoma progression, invasion and metastasis we are doing CRISPR epigenome library screening targeting more than 500 epigenetic genes and 4 guide RNAs targeting each for brain medulloblastoma cells (ONS76 and D425 cells). Initially we have transduced cells with epigenome CRISPR library with different titers to determine the effective viral titer to transfect 30% or lesser number of cells. So that each transduced cell has each guide RNA targeting each gene. Further the transduction was scaled up and after selection the cells were expanded. Further in vitro invasion assay the invaded and un-invaded cells were collected across the Matrigel matrix coated membrane. Along with further enrichment and expansion of these cells we isolated the genomic DNA and sequenced them. The present CRISPR epigenome sequencing analysis targeting invasion property of medulloblastoma cells may provide novel key epigenetic factors affecting the medulloblastoma invasion and eventually metastasis might be the novel and more effective therapeutic target for medulloblastoma treatment.

PRC2-independent actions of H3.3K27M in embryonic stem cell differentiation.

The histone H3 variant, H3.3, is localized at specific regions in the genome, especially promoters and active enhancers, and has been shown to play important roles in development. A lysine to methionine substitution in position 27 (H3.3K27M) is a main cause of Diffuse Intrinsic Pontine Glioma (specifically Diffuse Midline Glioma, K27M-mutant), a lethal type of pediatric cancer. H3.3K27M has a dominant-negative effect by inhibiting the Polycomb Repressor Complex 2 (PRC2) activity. Here, we studied the immediate, genome-wide, consequences of the H3.3K27M mutation independent of PRC2 activity. We developed Doxycycline (Dox)-inducible mouse embryonic stem cells (ESCs) carrying a single extra copy of WT-H3.3, H3.3K27M and H3.3K27L, all fused to HA. We performed RNA-Seq and ChIP-Seq at different times following Dox induction in undifferentiated and differentiated ESCs. We find increased binding of H3.3 around transcription start sites in cells expressing both H3.3K27M and H3.3K27L compared with WT, but not in cells treated with PRC2 inhibitors. Differentiated cells carrying either H3.3K27M or H3.3K27L retain expression of ESC-active genes, in expense of expression of genes related to neuronal differentiation. Taken together, our data suggest that a modifiable H3.3K27 is required for proper histone incorporation and cellular maturation, independent of PRC2 activity.

Abstract LB061: Evaluation of the in vivo efficacy of the B7-H3 targeting antibody-drug conjugate (ADC) DS7300a: A report fro the Pediatric Preclinical In Vivo Resting (PIVOT) program

Abstract Introduction: B7-H3 (encoded by the CD276 gene) is suggested to act as an immune checkpoint molecule and is highly expressed in some pediatric solid tumors. Monoclonal antibodies targeting B7-H3 (8H9 and MGA271) are well-tolerated but have demonstrated limited success in clinical trials. DS-7300a is a B7-H3 targeting ADC with a payload of DXd (an exatecan derivative that inhibits DNA topoisomerase I). DS-7300a has a drug-to-antibody ratio of 4 and it has shown promising early clinical activity in adults with advanced solid cancers (Johnson, Annals of Oncology 2021; 32:S583-S585). Methods: Xenograft models for several pediatric cancer types were selected for preclinical testing of DS-7300a based on the high rates of B7-H3 expression in these cancers. Models were dosed at 10 mg/kg administered intravenously every other week for two doses (Q2wk x 2). Different experimental designs were used for efficacy testing. For rhabdomyosarcoma, Ewing sarcoma, Wilms tumor, and other pediatric solid tumors a single mouse trial (SMT) design with 40 models was used. A cohort design (N=10) was used for efficacy testing in 6 osteosarcoma models, 3 patient-derived orthotopic xenograft glioblastoma (PDOX) models, and 3 PDOX ependymoma models. For neuroblastoma models, 10 models (2 mice per model) were tested. To evaluate treatment efficacy, objective response measures based on changes in relative tumor volume were used (Houghton, Pediatr Blood Cancer 2007;49:928-940). Results: As a single agent, DS-7300a induced a statistically significant prolongation of survival in 2 of 3 orthotopic glioblastoma and 1 of 3 ependymal tumor models. DS-7300a demonstrated high efficacy (maintained complete response, complete response, and partial response) in most non-CNS models tested: 17 of 21 rhabdomyosarcomas, 5 of 7 osteosarcomas, 7 of 10 neuroblastomas and 2 of 2 Wilms tumors. For Ewing sarcoma, most models were classified as progressive disease (9 of 15). For osteosarcoma models, the log cell kill per dose values ranged from 0.95 to 2.76 indicating high activity of the agent in these models. The activity for DS-7300a followed the general pattern of protein and RNA expression levels for B7-H3/CD276 in the models, with Ewing sarcoma models showing lower expression compared to the other models. Conclusions: DS-7300a shows tumor-regressing anticancer activity across a wide range of pediatric solid tumor models. The maintained complete remissions observed for osteosarcoma models are noteworthy, as this level of response is uncommon for these models and as osteosarcoma shows the highest B7-H3 expression among pediatric cancers. The high level of preclinical activity observed for DS-7300a combined with the promising early clinical activity observed for adult patients provide strong rationale for studying DS-7300a in children with B7-H3 expressing solid tumors. Citation Format: Richard Gorlick, E. Anders Kolb, Yifei Wang, Peter Houghton, Raushan Kurmasheva, Yael Mosse, John Maris, Matthew Tsang, David Groff, Kateryna Krytska, Xiao-Nan Li, Yuchen Du, Jun Hasegawa, Nanae Izumi, Steven Neuhauser, Anuj Srivastava, Tim Stearns, Vivek Philip, Emily L. Jocoy, Jeff Chuang, Carol J. Bult, Beverly Teicher, Malcolm Smith. Evaluation of the in vivo efficacy of the B7-H3 targeting antibody-drug conjugate (ADC) DS7300a: A report fro the Pediatric Preclinical In Vivo Resting (PIVOT) program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB061.

