Abstract Background Dordaviprone (ONC201) is the first FDA-approved therapy for H3K27M-mutant diffuse midline glioma (DMG). However, therapeutic response varies among patients, and no imaging biomarkers currently predict long-term benefit. Diffusion-weighted MRI and apparent diffusion coefficient (ADC) obtained during standard clinical imaging reflects tissue cellularity and necrosis, offering potential as an early response indicator. Methods Clinical and imaging data from patients treated with dordaviprone (n = 83) on completed studies underwent centralized neuroradiology review, ADC histogram analysis, and Functional Diffusion Mapping (fDM), which were compared and correlated to CSF tumor DNA (tDNA) and metabolomics when available. Final analysis included 38 patients receiving standardized dordaviprone dosing and treatment schedule. Results In the pre-progression cohort, early changes in volumetric or cross-sectional RAPNO criteria were not predictive of overall (OS) or progression-free survival (PFS). Importantly, in patients treated pre-progression with dordaviprone post-radiation, tumors with increased diffusion ADC (reduced cellularity) at cycle 3 demonstrated median OS of 732 days versus 360 days for those with decreased ADC (increased cellularity, p = 0.0149); with corresponding PFS of 334 versus 110 days (p = 0.0132). fDM analysis of spatio-temporal changes of ADC did not correlate with outcomes in a sub-cohort analysis but demonstrated predictive patterns in individual patient time course series. CSF metabolomics stratified by ADC change revealed differences in reductive stress metabolites among dordaviprone responders, linking ADC elevation to dordaviprone -driven metabolic effects. Conclusion Early increase in ADC correlates with improved response to dordaviprone in H3K27M-DMG and correlates with CSF metabolic changes, suggesting ADC warrants prospective validation as a biomarker.

PDGFRA alterations define a high-risk subset of high-grade glioma (HGG), yet targeted therapies have yielded limited and transient benefit. Here, we show that the CNS-penetrant PDGFRA inhibitor avapritinib induces sustained MAPK pathway activation at supratherapeutic dosing, revealing a therapy-induced adaptive vulnerability. High-dimensional kinome profiling (>900 nodes) and in vivo studies demonstrate robust, dose-dependent ERK activation following avapritinib treatment. This response is enriched in cycling oligodendrocyte precursor cell-like (OPC-like) tumor populations and promotes survival through ERK-dependent stabilization of the anti-apoptotic protein MCL-1. Modeling of clinically relevant PDGFRA variants reveals that D842V-mutant tumor cells exhibit heightened MAPK activation and potential for MAPK co-targeting. Rational combination strategies suppress this adaptive signaling, with MEK inhibition producing durable pathway suppression and significant survival benefit in vivo. Translation to patients demonstrates feasibility and early clinical activity, including a sustained complete regression in an unresected PDGFRA-mutant HGG treated with avapritinib and the MEK1/2 inhibitor selumetinib. Together, these findings identify adaptive MAPK reactivation as a targetable liability and support combined PDGFRA-MAPK inhibition as a therapeutic strategy.

Traditional cell culture models are often infeasible and fail to adequately capture the tumor microenvironment of pediatric brain tumors. To address this gap, we developed a live bio-bank of patient-derived tumor-oids (PDTs) without single-cell dissociation to maintain the 3D architecture and microenvironment of pediatric brain tumors. Within multiple sites at the Children’s Brain Tumor Network (CBTN), we successfully established 125 PDTs from over 30 pediatric brain tumor types that underwent tumor resection, biopsy, or autopsy. The vast majority (96%) of PDTs from surgical resections demonstrated feasibility, defined as rounded viable organoids after 4 weeks, including previously difficult or impossible to culture low grade brain tumor types. Feasibility was optimized for: (1) tissue direct from OR, (2) storage in cold transport media for up to 48 hours, and (3) freeze/thaw with subsequent re-growth. Assessment of low-grade glioma PDTs demonstrated CD3 positive T cells in early passage PDTs (<4 weeks) and non-tumor neurons (MAP2+) and microglia (IBA1+) in late passage PDTs (4-8 weeks+). We performed personalized panel drug screens to rapidly identify tumor-specific drug sensitivities. To date, 15 PDTs have undergone individualized drug screens within 4 weeks of resection, revealing actionable vulnerabilities that correlated with molecular findings. In one case, a H3K27M-DMG PDT showed unique low nM sensitivity to selinexor; the patient was independently treated with selinexor for 12 months+ with ongoing remission. Ongoing work includes: (1) multi-omic comparisons of tumor tissue, early and late passage PDTs, and subsequent (tumor only) cell lines in DMG, CNS sarcoma, ependymoma and LGG/DNET models, and (2) integration of PDT-drug screen across homogenously treated target validation cohorts of patients in multi-site clinical trials (e.g. PNOC041). In summary, our work establishes the technical and biological feasibility of a precision medicine pipeline, allowing unprecedented access to living tumor tissue across the entire spectrum of pediatric brain tumors.

