Central Nervous System Tumor Patients Research Articles

Abstract 6372: Germline pathogenic variants are enriched in pediatric central nervous system tumor patients and associate with somatic functional consequences

Abstract The genetic contribution of rare pathogenic germline variation to central nervous system (CNS) tumor formation in pediatric patients without a family history of cancer remains unclear. We sought to characterize the prevalence, spectrum, and clinical significance of pathogenic germline variation in cancer predisposition genes (CPGs) in 838 patients profiled in the Pediatric Brain Tumor Atlas (PBTA) by whole genome sequencing (n=821) or whole exome sequencing (n=17). Rare variants in 214 CPGs were annotated as pathogenic (P) or likely pathogenic (LP) consistent with American College of Medical Genetics criteria using AutoGVP, our open-source automated pathogenicity assessment tool. To assess enrichment of P/LP variants in the PBTA, we compared the frequency of CPG P/LP variants in cases to a tumor-free control cohort from Penn Medicine BioBank (PMBB; n=6,295). Somatic alterations, mutational signatures, and gene set enrichment analyses from matched tumor sequencing were integrated to characterize functional consequences associated with germline pathogenic variation. We observed 215 germline P/LP variants within 76 distinct CPGs in 189 (22.6%) PBTA patients. CPG P/LP variants were most prevalent among patients with a plexiform neurofibroma diagnosis (9/14, 64.3%), and least prevalent among patients with craniopharyngioma (3/39, 7.8%). We detected syndrome-associated P/LP variants in 45/58 (77.6%) patients with a clinically reported cancer predisposition syndrome and incidentally in another 37 patients. CPG P/LP variants were significantly enriched in the PBTA cohort relative to the PMBB control cohort (OR=2.0, p=1.0E-12, CI=1.6-2.4), and NF1 harbored the most significant excess P/LP variant burden (OR=30.7, p=4.57E-13, CI=11.2-105.1). We observed a significant enrichment of mismatch repair (MMR) gene P/LP variants in patients with high-grade gliomas (HGG) and tumors from MMR P/LP carriers exhibited significantly higher MMR-deficiency mutational signature exposures relative to tumors from non-carrier HGG patients (SBS14: W=478, p=1.71E-05). Overall, 90/215 (41.9%) of identified germline P/LP variants co-occurred with at least one corresponding somatic alteration in matched tumors, including oncogenic SNV (n=5), copy number variation (n=28), loss of heterozygosity (n=54), differential gene expression (n=23), and proximal alternative splicing (n=19). In summary, rare germline P/LP variants are enriched in PBTA patients and are associated with somatic events in the same gene, resulting in gene- and pathway-specific functional consequences. Our work emphasizes a role for pathogenic germline variation in pediatric CNS tumor development and frequent under-identification of cancer predisposition syndromes, underscoring the importance of germline sequencing in personalized patient care strategies. Citation Format: Ryan J. Corbett, Rebecca Kaufman, Shelly W. McQuaid, Zalman Vaksman, Saksham Phul, Miguel A. Brown, Jennifer L. Mason, Heena Desai, Ryan Hausler, Ammar S. Naqvi, Antonia Chroni, Zhuangzhuang Geng, Bo Zhang, Chuwei Zhong, Yuankun Zhu, Allison P. Heath, Marilyn Li, Penn Medicine BioBank, Phillip B. Storm, Adam C. Resnick, Kara N. Maxwell, Miriam Bornhorst, Kristina A. Cole, Angela J. Waanders, Suzanne P. MacFarland, Jo Lynne Rokita, Sharon J. Diskin. Germline pathogenic variants are enriched in pediatric central nervous system tumor patients and associate with somatic functional consequences [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6372.

Identifying brain tumor patients' subtypes based on pre-diagnostic history and clinical characteristics: a pilot hierarchical clustering and association analysis.