QOL-01. Inflammatory biomarkers and psychological sequela in pediatric brain tumor survivors

BACKGROUND AND AIMS: Pediatric brain tumors are the second most common type of pediatric cancer, and these patients face the worst health related quality of life (HRQOL) outcomes. Adult studies show increased inflammation association with lower HRQOL in adult brain tumor survivors. This relationship has not been explored in pediatric brain tumor survivors (PBTS). We conducted a case-control study to explore the relationship between inflammatory biomarkers and psychological sequela (i.e., sleep disturbance, fatigue, pain, negative affect) in PBTS. METHODS: Survivors aged 7-14 years with a primary brain tumor diagnosis were recruited from UMMC (N=29) and UAB (N=4) between 2016-2019. A control group (N=12) was recruited from UMMC well-child appointments. Parents and children completed self-reported surveys of pain, sleep, fatigue, and mood. The primary aims were to: (1) examine levels of C-reactive protein (CRP) inflammation in PBTS compared to controls (2) examine if higher CRP and SOX2 (from tumor tissue) were associated with psychological sequela. Independent samples T-Tests and spearman correlations evaluated aims. RESULTS: The final sample included 33 PBTS: median age=12.42 years; sex=51.5% female; race=63.6% Caucasian, 33.3% African American; pathologic diagnoses=67% astrocytoma/glioma, 11% medulloblastoma, 6% ependymoma, 12% other. Twelve controls had a median age=11.98 years; sex=41.7% female; race=16.7% Caucasian, 83.8% African American. There were marginal elevations in CRP for PBTS (44%, n=13) compared to controls (13%, n=1) (p=0.06). In PBTS, higher CRP levels were associated with greater parent-reported fatigue (p=0.035), sleep-wake disorders (p=0.017), excessive somnolence (p=0.042) and longer pain duration (p=0.037). From 13 tumor samples, positive SOX2 (69% of samples) was associated with increased parent-reported sleep-wake disorders (p=0.016), excessive somnolence (p=0.036), and both child and parent-reported sleep disturbance (child: p=0.014; parent: p=0.034). CONCLUSIONS: Elevated inflammation in PBTS, up to 9 years post-treatment, is consistently associated with increased sleep disturbance and fatigue. These relationships warrant further investigation in PBTS.