Diffuse intrinsic pontine glioma (DIPG) is a rare but extremely malignant central nervous system tumor primarily affecting children that is almost universally fatal with a devastating prognosis of 8-to-12-month median survival time following diagnosis. Traditionally, DIPG has been diagnosed via MR imaging alone and treated with palliative radiation therapy. While performing surgical biopsies for these patients has been controversial, in recent years, advancements have been made in the safety and efficacy of surgical biopsy techniques, utilizing stereotactic, robotics, and intraoperative cranial nerve monitoring as well as the development of liquid biopsies that identify tumor markers in either cerebrospinal fluid or serum. With more molecular data being collected from these tumors due to more frequent biopsies being performed, multiple treatment modalities including chemotherapy, radiation therapy, immunotherapy, and epigenetic modifying agents continue to be developed. Numerous recent clinical trials have been completed or are currently ongoing that have shown promise in extending survival for patients with DIPG. Focused ultrasound (FUS) has also emerged as an additional promising adjunct invention used to increase the effectiveness of therapeutic agents. In this review, we discuss the current evidence to date for these advancements in the diagnosis and treatment of DIPG.

Blood-based liquid biopsies are poised to revolutionize brain cancer diagnosis and monitoring. Recent studies have shown that circulating tumor DNA (ctDNA) can be detected in blood via droplet digital PCR (ddPCR), but due to low ctDNA burden (generally <0.5%) and difficulty multiplexing, timely detection and precise quantification of clinically relevant ctDNA changes using ddPCR remains challenging. To solve these problems, we have developed tail-ligated dsDNA recombinase polymerase amplification sequencing (TLDRseq), a sequencing-based assay using (1) targeted PCR, (2) intra-dsDNA 5’-to-3’ tail ligation, (3) isothermal hairpin amplification, (4) Oxford Nanopore sequencing, and (5) informatic error correction. We designed TLDRseq with the goal of developing an easy-to-perform, low error rate, multiplexable assay that is easily adoptable by clinical/academic labs even in low-resource settings. We established a biobank of 165 serial blood samples for analysis from 22 pediatric patients with primary brain tumors (4-24 timepoints and 2-8 targets/patient) undergoing treatment. Serial samples from a cohort of five patients with known H3K27M+TP53/BRAF disease (n=32/64 samples/targets) were sequenced and compared to prior ddPCR results. TLDRseq generates ultra-deep (>100,000 reads/target) and highly sensitive results (75% for all targets (n=48/64) and 100% per timepoint). Results strongly correlated with ddPCR (r=.9957; P (two tailed) <.0001), while offering an exceptionally low limit of detection for H3.3K27M (~0.004% LOD). As an example of multiplex tumor evolution tracking, a patient with H3K27M/BRAFV600E DMG showed consistently positive BRAFV600E signal and negative H3K27M signal in blood. After targeted dabrafenib therapy BRAF signal was eventually lost (later confirmed lost at autopsy), while H3K27M signal spiked ~1-month prior to radiographic progression. Ongoing work will complete cohort analysis of 83 unique mutations over time with clinical/radiographic and paired tumor sequencing comparisons. This work represents exciting progress towards routine, low-cost, rapid, sensitive detection, and precise quantification of ctDNA in brain tumor plasma, and will increase our understanding of disease evolution under treatment.