Central nervous system (CNS) tumors are severe health conditions with increasing incidence in the last years. Different biological, environmental and clinical factors are thought to have an important role in their epidemiology, which however remains unclear. The aim of this pilot study was to identify CNS tumor patients' subtypes based on this information and to test associations with tumor malignancy. 90 patients with suspected diagnosis of CNS tumor were recruited by the Neurosurgery Unit of IRCCS Neuromed. Patients underwent anamnestic and clinical assessment, to ascertain known or suspected risk factors including lifestyle, socioeconomic, clinical and psychometric characteristics. We applied a hierarchical clustering analysis to these exposures to identify potential groups of patients with a similar risk pattern and tested whether these clusters associated with brain tumor malignancy. Out of 67 patients with a confirmed CNS tumor diagnosis, we identified 28 non-malignant and 39 malignant tumor cases. These subtypes showed significant differences in terms of gender (with men more frequently presenting a diagnosis of cancer; p = 6.0 ×10-3) and yearly household income (with non-malignant tumor patients more frequently earning ≥25k Euros/year; p = 3.4×10-3). Cluster analysis revealed the presence of two clusters of patients: one (N=41) with more professionally active, educated, wealthier and healthier patients, and the other one with mostly retired and less healthy men, with a higher frequency of smokers, personal history of cardiovascular disease and cancer familiarity, a mostly sedentary lifestyle and generally lower income, education and cognitive performance. The former cluster showed a protective association with the malignancy of the disease, with a 74 (14-93) % reduction in the prevalent risk of CNS malignant tumors, compared to the other cluster (p=0.026). These preliminary data suggest that patients' profiling through unsupervised machine learning approaches may somehow help predicting the risk of being affected by a malignant form. If confirmed by further analyses in larger independent cohorts, these findings may be useful to create potential intelligent ranking systems for treatment priority, overcoming the lack of histopathological information and molecular diagnosis of the tumor, which are typically not available until the time of surgery.

Classification and Grading of Central Nervous System Tumors According to the World Health Organization 5th Edition.

The World Health Organization (WHO) released the 5th edition of its classification of central nervous system (CNS) tumors in 2021. Advances in the landscape of molecular tumor pathophysiology prompted major revisions to the previous edition released in 2016, some of which were first introduced by the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy-Not Official WHO (cIMPACT-NOW). The 2021 classification system integrates newly gained molecular insights to guide changes in tumor taxonomy and nomenclature, introduces several new types of tumors, and expands the use of molecular testing for diagnosis and grading, with a particular impact on adult-type and pediatric-type gliomas, ependymomas, and embryonal tumors. These updates aim to promote clear and accurate diagnoses, yield more reliable prognostic information, and enable the selection of optimal therapies. Familiarity with these changes will be of great importance for clinicians involved in the management of CNS tumor patients.

Automated Capture and Analysis of Circulating Tumor Cells in Pediatric, Adolescent and Young Adult Patients with Central Nervous System Tumors.

Tumors of the central nervous system (CNS) are the most common and lethal childhood malignancy. Detection of residual disease and longitudinal monitoring of treatment response in patients are challenging and rely on serial imaging. This current standard of care fails to detect microscopic disease or provide molecular characteristics of residual tumors. As such, there is dire need for minimally invasive liquid biopsy techniques. We have previously shown the high specificity of using cell surface vimentin (CSV) to identify circulating tumor cells (CTCs) from patients bearing various types of cancers. Here, we describe the first report of CTCs captured from peripheral blood samples in 58 pediatric CNS tumor patients. In this study, we used a CSV-coated cell capture chip, the Abnova CytoQuest automated CTC isolation system, to boost the CTC capture from pediatric patients with CNS tumors. We successfully isolated CTCs in six glioma patients using immunostaining of histone H3 lysine27-to-methionine (H3K27M) mutations which are highly expressed by this tumor. We show that CSV is a viable marker for CNS CTC isolation and that this is a feasible method for detecting microscopic disease. Larger-scale studies focusing on CTCs in pediatric CNS tumors to explore their diagnostic and prognostic value are warranted.

OTHR-10. COLLABORATIVE EFFORTS FOR HOSPITAL-BASED BIOBANK DEVELOPMENT AND OPTIMIZATION TO ENSURE QUALITY PEDIATRIC SPECIMENS FOR TRANSLATIONAL RESEARCH

Abstract In recent years, drastic improvements in sequencing technologies have led to a better understanding of the specific genetic drivers of cancer. However, one of the greatest barriers to these discoveries is access to primary patient tumor tissue, especially in cancers of the Central Nervous System (CNS). In many cases tissue from surgery is processed and stored in Formalin-Fixed Paraffin-Embedded (FFPE) blocks, which sets limitations on downstream applications. In 2015, our team launched an initiative to collect and efficiently store samples from pediatric CNS tumor patients during surgeries and autopsies. Through extensive collaboration between Neuro-Oncology, Neurosurgery, Pathology, and our Translational Laboratory, we have optimized the collection process to effectively preserve sample integrity and therefore maximize their potential usage. To date, we have collected over 4,509 samples from 293 patients (636 flash-frozen tissue, 1,026 plasma & buffy coat, 748 serum, 996 whole blood, 316 Cerebrospinal Fluid (CSF), and 787 parental blood samples). In addition, we have cultured 31 primary cell lines and collected over 242 dissociated tumor specimens for implantation into patient-derived xenograft (PDX) mouse models. Through our partnership with neurosurgery and pathology, we are permitted to attend surgeries and autopsies to immediately flash-freeze tumor tissue in liquid nitrogen, typically within minutes of the tissue being removed from the body. This immediate flash-freezing process aims to ensure specimen integrity and preserve molecular profiles. From there, the tissue is either stored in our -140oC freezer for future use or sent to partnered consortiums, including the Children’s Brain Tumor Network (CBTN) and Gift from a Child. Since the optimization of the collection process, the focus has now shifted to investigating the efficacy of our storage methods by quantifying DNA/RNA integrity over time. By ensuring the successful preservation of the samples, we can maximize their impact in the pediatric neuro-oncology research field.