MODL-09. Exploring the role of Magmas (mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction) inhibition as a potential therapeutic intervention in medulloblastoma

Abstract BACKGROUND: Brain tumors are the second most common type of pediatric cancer and the leading cause of all cancer-related deaths in children. Medulloblastoma (MB) is the most common type of malignant pediatric brain tumor and has a five-year overall survival ranging from 40-75%, depending on the patient’s age and other prognostic features. There are various anti-cancer therapies against medulloblastoma, but the treatment of recurrent and refractory disease remains a challenge. As a result, the need for new and novel therapies remain a top priority. One area of interest in CNS tumors are targets within mitochondria. Magmas overexpression has been reported in multiple types of metabolically active tissue and cancer cells, including prostate cancer, pituitary adenoma, and glioma. Some new data suggest that specific subgroups of medulloblastoma may also overexpress Magmas. This ongoing study aims to examine whether Magmas inhibition by compound “BT9” could be beneficial in the treatment of medulloblastoma. METHODS: We continue to study the ability of a Magmas inhibitor (BT#9) as a therapeutic agent in stable medulloblastoma cell lines and patient-derived primary cultures by performing MTT assays, tunnel assays, flow cytometry, migration assays, and invasion assays. RESULTS: Similar to the adult GBM studies, Magmas inhibition by BT#9 had significant cytotoxic effects, causing both decreased cell proliferation, increased apoptosis, and blocked cell migration in medulloblastoma cell lines DAOY, D283, and D425. IC50s determined for each during different time points demonstrated an average range 2-5μM compared to the average range seen in adult glioblastoma cell cultures which could range up to 10 μM. These findings suggest that the inhibition of Magmas could potentially optimize clinical outcomes in recurrent/refractory medulloblastoma and warrants further investigation. Our future studies will include the determination of IC50s for primary cell cultures and in vitro testing with patient-derived xenograft models.

TBIO-04. Comprehensive analysis of mutational signatures in pediatric cancers

Abstract Analysis of mutational signatures can reveal the underlying molecular processes that have caused the somatic mutations found in genomes. We performed an extensive analysis encompassing mutational signatures of single base substitution and, for the first time, signatures of small insertions/deletions in ~800 whole-genome-sequenced tumor-normal pairs from 25 molecularly defined pediatric cancer types. More than half of this cohort consists of pediatric CNS-tumors (n=416), including high- and low-grade gliomas, ependymomas, and embryonal tumors. These were subsequently compared with COSMIC v.3 signatures to identify overlap with the latest set of known mutational signatures. We identified only a small number of mutational signatures active in pediatric cancers when compared to previously analyzed adult cancers. Amongst these, SBS1 and SBS5 were present in nearly all pediatric tumors analyzed, with a significant correlation of signature activity with age at diagnosis, as expected. Further, we found SBS21 activity in a fraction of high-grade gliomas, which is the result of defective DNA mismatch repair, SBS36 in fractions of ETMR and Group4-medulloblastoma, the result of defective base excision repair, and SBS44 in Group4-medulloblastomas, caused by defective DNA mismatch repair. For these signatures, no consistent genetic alteration was identified. We report a significantly smaller proportion of pediatric tumors which show homologous-recombination repair defect signature SBS3 compared to previously published analyses. Additionally, the previous mutational signature analysis of this cohort based on COSMIC v.2 reference signatures had identified a novel substitution signature (Signature.P1), active in the brain tumors ATRT and ependymoma. Our updated results suggest that Signature.P1 is not a pediatric specific mutational signature, but a treatment-associated signature identified in a small fraction of brain tumors that were annotated as treatment-naïve. This analysis provides a systematic overview of mutational signatures in pediatric cancers, which is relevant for understanding tumor biology and future research in defining biomarkers for treatment response.

HGG-50. Specific sensitivity of pediatric high-grade glioma with ATRX inactivation to PARP inhibitor combinations