Abstract PDGFRA is a frequently altered gene in pHGG, driving aggressive behavior and worse prognoses. Avapritinib, a potent CNS-penetrant PDGFRA inhibitor, has shown promise in vitro, in vivo, and in pHGG patients. Given the failure of single-agent trials in targeting PDGFRA-altered HGG, combinatorial therapy is likely needed with other targetable pathways for treatment. We performed a high-throughput kinase-activity mapping (HT-KAM) screen to detect the catalytic activity of > 900 kinase-substrate nodes in our pHGG models. These results demonstrated that supraphysiological doses of > =1uM avapritinib treatment of PDGFRA-altered pHGG cells in vitro results in sustained activation of the MAPK pathway. Specifically, short-term avapritinib treatment with > =1uM doses resulted in MEK/ERK (MEK2) and MEK/JNK (MKK4/7) activation, and long-term treatment resulted in sustained MEK/ERK (MEK2) activation across all models. Dose-dependent pERK upregulation in response to avapritinib was confirmed in multiple pHGG in vitro and in vivo models. Single-cell RNA-seq analysis of avapritinib-treated pHGG tumors in vivo demonstrated that cycling OPC-like cells were primarily responsible for increased expression of MAPK pathway genes. Furthermore, upregulation of the ERK-driven anti-apoptotic protein MCL-1 was found in short-term avapritinib-treated pHGG cells in vitro. Combinatorial treatment of pHGG models with MEK (trametinib), ERK (ulixertinib) and integrated stress/ERK inhibitors (ONC201, ONC206) in vitro eradicated pERK activity. Trametinib demonstrated the strongest combinatorial survival benefit among preliminary results in PDGFRA-driven pHGG models in vivo. We subsequently showed in vitro synergy between avapritinib and trametinib in a tumor-derived organoid from a pediatric patient with PDGFRA D842V-mutant metastatic CNS sarcoma that grew on avapritinib. This patient was later treated with this combination and demonstrated stability for five months. Combinatorial therapy with avapritinib and a MEK inhibitor was performed in four additional PDGFRA-driven pHGG patients. In light of sustained MAPK activation identified in our study, dual avapritinib-MAPK targeted treatment may be an effective approach for PDGFRA-driven pHGG.

PDGFRA is crucial to tumorigenesis and frequently genomically altered in high-grade glioma (HGG). In a comprehensive dataset of pediatric HGG (n = 261), we detect PDGFRA mutations and/or amplifications in 15% of cases, suggesting PDGFRA as a therapeutic target. We reveal that the PDGFRA/KIT inhibitor avapritinib shows (1) selectivity for PDGFRA inhibition, (2) distinct patterns of subcellular effects, (3) in vitro and in vivo activity in patient-derived HGG models, and (4) effective blood-brain barrier penetration in mice and humans. Furthermore, we report preliminary clinical real-world experience using avapritinib in pediatric and young adult patients with predominantly recurrent/refractory PDGFRA-altered HGG (n = 8). Our early data demonstrate that avapritinib is well tolerated and results in radiographic response in 3/7 cases, suggesting a potential role for avapritinib in the treatment of HGG with specific PDGFRA alterations. Overall, these translational results underscore the therapeutic potential of PDGFRA inhibition with avapritinib in HGG.

The analysis of cell-free tumor DNA (ctDNA) and proteins in the blood of patients with cancer potentiates a new generation of non-invasive diagnostic approaches. However, confident detection of tumor-originating markers is challenging, especially in the context of brain tumors, where these analytes in plasma are extremely scarce. Here, we apply a sensitive single-molecule technology to profile multiple histone modifications on individual nucleosomes from the plasma of patients with diffuse midline glioma (DMG). The system reveals epigenetic patterns unique to DMG, significantly differentiating this group of patients from healthy subjects or individuals diagnosed with other cancer types. We further develop a method to directly quantify the tumor-originating oncoproteins, lysine 27 to methionine substitution in histone H3 (H3-K27M) and mutant p53, from <1 mL of plasma, allowing for the accurate molecular classification of patients with DMG. We show that our strategy correlates with MRI and droplet-digital PCR (ddPCR) measurements of ctDNA, highlighting the clinical potential of single-molecule-based, multi-parametric assays for DMG diagnosis and treatment monitoring.