Factors affecting administration of palliative care in primary central nervous system tumors: An NCDB analysis.

e24146 Background: Primary central nervous system (CNS) tumors are a diverse group of neoplasms that can vary greatly in terms of their cellular origin, histological features, clinical behavior, and prognosis. Many diagnoses leave patients with a poor prognosis and an impaired quality of life. Palliative care (PC) has long been used as a means to improve quality of life and symptoms associated with debilitating malignancies; however, its use in CNS tumors is not well characterized. Yet, the plethora of life-altering symptoms such as gait impairment, cognitive decline, motor deficits, and seizures provide a need for further multidisciplinary approaches to integrating PC with primary CNS tumor diagnoses. We aim to assess factors that influence the receipt of PC in the current data on central nervous system tumor patients and assess differences in overall survival. Methods: This study retrospectively evaluated the use of PC in patients with single primary malignant central nervous system tumors (gliomas, neuroepitheliomatous neoplasms, and meningiomas) diagnosed and recorded in the National Cancer Database (NCDB) between 2004 and 2020. Patients were identified by ICD-O-3 coding and patients with other malignancies were excluded. Multivariable logistic regression was performed to evaluate associations between patient characteristics and the use of PC, and Kaplan-Meier curves with log-rank analysis were used to determine survival for each group. Results: After excluding patients without information on utilization of PC, a total of 9295 patients with primary CNS tumors were considered in the final analysis. Overall, 1.31% received palliative care. From patients receiving palliative care, 81.1-93.4% received non-curative treatment with surgery, radiation, chemotherapy, or a combination of one or more of these modalities with additional pain management. Odds of receiving palliative care were decreased in black patients, patients who received non-palliative surgical treatment, patients with a primary tumor site at the optic chiasm, patients with unilateral tumors, and patients with private, medicaid, other government, or unknown insurance status. Odds of receiving palliative care were increased with increased Charlson-Deyo Score, in patients receiving non-palliative systemic or radiation treatment, and patients with a primary site at the spinal dura. Utilization of PC was associated with a decreased 1-year and 5-year overall survival. Conclusions: PC use in CNS tumors was influenced by tumor location, treatment type, insurance status, patient race, and comorbidity index. This study demonstrates the areas in which use of PC utilization is still lacking across a diverse set of CNS tumors and patients. Further exploration is needed into the integration of palliative care broadly across these malignancies as it still remains low despite the abundance of quality-of-life altering symptoms.

A phase 2 study of sirolimus in combination with metronomic chemotherapy (CHOAnome) in children with recurrent and/or refractory solid and CNS tumors.

10024 Background: Multiple low-dose, anti-angiogenic (metronomic) chemotherapy regimens have demonstrated activity in pediatric solid and central nervous system (CNS) tumors. The mTOR pathway is activated in many human cancers and provides an attractive therapeutic target. We previously conducted a phase 1 trial establishing the dose (2 mg/m2), safety and tolerability of sirolimus in combination with metronomic chemotherapy. In the current study, we conducted a prospective, multi-institutional phase 2 trial to determine the objective response rate (ORR) in pediatric patients with recurrent and/or refractory (R/R) solid and CNS tumors treated with this regimen (NCT02574728); herein we report the results of the CNS tumor stratum. Methods: Patients aged 12 months to 30 years with R/R CNS tumors of all world health organization (WHO) grades were eligible. After completion of the trial’s first stage, patients with diffuse intrinsic pontine glioma (DIPG) were excluded due to lack of efficacy. Patients were required to have measurable disease and no known curative therapeutic options. Treatment consisted of continuous sirolimus (2 mg/m2/dose PO daily), celecoxib (100 mg PO BID), and oral etoposide (50 mg/m2/day; max dose: 100 mg) alternating every 21 days with oral cyclophosphamide (2.5 mg/kg/day; max dose: 100 mg) in 42-day cycles. Sirolimus was dose-adjusted to maintain a serum trough concentration of 10-15 ng/ml. Response was determined using a modified Response Assessment in Neuro-Oncology (RANO) criteria. Enrollment proceeded using a Fleming’s two-stage design based on best overall response (BOR), which required two objective disease status determinations. Results: Twenty-two CNS tumor patients were enrolled; 20/22 were evaluable for response. Median age was 11.2 years (range: 5.9 – 18.1); 6 (30%) were female. Diagnoses included DIPG (N = 5), medulloblastoma (N = 3), high-grade (HG) glioma (N = 5), HG/malignant neuroepithelial tumor (N = 3) and one each of diffuse midline glioma, atypical teratoid/rhabdoid tumor (ATRT), choroid plexus carcinoma, and pineoblastoma. Median number of cycles was 2 (range: 1-6). Best response after any cycle was partial response (PR) in 4 [medulloblastoma (N = 3); pineoblastoma (N = 1)], stable disease (SD) in 4, and progressive disease (PD) in 12 for an ORR of 20%; all patients with DIPG had PD at or before their first disease evaluation. BOR was PR in 3 (15%), SD in 2 (10%), PD in 14 (70%), and unknown in 1 (5%). Four patients had SD or response for ≥ 6 months [medulloblastoma (n = 1), pineoblastoma (N = 1), and HG glioma (N = 2)]. Conclusions: The combination of sirolimus with metronomic chemotherapy showed activity in pediatric patients with some R/R CNS tumors, producing objective responses in medulloblastoma and pineoblastoma. Patients with DIPG did not benefit from this regimen and should be offered alternative treatments. Clinical trial information: NCT02574728 .