Pediatric high-grade gliomas (pHGG) account for approximately 12% of pediatric brain tumors. Despite advances in molecular diagnosis and identification of discrete molecular subtypes, pHGG are the leading cause of cancer-related death in children. Thus, current research focuses on identifying novel therapeutic targets. Sequencing analyses across pediatric cancer types identified DNA repair perturbations as potentially targetable events in certain types of pediatric brain tumors. Herein, we investigated the potential of PARP inhibitors (PARPi), impeding the central role of PARP in DNA damage repair, in pHGG. We screened a patient-derived primary pHGG cell line panel (n=7) for their sensitivity towards 6 different PARPi (niraparib, olaparib, pamiparib, rucaparib, talazoparib, veliparib) using cell viability assays. Basal expression of DNA repair related proteins was assessed by immunoblot, and propidium iodide-based flow cytometry was used for cell cycle analysis. All pHGG were resistant towards single compound PARP inhibition. Interestingly, two H3F3A-G34R mutant pHGG models harboring inactivating ATRX mutations were characterized by elevated basal levels of pH2AX, suggesting increased stress resulting from DNA damage. Consequently, simultaneous targeting of PARP and other components of DNA repair in the respective models showed strong synergistic effects on cell viability, which was not observed to a comparable extent in other models such as BRAFV600E/TERT promotor mutant pHGG. Combination of talazoparib and irinotecan resulted in S-phase arrest. Within a precision oncology approach, we treated a 11-year-old child suffering from H3F3A-G34R mutant pHGG with ATRX mutation, that progressed during radiation, with niraparib and topotecan. The patient achieved partial remission and disease stabilization for 1 year. Taken together, PARPi combinations show potential for the treatment of pHGG with ATRX mutations. Currently, all cell models are characterized for DNA repair signatures by DNA sequencing. Further, in depth characterization of DNA damage responses upon concomitant PARP and topoisomerase inhibition in ATRX-mutated pHGG are ongoing.

A Review of Proton Therapy - Current Status and Future Directions.

The original rationale for proton therapy was the highly conformal depth-dose distributions that protons are able to produce, compared to photons, which allow greater sparing of normal tissues and escalation of tumor doses, thus potentially improving outcomes. Additionally, recent research, which is still ongoing, has revealed previously unrecognized advantages of proton therapy. For instance, the higher relative biological effectiveness (RBE) near the end of the proton range can be exploited to increase the difference in biologically effective dose in tumors vs. normal tissues. Moreover, the smaller "dose bath", i.e., the compact nature of proton dose distributions has been found to reduce exposure of circulating lymphocytes and the immune organs at risk. There is emerging evidence that the resulting sparing of the immune system has the potential to improve outcomes. Protons, accelerated to therapeutic energies ranging from 70 to 250 MeV, are transported to the treatment room where they enter the treatment head mounted on a rotating gantry. The initially narrow beams of protons are spread laterally and longitudinally and shaped appropriately to deliver treatments. Spreading and shaping can be achieved by electro-mechanically for "passively-scattered proton therapy' (PSPT); or using magnetic scanning of thin "beamlets" of protons of a sequence of initial energies. The latter technique is used to treat patients with optimized intensity modulated proton therapy (IMPT), the most powerful proton therapy modality, which is rapidly supplanting PSPT. Treatment planning and plan evaluation for proton therapy require different techniques compared to photon therapy due, in part, to the greater vulnerability of protons to uncertainties, especially those introduced by inter- and intra-fractional variations in anatomy. In addition to anatomic variations, other sources of uncertainty in the treatments delivered include the approximations and assumptions of models used for computing dose distributions and the current practice of proton therapy of assuming the RBE to have a constant value of 1.1. In reality, the RBE is variable and a complex function of proton energy, dose per fraction, tissue and cell type, end point, etc. Despite the high theoretical potential of proton therapy, the clinical evidence supporting its broad use has so far been mixed. The uncertainties and approximations mentioned above, and the technological limitations of proton therapy may have diminished its true clinical potential. It is generally acknowledged that proton therapy is safe, effective and recommended for many types of pediatric cancers, ocular melanomas, chordomas and chondrosarcomas. Promising results have been and continue to be reported for many other types of cancers as well; however, they are based on small studies. At the same time, there have been reports of unforeseen toxicities. Furthermore, because of the high cost of establishing and operating proton therapy centers, questions are often raised about the value of proton therapy. The general consensus is that there is a need for continued improvement in the state of the art of proton therapy. There is also a need to conduct randomized trials and/or collect outcomes data in multi-institutional registries to generate high level evidence of the advantages of protons. Fortuitously, such efforts are taking currently place. Ongoing research is aimed at better understanding the biological and immunomodulatory effects of proton therapy and the consequences of the physical uncertainties on proton therapy and reducing them through image-guidance and adaptive radiotherapy. Since residual uncertainties will remain despite our best efforts, in order to increase the resilience of dose distributions in the face of uncertainties and improve our confidence in dose distributions seen on treatment plans, robust optimization techniques are being developed and implemented and continue to be perfected. Such research and continuing technological advancements in planning and delivery methods are likely to help demonstrate the superiority of protons.