Abstract BACKGROUND TP53 and IDH mutations are common in brain tumors and are highly relevant biomarkers for diagnosis and treatment. Thus, biopsy tissue is commonly tested for these mutations via immunohistochemistry (IHC), which is rapid and generally considered highly sensitive. However, IHC only tests for IDH1R132H (80%-90% of IDH mutant disease) and rarer IDH mutations are not covered. p53 nuclear staining is used to identify non-specific p53 upregulation and loss of function but does not test for specific mutations and has a relatively lower overall sensitivity/specificity of ~80%/80-97%. Sequencing can resolve all mutations and improve over IHC, but long turnaround time and high cost prevents this. New advances in rapid sequencing technologies allow same-day, low-cost panel sequencing. However, access to biopsy tissue is zero-sum and obtaining additional tissue for molecular diagnostics confers increased morbidity. We investigate whether tumor DNA recovered from surgical waste (specifically core biopsy needle fluid and non-adherent gauze) is sufficient to diagnose IDH/p53 status of tumors. METHODS Surgeons were instructed to save biopsy needle rinse saline, and/or to recover any non-adherent gauze used in specimen handling. Cell-free DNA was extracted and sequenced using a same-day targeted panel protocol and/or TruSight Oncology 500 panel sequencing. RESULTS 100% of recovered samples yielded enough DNA for targeted sequencing (17/17). In all IDH1R132H IHC positive cases, waste sequencing recapitulated the mutation (4/4). In 7/9 p53 positive cases via IHC (>10% staining), waste sequencing of common hotspot loci was able to identify a specific TP53 mutation. Importantly, sequencing identified alternate IDH mutations in three IHC negative cases (3/13), discovered a rarer TP53 mutation (E286K) in one IHC negative case, and confirmed TP53 negative status in one indeterminate p53 report. CONCLUSIONS Surgical waste is an important diagnostic resource that should be leveraged for rapid diagnostic sequencing. Sufficient DNA can be easily recovered from surgical waste and sequenced within the same time-frame–but with increased sensitivity–to IHC.

All supplementary data except table S3A and S3B

Abstract BACKGROUND Brain tumor classification has increasingly integrated molecular diagnostics into official criteria. However, biopsy tissue is limited due to the small quantity acquired in eloquent areas of the brain and is, therefore, usually earmarked for histology. We have identified that the fluid used to wash biopsy needles contains excess tumor material that can be diagnostically useful. Characterization of this non-zero-sum waste material provides an opportunity for improvement in the diagnosis and treatment for brain tumor patients. METHODS For suspected or established high grade brain tumor biopsies (n = 37 across a multi-institutional cohort at submission), neurosurgeons were instructed to save fluid used to wash biopsy needles. Samples were processed and characterized in a variety of methods to establish diagnostic utility including (1) cell-free DNA isolation and targeted panel sequencing (n = 13), (2) isothermal amplification of cell-free DNA from raw wash water for rapid diagnostics (n = 2), (3) cell-line generation (n = 24) and, (4) cell-free mutant protein capture and quantification (n = 9). RESULTS (1) 13 wash samples were considered for cf-tDNA extraction and Illumina TruSight Oncology 500 panel sequencing (with extracted DNA ranging from 20ng-40ug). In 2 returned cases (11 pending), concordance was 100% with clinical targeted panel sequencing where panels overlapped. (2) One case suspected of harboring H3K27M was subjected to targeted, loop-mediated isothermal amplification and Oxford Nanopore real-time sequencing, confirming an H3.3K27M diagnosis (11.8% allele fraction) within 1hr35min of receipt of sample from the operating room. (3) 5 cases resulted in primary cell culture generation (4) A novel single-molecule mutant protein quantification technique was performed on 9 cases. RESULTS from all analyses and concordance with outstanding sequencing will be presented. CONCLUSION We demonstrate that biopsy byproducts contain enough high-quality tumor-derived material to perform a variety of useful diagnostics with the potential to have immediate translational impact on standard of care.