Interventions to improve neurocognitive late-effects in pediatric and adolescent CNS tumor patients and survivors - a systematic review.

Survival of children and adolescents diagnosed with central nervous system (CNS) tumors massively improved over the last decades due to better diagnostics, treatment, and supportive care. However, morbidity is still the highest of all cancer entities in this age group with neurocognitive late-effects being one of the most severe. With this systematic review, we aim to summarize interventions designed to prevent or improve neurocognitive late-effects in CNS tumor patients. We searched PubMed on August 16th 2022 and included publications studying interventions for neurocognitive late-effects in pediatric and adolescent patients and survivors diagnosed with a CNS tumor. We included any form of neurocognitive intervention during treatment or following treatment completion. We considered all types of studies except for expert opinions and case reports. The literature search resulted in 735 publications. We included 43 publications in the full text screening and 14 met our inclusion criteria. Of those, two assessed the impact of pharmacological interventions, three of exercise interventions, five of online cognitive training, and four assessed behavioral interventions. Different neuropsychological test batteries and imaging were used to measure the impact of the respective interventions. Most studies showed a positive impact of the interventions in single to several of the subtests used. We found several intervention studies indicating improvement of neurocognitive problems in children and adolescent CNS tumor survivors. In this population exercise interventions or online cognitive training might mitigate or improve neurocognitive late-effects.

Multi-institution analysis of tumor mutational burden and outcomes in pediatric central nervous system tumor patients.

Pediatric central nervous system (CNS) tumors are the leading cause of pediatric cancer mortality. Research addressing genomic biomarkers and clinical outcomes is needed to inform therapeutic decision-making. We conducted a retrospective analysis of pediatric patients (age <21) diagnosed with a primary CNS tumor at four upstate New York hospitals from 2008 to 2021. Clinical and histopathologic data were identified from each patient, including genomic analysis of somatic mutations and tumor mutational burden (TMB) where available. These variables were each compared with overall survival using Cox regression analyses. Multivariable analysis was conducted to identify patient characteristics that may independently predict survival. We identified 119 patients. Common tumor types included low-grade glioma (N=51), high-grade glioma (N=29), and medulloblastoma (N=11). Common driver mutations included TP53 inactivation (N=16), BRAF-KIAA1549 fusion (N=16), FGFR1 amplification (N=12), BRAF V600E mutation (N=12), NF1 loss (N=12), and H3F3A K28M mutation (N=6). Median TMB was one mutation/megabase (mut/Mb, range=0-132). Overall survival was 79.9%. Variables associated with poorer survival on univariable analysis were higher TMB (p=.002, HR 4.97), high-grade tumors (p=.009, HR 84.3), and high-grade glioma histology (p=.021, HR 3.14). Multivariable analyses further identified TMB (p=.011, HR 4.46) and high-grade histology (p=.015, HR 5.28) as independently predictive of worse survival. Tumor progression was more common in high-TMB (N=15, 44%) than in low-TMB tumors (N=19, 35%). High TMB is correlated with higher rates of progression and death as compared to low-TMB tumors. These findings may help identify patients who may benefit from alternative treatments, such as immunotherapies.

Evaluation of Cellular miR-122 Expression in Association with the Presence of Varicella-Zoster Virus among Central Nervous System Tumors

Background: Brain tumors are all primary central nervous system (CNS) tumors with unclear etiologies and viral infections, especially human herpesviruses, which have emerged as a hot topic for comprehensive research. Objectives: The present study aimed at assessing the molecular epidemiology of varicella-zoster virus (VZV) and its association with microRNA 122 (miR-122) expression in CNS tumor samples. Methods: Fresh frozen tissue samples were collected from 60 CNS tumor patients and 45 healthy controls. A nested PCR assay was performed to detect the VZV-DNA. Subsequently, the expression level of miR-122 was evaluated in the CNS tumor tissue samples of patients and the brain tissue samples were obtained from healthy controls, using a real-time PCR assay. Results: Of 60 patients with CNS tumors, 29 were men and 31 were women. VZV-DNA was detected in 13.3% of the CNS tumor tissue specimens. There was no statistically significant association between the presence of VZV-DNA and different types of CNS tumors (P &gt; 0.05). Furthermore, the expression level of miR-122 was significantly downregulated in the CNS tumor tissue samples obtained from the patients compared with those of the healthy controls (P &lt; 0.05). Additionally, the expression level of miR-122 was significantly lower in the VZV-positive tumor samples as compared with those of the VZV-negative tumor samples and the healthy controls. Conclusions: Although VZV plays no direct role in the development of CNS tumors, the virus may affect the biology of CNS tumors by decreasing the expression levels of miR-122, which consequently leads to an increased risk of malignancy. However, the experimental data are not conclusive enough; so, further investigations are needed.

NCOG-27. A SINGLE-INSTITUTION RETROSPECTIVE SERIES OF SARS-COV-2 INFECTION IN ADULT GLIOMA PATIENTS

Abstract A subset of cancer patients is particularly vulnerable to SARS-Cov-2 infection; however, real-world outcomes-based data on primary central nervous system tumor patients is sparse. This retrospective case series describes a cohort of adult glioma patients seen at Stanford Cancer Center between 1/20/2020 through 5/24/2022 who contracted SARS-Cov-2. We identified 18 patients with a diagnosis of glioma, with a median age of 54 years, who were infected with Covid-19. One patient contracted Covid-19 twice during this two-year period. Of these patients, four had pathology confirmed low-grade glioma, defined as oligodendroglioma or astrocytoma WHO grade 2, and 14 patients had high-grade glioma, defined as astrocytoma or glioblastoma WHO grade 4. Median KPS at time of infection was 70. Six individuals had notable cardiovascular comorbidities including coronary artery disease, obesity, and/or diabetes. All but one patient was vaccinated against Covid-19, and 6 were taking dexamethasone at the time of infection. Three patients required hospital admission for management of Covid-19 symptoms, although none required ICU-level of care. None died from Covid-related complications; however, two died from complications of their underlying cancer at the end of study. Our data suggest that glioma patients seen at Stanford Cancer Center do not experience an exceptionally high Covid-19 infectivity, hospitalization, or mortality rate, especially when compared to other vulnerable populations such as lung cancer patients. High vaccination rates, fairly low prevalence of cardiovascular comorbidities, and adherence to pandemic precautions among this cohort may have contributed to these results.

Quality of life, out-of-pocket expenditures, and indirect costs among patients with the central nervous system tumors in Thailand.

The aim of this study was to investigate out-of-pocket (OOP) expenditures, indirect costs, and health-related quality of life (HRQoL) associated with the central nervous system (CNS) tumors in Thailand. A prospective study of CNS tumor patients who underwent first tumor resection at a tertiary care institution in Thailand was conducted. Patients were interviewed during hospitalization for undergoing first surgery. Within 6 months, they were interviewed once more if the disease continued to progress. Costs collected from a patient perspective and converted to 2019 US dollars. For dealing with these skewed data, a generalized linear model was used to investigate the effects of disease severity (malignancy, progressive disease, Karnofsky performance status score, and histology) and other factors on costs (OOP, informal care, productivity loss, and total costs). P < 0.05 was considered statistical significant for all analysis. Among a total of 123 intracranial CNS tumor patients, there were 83 and 40 patients classified into benign and malignant, respectively. In the first brain surgery, there was no statistical difference in HRQoL between patients with benign and malignant tumors (P = 0.072). However, patients with progressive disease had lower HRQoL mean scores at pre-operative and progressive disease periods were 0.711 (95% confidence interval [CI]: 0.662-0.760) and 0.261 (95% CI: 0.144-0.378), respectively. Indirect expenditures were the primary cost driver, accounting for 73.81% of annual total costs. The total annual costs accounted for 59.81% of the reported patient's income in malignant tumor patients. The progressive disease was the only factor that was significantly increases in all sorts of costs, including the OOP (P = 0.001), the indirect costs (P = 0.013), and the total annual costs (P = 0.001). Although there was no statistical difference in HRQoL and costs between patients with benign and malignant tumor, the total costs accounted for more than half of the reported income in malignant tumor patients. The primary cause of significant increases in all costs categories was disease progression.