ReadUntil enables Oxford Nanopore Technology’s (ONT) sequencers to selectively sequence reads of target species in real-time. This enables efficient microbial enrichment for applications such as microbial abundance estimation and is particularly beneficial for metagenomic samples with a very high fraction of non-target reads (> 99% can be human reads). However, read-until requires a fast and accurate software filter that analyzes a short prefix of a read and determines if it belongs to a microbe of interest (target) or not. The baseline Read Until pipeline uses a deep neural network-based basecaller called Guppy and is slow and inaccurate for this task (∼60% of bases sequenced are unclassified). We present RawMap, an efficient CPU-only microbial species-agnostic Read Until classifier for filtering non-target human reads in the squiggle space. RawMap uses a Support Vector Machine (SVM), which is trained to distinguish human from microbe using non-linear and non-stationary characteristics of ONT’s squiggle output (continuous electrical signals). Compared to the baseline Read Until pipeline, RawMap is a 1327X faster classifier and significantly improves the sequencing time and cost, and compute time savings. We show that RawMap augmented pipelines reduce sequencing time and cost by ∼24% and computing cost by ∼22%. Additionally, since RawMap is agnostic to microbial species, it can also classify microbial species it is not trained on. We also discuss how RawMap may be used as an alternative to the RT-PCR test for viral load quantification of SARS-CoV-2. Availability and implementation Software is released with MIT License and available on GitHub: https://github.com/harisankarsadasivan/RawMap

Background: Over the past decade, the WHO classification of brain tumors has increasingly integrated molecular diagnostics into official classification criteria. However, biopsy tissue is precious due to high morbidity associated with repeat biopsies, and usually earmarked for histology, leaving little or no tissue for clinical molecular diagnostics. We have identified that the fluid used to wash biopsy needles after cores contains excess tumor DNA and cells that can be diagnostically useful. Additional research of tumor DNA and tumor tissue recovered from biopsy needle washes provides an opportunity for improvement in the diagnosis and treatment for brain tumor patients, while utilizing material that is otherwise thrown away. Methods: In two cases, surgeons were instructed to save 5-15ml of fluid used to wash biopsy needles. Isothermal targeted amplification of H3.3 and H3.1 histone genes was performed on raw wash fluid. Resulting amplicons were sequenced using an Oxford Nanopore MinION. DNA extracted from wash fluid was subjected to NGS sequencing via the Illumina TruSight Oncology 500 panel and compared to a CLIA certified NGS sequencing results from clinically indicated biopsy tissue. Results: For case 1, 5ul of raw wash fluid was subjected to rapid Loop-mediated Isothermal Amplification (LAMP) targeting the H3.1 and H3.3 K27M histone mutations and immediately sequenced using a MinION sequencer via rapid library preparation. Sequencing revealed a H3.3 K27M histone mutation present in wash fluid at 12% variant allele fraction (VAF). The variant call (99.99% CI) was available 1hr5mins after acquisition of material. For case 2, 5ml of wash fluid was processed using a cell-free DNA extraction kit resulting in ~250ng of recovered DNA. A LAMP assay targeting the H3.3 K27M mutation was performed on extracted DNA from wash fluid. Sequencing revealed an H3.3 K27M histone mutation present at 55% VAF, which was later corroborated using digital droplet PCR. Clinically indicated tissue from both biopsies was submitted for cancer panel sequencing in a CLIA certified setting. DNA extracted from wash fluid from both cases was subjected to Illumina TruSight Oncology 500 panel sequencing. In both cases, the panel was able to corroborate all SNV mutations reported by clinical sequencing that were also covered by the panel. Accrual of needle wash specimens is ongoing, with multiple monthly procedures. Conclusions: Large amounts of diagnostically useful material is present in biopsy needle wash fluid, which is usually discarded. Isothermal assays coupled with MinION sequencing can rapidly identify clinically relevant point mutations directly from wash fluid (e.g. IDH1), and enough DNA was recovered from wash fluid from both case-studies to submit for targeted cancer panel sequencing. These results suggest biopsy needle wash fluid should be considered a first-class diagnostic resource in any cancer. Citation Format: Jack Wadden, Vishal John, Seongbae Kong, Kait Verbal, Amy K. Bruzek, Wajd Al-Holou, Jason A. Heth, Hugh Garton, Carl Koschmann. Zero-waste molecular diagnostics from biopsy needle wash water [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 2115.