Changes in survival over time for primary brain and other CNS tumors in the United States, 2004–2017

PurposeDespite advances in cancer diagnosis and clinical care, survival for many primary brain and other central nervous system (CNS) tumors remain poor. This study performs a comprehensive survival analysis on these tumors.MethodsSurvival differences were determined utilizing the National Program of Cancer Registries Survival Analytic file for primary brain and CNS tumors. Overall survival and survival of the 5 most common histopathologies, within specific age groups, were determined. Overall survival was compared for three time periods: 2004–2007, 2008–2012, and 2013–2017. Survival differences were evaluated using Kaplan–Meier and multivariable Cox proportional hazards models. Models were adjusted for sex, race/ethnicity, and treatment. Malignant and non-malignant brain tumors were assessed separately.ResultsAmong malignant brain and CNS tumor patients overall, there were notable differences in survival by time period among all age groups. Similar differences were noted in non-malignant brain and CNS tumor patients, except for adults (aged 40–64 years), where no survival changes were observed. Survival differences varied within specific histopathologies across age groups. There were improvements in survival in 2008–2012 and 2013–2017, when compared to 2004–2007, in children, AYA, and older adults with malignant tumors, and among older adults with non-malignant tumors.ConclusionOverall survival for malignant brain and other CNS tumors improved slightly in 2013–2017 for all age groups as compared to 2004–2007. Significant changes were observed for non-malignant brain and other CNS tumors among older adults. Information regarding survival over time can be utilized to identify population level effects of diagnostic and treatment improvements.

Abstract 5110: Development and validation of a liquid biopsy assay with cerebrospinal fluid derived cell free DNA using droplet digital PCR for clinical applications

Abstract Background: Cerebrospinal fluid (CSF) has been recognized as an important biofluid for biomarker analysis of central nervous system (CNS) tumors due to the proximity to the disease/tumor site as compared to serum or plasma. CNS tumors have significantly enriched tumor-derived cfDNA/circulating tumor DNA (ctDNA) in CSF, however, the absolute quantities are low. Therefore, highly sensitive targeted methods such as next generation sequencing or digital droplet PCR are required to efficiently detect mutations. Recent studies have suggested the feasibility of detecting H3K27M in CSF and the potential of these mutations as biomarkers in CNS malignancies. We report the establishment of a protocol for CSF collection, cfDNA extraction and proof of concept study for the analysis of H3K27 mutations as potential clinical biomarkers for CNS tumors. Methods: CSF was collected from healthy donors (n=10) via lumbar puncture. cfDNA was isolated from approximately 2-3mL CSF from each donor using the Qiagen Circulating Nucleic Acid kit and quantified using the Qubit High Sensitivity DNA kit. Five normal donors were first tested for cfDNA recovery from CSF and then the remaining five were pooled and tested via a spike-in recovery experiment of KRAS G12D mutation ddPCR detection assay. Primer/probes specific to wildtype H3F3A/HIST1H3B and mutant to K27M were designed for ddPCR testing with cfDNA from the CSF of CNS tumor patients (n=10). Results: cfDNA was successfully isolated from CSF of five healthy samples. The mean cfDNA concentration was 7.6ng DNA per mL CSF with a range of 3.89-11.27 ng/mL CSF. The low yield of cfDNA further confirm the need for an extremely sensitive detection method for biomarkers in CSF. Synthetic DNA for the KRAS G12D mutation was spiked into a pool of healthy volunteer CSF at quantities as low as 1fg and then measured successfully downstream in ddPCR, illustrating the ability of this workflow to recover and measure very low levels the mutant DNA. The 1fg spike in amount yielded an average of 0.77copies/uL. Multiple ddPCR assays were designed for the HIST1H3B assay but after several iterations, no successful assay could be established for this mutation: a result consistent with other reported findings. However, an assay to measure H3F3A p.K27M was identified and is currently being optimized. Conclusion: Collectively, we demonstrated the use of CSF as a matrix for biomarker testing in a clinical setting. Using commercial kits, ctDNA can be extracted from CSF and used downstream in biomarker detection assays. On-going work includes expanding the tested cohort to CNS tumor samples using a developed ddPCR assay to detect the H3F3A p.K27M mutation. We anticipate the establishment of a reliable CSF cfDNA detection method to enable the study of CNS tumor for genomic evolution, minimal residual disease monitoring and the characterization of tumors at recurrence. Citation Format: Dennis J. O'Rourke, Danyi Wang, Zheng Feng. Development and validation of a liquid biopsy assay with cerebrospinal fluid derived cell free DNA using droplet digital PCR for clinical applications [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 5110.

ETMR-11. Transcriptional changes upon knockdown of alteredBCOR/BCORL1 transcripts in preclinical models of CNS embryonal tumors with BCOR-related alterations

BCL-6 transcriptional corepressor (BCOR) is an epigenetic regulator that silences gene expression mainly via the polycomb repressive complex 1.1 (PRC1.1). BCOR genomic alterations are found in a variety of different tumors and recently central nervous system (CNS) tumors with BCOR internal tandem duplication (ITD) were classified as a distinct molecular subgroup. We established and characterized two cell models derived from BCOR altered CNS tumor patients. One model is characterized by a frameshift mutation in the BCOR gene resulting in the expression of a truncated protein lacking the C-terminal PUFD domain required for correct assembly of the PRC1.1. Additionally, this model harbors a translocation of the BCOR homologue BCORL1. The second model has a characteristic internal tandem duplication (ITD) within the BCOR gene. To study the effects of mutated BCOR/BCORL1 on gene expression, we performed siRNA mediated knockdown of altered BCOR/BCORL1 transcripts in both models and analyzed transcriptional changes by mRNA expression array. Differentially expressed genes in BCOR/BCORL1 knockdown versus wild type conditions were enriched for signaling pathways involved in cell cycle progression, cell growth, DNA replication and cancer. This suggests that the alterations in BCOR/BCORL1 might have pro-oncogenic effects and thereby contribute to the aggressive phenotype of this disease. Especially in the BCOR ITD model knockdown of BCOR led to transcriptional downregulation of genes associated with the development of brain tumors such as FGF18, PDGFA and PDGFRA. Our results indicate that specific BCOR/BCORL1 alterations might impair its endogenous function as transcriptional repressor and deregulate the expression of multiple PRC1.1 target genes. An in depth characterization of epigenetic and transcriptional changes in BCOR/BCORL1 altered CNS tumors could lead to the identification of critical downstream effectors and ultimately reveal new therapeutic vulnerabilities.

Major Features of the 2021 WHO Classification of CNS Tumors.

Advances in the understanding of the molecular biology of central nervous system (CNS) tumors prompted a new World Health Organization (WHO) classification scheme in 2021, only 5years after the prior iteration. The 2016 version was the first to include specific molecular alterations in the diagnoses of a few tumors, but the 2021 system greatly expanded this approach, with over 40 tumor types and subtypes now being defined by their key molecular features. Many tumors have also been reconceptualized into new "supercategories," including adult-type diffuse gliomas, pediatric-type diffuse low- and high-grade gliomas, and circumscribed astrocytic gliomas. Some entirely new tumors are in this scheme, particularly pediatric tumors. Naturally, these changes will impact how CNS tumor patients are diagnosed and treated, including clinical trial enrollment. This review addresses the most clinically relevant changes in the 2021 WHO book, including diffuse and circumscribed gliomas, ependymomas, embryonal tumors, and meningiomas.

BIOM-19. DECIPHERING THE METHYLATION SIGNATURE OF CIRCULATING EXTRACELLULAR VESICLE DNA FOR CNS TUMOR CLASSIFICATION

Abstract Genome-wide methylation profiling has recently been developed into a tool that allows subtype tumor classification in central nervous system (CNS) tumors. We previously showed that extracellular vesicle (EV) DNA faithfully reflects the tumor methylation class, including information on the IDH mutation and MGMT promoter methylation status. Furthermore we showed that circulating plasma EVs are elevated in CNS tumor patients in comparison to non-tumor donors (HD) controls with tumor related protein profiles. We now investigated, whether the methylation signatures of circulating DNA (both EV and cfNDA) can be used in liquid biopsy approaches for CNS tumor detection and classification. We isolated DNA from circulating EVs (n=27), cfDNA (n=27) and tumor tissue DNA (n=90) of patients with glioblastoma (GBM), meningioma (MGN) and cerebral metastases (CM). Patients undergoing epilepsy surgery as well as aneurysm clipping were used as non-tumor controls (HD, n= 7). EVs were classified by nanoparticle analysis, immunoblotting, imaging flow cytometry and electron microscopy. Isolated EV-DNA comprised many sorts of molecular weight (up tp &amp;gt;10Kb) in comparison to cfDNA (130-140bp). Healthy donors and tumor patients showed not differences in their DNA size profiles. We performed genome-wide methylation profiling by 850k Illumina EPIC arrays for all DNA analytes and tumor entities. Linear models and empirical Bayes methods identified significant differentially methylated CpGs (GBM vs. HD, MGN, vs HD, CM vs. HD), that revealed tumor specific signatures to detect and discriminate different CNS tumor entities. Visualization of differentially methylated CPGs by dimension reduction (PCA, t-SNE, Umap) verified tumor specific clusters. cfDNA and EV-DNA exhibited distinctive individual CpG profiles. Our study shows that the methylation signature of circulating EV DNA and cfDNA can be used to separate healthy individuals from tumor patients and could potentially complement standard-of-care imaging to improve tumor detection, classification and surveillance.