Abstract BACKGROUND Diffuse midline gliomas (DMG) are aggressive and universally fatal with a median survival rate of only 9-12 months. MRIs can be difficult to interpret due to diffuse disease and radiation-induced swelling. Recent work has shown that liquid biopsy of cell-free tumor DNA (cf-tDNA) in patient plasma using digital droplet PCR (ddPCR) might help supplement radiographic monitoring, and enable more quantitative, low-cost, rapid tests. Nanopore sequencing has been employed to quickly and affordably measure and track treatment response in DMG patient cerebrospinal fluid (CSF) where cf-tDNA variant allele fractions are high ( >1%). However, plasma cf-tDNA signal is much lower than CSF (typically < 0.5%), and no platforms have demonstrated the feasibility of quickly (< 1day) sequencing brain tumor cf-tDNA in plasma samples to our knowledge. METHODS To solve these problems, we have developed a same-day, multiplexed, concatemeric consensus error corrected (CCEC-Seq) assay approach for Nanopore sequencing. We combine multiplex PCR and loop-mediated isothermal amplification (LAMP) to capture short cf-tDNA fragments and concatemerize them. Sequenced concatemer segments should agree, and allow informatic consensus error correction, reducing Nanopore error rates. RESULTS We generated a multiplex (n = 8) DMG hotspot loci panel, which was able to reduce sequencing false positive call rates by 3.1x-10.8x. We were able to employ the CCEC-Seq technique on serial plasma samples from a patient with DMG with H3.3K27M mutation undergoing treatment with ONC201 with results comparable to a previously validated ddPCR assay. Ongoing work will apply this multiplex CCEC-Seq panel to a larger cohort of serial DMG plasma samples. CONCLUSION To our knowledge, this is the first demonstration of same-day, sequencing-based, liquid biopsy protocol for a plasma sample from a brain tumor patient. This approach has the potential to greatly reduce resource requirements, and ease of operation for liquid biopsy for DMG and other brain tumor disease monitoring.

Diffuse midline glioma (DMG) with H3K27M mutation is an aggressive and difficult to treat pediatric brain tumor. Recurrent gain of function mutations in H3.3 (H3.3A) and H3.1 (H3C2) at the 27th lysine to methionine (H3K27M) are seen in over 2/3 of DMGs, and are associated with a worse prognosis. Due to the anatomical location of DMG, traditional biopsy carries risk for neurologic injury as it requires penetration of vital midline structures. Further, radiographic (MRI) monitoring of DMG often shows nonspecific changes, which makes therapeutic monitoring difficult. This indicates a critical need for more minimally invasive methods, such as liquid biopsy, to understand, diagnose, and monitor H3K27M DMG. Here, we review the use of all modalities to date to detect biomarkers of H3K27M in cerebrospinal fluid (CSF), blood, and urine, and compare their effectiveness in detection, diagnosis, and monitoring treatment response. We provide specific detail of recent efforts to monitor CSF and plasma H3K27M cell-free DNA in patients undergoing therapy with the imipridone ONC201. Lastly, we discuss the future of therapeutic monitoring of H3K27M-DMG, including biomarkers such as mitochondrial DNA, mutant and modified histones, and novel sequencing-based approaches for improved detection methods.

Nanopore Sensitivity/Specificity details

Malignant primary brain tumors are the most common cancer in children aged 0–14 years, and are the most common cause of death among pediatric cancer patients. Compared to other cancers, pediatric brain tumors have been difficult to diagnose and study given the high risk of intracranial biopsy penetrating through vital midline structures, where the majority of pediatric brain tumors originate ( Ostrom et al., 2015 ). Furthermore, the vast majority of these tumors recur. With limitations in the ability to monitor using clinical and radiographic methods alone, minimally invasive methods such as liquid biopsy will be crucial to our understanding and treatment. Liquid biopsy of blood, urine, and cerebrospinal fluid (CSF) can be used to sample cfDNA, ctDNA, RNA, extracellular vesicles, and tumor-associated proteins. In the past year, four seminal papers have made significant advances in the use of liquid biopsy in pediatric brain tumor patients ( Liu et al., 2021 ; Cantor et al., 2022 ; Miller et al., 2022 ; Pagès et al., 2022 ). In this review, we integrate the results of these studies and others to discuss how the newest technologies in liquid biopsy are being developed for molecular diagnosis and treatment response in pediatric brain tumors.

Recent clinical trials for H3K27-altered diffuse midline gliomas (DMGs) have shown much promise. We present a consensus roadmap and identify three major barriers: (1) refinement of experimental models to include immune and brain-specific components; (2) collaboration among researchers, clinicians, and industry to integrate patient-derived data through sharing, transparency, and regulatory considerations; and (3) streamlining clinical efforts including biopsy, CNS-drug delivery, endpoint determination, and response monitoring. We highlight the importance of comprehensive collaboration to advance the understanding, diagnostics, and therapeutics for DMGs.