Residential distance from the reporting hospital and survival among adolescents, and young adults diagnosed with CNS tumors.

Prior research shows that residential distance to a treatment facility may be an important factor in central nervous system (CNS) tumor outcomes. Our goal was to examine residential distance to the reporting hospital and overall survival in adolescents and young adults (AYA) diagnosed with CNS tumors. National Cancer Database data on AYA 15-39years old diagnosed with CNS and Other Intracranial and Intraspinal Neoplasms (CNS tumors) from 2010 to 2014 were obtained. Distance between the case's residence at diagnosis or initial treatment and the reporting hospital was classified in miles as short (≤ 12.5), intermediate (> 12.5 and < 50), and long (≥ 50). Cox proportional hazards regression models were used for analyses. Among 9335 AYA diagnosed with CNS tumors, hazard ratios (HRs) were 1.06 (95% CI 0.96-1.17) and 0.82 (95% CI 0.73-0.93) for those with residences at intermediate and long vs. short distances, respectively, after adjusting for age, sex, race/ethnicity, and zip-code level education and income. After adjusting for the facility volume of CNS tumor patients, the association was attenuated for long vs. short distance residences (HR 0.92, 95% CI 0.81-1.04). The HRs varied by tumor type, race/ethnicity, and zip-code level income with significantly lower hazards of death for those with residences at long vs. short distances for low-grade astrocytic tumors, ependymomas, non-Hispanic Whites, and those from higher-income areas. Living at long distances for CNS tumor care may be associated with better survival in AYA patients. This may be explained by travel to facilities with more experience treating CNS tumors.

Unexpected Binding of Tozuleristide "Tumor Paint" to Cerebral Vascular Malformations: A Potentially Novel Application of Fluorescence-Guided Surgery.

Fluorescence-guided surgery (FGS) is under investigation as a means to improve the extent of resection for primary central nervous system (CNS) tumors. Tozuleristide, known also as "Tumor Paint," is an investigational tumor-targeting agent covalently conjugated to a derivative of the fluorescent dye indocyanine green. To report the finding of avid intraoperative fluorescence of tozuleristide on cerebral vascular malformations. Our institution is participating in a phase 2/3 study of intraoperative near-infrared fluorescence detection of pediatric primary CNS tumors in patients receiving intravenous tozuleristide and imaged with the Canvas system. Our site enrolled 2 patients with intracranial lesions, suspected preoperatively of possibly being gliomas that proved to be cavernous vascular malformations after resection. Each lesion had a dark blue mulberry appearance and each fluoresced avidly with tozuleristide. Each was completely resected, and the patients recovered without deficit. Pathological assessment showed cavernous angioma for both cases. Tozuleristide fluorescence is postulated to result from binding to matrix metalloproteinase-2 and annexin A2, and literature review demonstrates expression of both these ligands on multiple cerebrovascular lesions, including cavernous malformations. This finding deserves further investigation to determine if tozuleristide "Tumor Paint" may have a wider role in the identification of non-neoplastic intracranial pathologies.

Profile of Coagulation Marker and The Influence Factors in Central Nervous System Tumor

Brain tumors have the highest percentage of thrombosis compared to other types of malignancies. Hypercoagulation is a risk for thromboembolic events in patients with malignancy. Hypercoagulation conditions are frequently found in patients with malignancy arising from the aptitude of tumor cells to activate the coagulation system. This condition can be detected through coagulation markers in the blood. This marker is PT, APTT, INR, Fibrinogen and D-Dimer. This research is a descriptive analytic study using secondary data from medical records of patients with central nervous system (CNS) tumors. The inclusion criteria were CNS tumor patients &gt; 18 years old and had been diagnosed with CNS tumor. Coagulation markers are recorded based on the results of the initial laboratory examination upon entering the hospital, consisting of D-Dimer, PT, APTT, Fibrinogen, INR. There were 124 research subjects, consisting of 60 men and 64 women. Most research subjects are in the age range of 40-49 years (54.8%). Hypercoagulation conditions were found in 92.74% of study subjects. The most common accompanying condition was hypertension (22.6%). There were no significant differences in the hypercoagulable conditions in groups with and without accompanying conditions. Hypercoagulation was found in most research subjects. There are no factors that influence the hypercoagulation condition in this study.