Citations

Abstract Diffuse leptomeningeal glioneuronal tumors (DLGNT) are rare central nervous system tumors. Our understanding of the clinical course, prognostic factors, and optimal treatment strategies remains limited. Histologic, molecular, and clinical data were collected and analyzed for 25 patients with DLGNT reviewed at St. Jude Children’s Research Hospital. The median age at diagnosis was 6 years (11 months-20 years). Nineteen (72%) patients were male. The disease was localized at diagnosis in 12 (48.0%) patients, with most tumors localized in the spinal cord. BRAF fusion was present in 22 (95.6%) of 23 patients who had material for testing. Of 18 patients who had tissue for DNA methylation profiling, 3 (16.7%) matched to DLGNT MC-1, 3 (16.7%) to DLGNT MC-2, and 12 (66.7%) did not match to a DLGNT subclass. Median follow-up was 63 months. Twenty-four (96.0%) patients received adjuvant therapy after initial diagnosis or at progression. Thirty-five cytotoxic chemotherapy and 24 single-agent targeted therapy regimens were used in 22 and 13 patients, respectively. With targeted therapy, 3 (12%) patients had partial responses. Eleven (44.0%) received radiotherapy. Median progression-free survival (PFS) after a patient’s first chemotherapy, targeted therapy, or radiotherapy regimen was 44 months (1-77), 11 months (4-39), and 16.5 months (9-23), respectively. The 5-year PFS was 18.7%±9.2, while the 5-year overall survival (OS) was 82.4%±9.2. The 5-year OS was lower in patients older than 9 years at diagnosis (60.0%±21.9 vs 91.7%±8.0, p = 0.02). Ki67 >4% was associated with worse PFS (0%±0 vs 24.6%±11.9 at 5 years, p = 0.03). Initial disease dissemination (localized vs. disseminated) and 1q gain status do not influence PFS or OS. PFS for DLGNT remains poor across all treatment modalities, including targeted therapies, despite the high frequency of BRAF fusions. Due to its rarity, establishing optimal therapeutic strategies for DLGNT requires additional treatment and outcome data and prospective multi-institutional trials.

Abstract Background Brain cells secrete extracellular vesicles (EVs) containing signaling and pathological proteins related to Alzheimer's disease (AD) and related disorders. Recent studies demonstrate brain‐derived EVs (BDEVs) contain misfolded tau and are transmissible of tau pathology in the brain. However, the underlying mechanism regarding EV‐mediated tau pathology is still largely uncharacterized. Method We conducted the immuno‐affinity purification of tau in human brain‐derived EVs (BDEVs) isolated from age and sex‐matched 14 AD and 14 CTRL cases and performed tandem mass‐tag mass spectrometry to profile unbiased tau interactome in BDEVs. We used Nanotemper Monolith to validate the interaction of EV‐tau interactors with tau, and super‐resolution microscopy (Nanoimager) to detect their colocalization in a single‐EV level. We next designed siRNAs to silence EV‐tau interactors in SH‐SY5Y cells overexpressing human P301L tau (SH‐SY5Y‐P301Ltau) and assessed tau loading to EVs by Flow Nanoanalyzer. These EVs were tested for their uptake and tau seeding by human iPSC‐derived neurons (iNeurons). We finally tested the inhibitors of identified molecules on BDEV‐mediated tau propagation in vivo . Result A total of 764 proteins were identified in BDEV‐associated tau interactome from CTRL and AD patients. Sixty‐five proteins were significantly downregulated in AD BDEVs; whereas 5 proteins were significantly upregulated in AD BDEVs compared to CTRL BDEVs. The most enriched tau‐interacting proteins were significantly positively correlated with Braak stage. We confirmed the direct binding of candidate proteins to tau with micromolar binding affinities by Monolith, and their colocalization with tau in human BDEVs using Nanoimager. Silencing of candidate molecules in human SH‐SY5Y‐P301Ltau cells showed reduced loading of tau in EVs, their reduced uptake by iNeurons, and tau seeding activities. Finally, intracerebroventricular injection of neutralizing antibody against the most promising target results in reduction of tau pathology in PS19 mice expressing P301S human tau mutant. Conclusion Our study identified tau‐interacting molecules highly enriched in AD BDEVs compared to CTRL BDVs. Silencing of candidate molecule reduced tau loading to the EVs and their uptake by iNeurons and neutralizing antibody suppressed tau dissemination, highlighting them as promising therapeutic targets to halt tau pathology in AD and related tauopathy.

Abstract Background Extracellular vesicles (EVs) are key mediators in transferring pathological proteins associated with Alzheimer's disease (AD). The apolipoprotein E (APOE) gene, particularly the ε4 allele, is a major genetic risk factor for late‐onset AD. However, the influence of APOE genotype on the biological characteristics and cargo composition of brain‐derived extracellular vesicles (BDEVs) in AD remains poorly understood. Method In this study, BDEVs were isolated from human AD temporal cortex with APOE3/3 and APOE4/4 genotypes ( N = 20 / group). Nanoflow cytometry and super‐resolution microscopy were used to evaluate the tau load within these BDEVs. EV‐mediated tau transmission was assessed in aged human Tau KI and APP NL‐G‐F : Tau KI mice in vivo . BDEV uptake and tau seeding were evaluated in iPSC‐derived neurons through live imaging. Additionally, shotgun lipidomics and data independent acquisition proteomics were analyzed lipidome and proteome of BDEVs, respectively. Key molecules linked to APOE genotype were identified through integrated weighted gene co‐expression network and trait correlation analysis. Result APOE4/4 BDEVs exhibited a higher proportion of phosphorylated tau (pS396) at single EV levels. Upon injection of 300pg tau of BDEVs into aged Tau KI and APP NL‐G‐F : Tau KI mice, APOE4/4 BDEVs significantly increased AT8 + phosphorylated tau levels, and induced neuroinflammation in vivo . Moreover, APOE4/4 BDEVs demonstrated stronger neuronal uptake and tau transfer compared to APOE3/3 BDEVs, impairing the neuronal activity of iPSC‐derived neurons. Lipidomic revealed that APOE4/4 BDEVs were significantly more enriched in pro‐inflammatory long‐chain polyunsaturated fatty acids (PUFA) compared to APOE3/3 with Braak stage association. Furthermore, gene ontology pathway analysis using differentially expressed proteins in APOE4/4 BDEVs showed increased activity in oxidative phosphorylation and sterol‐binding. Multi‐omics integrated data revealed that upregulation of PUFA was significantly associated with elevated cell adhesion molecules and ATPase transporter activity with neural cell adhesion molecule as one of key molecule. Notably, neutralizing antibody against the molecule effectively reduced APOE4/4 BDEV internalization and mitigated EV‐mediated tau pathology in recipient cells. Conclusion Our comprehensive analysis suggested that AD APOE4/4 BDEV may enhance transmissibility of tau pathology and neuroinflammation via enriched pro‐inflammatory PUFA and cell adhesion molecules. Targeting these pathways presents a promising therapeutic strategy to mitigate disease progression, particularly in APOE4 individuals.

Abstract Brain-derived extracellular vesicles (BDEVs) carry tau filaments and promote tau transmission in Alzheimer’s disease (AD). However, how APOE ε4 allele, a key genetic risk factor for AD, may change BDEV molecular structures thereby facilitate disease progression is poorly understood. Here we report comprehensive analyses of BDEVs isolated from human E3/3 and E4/4 AD brains with a biological multi-omics approach. E4/4 BDEVs significantly enhanced tau propagation in aged human MAPT (Tau) knock-in and APPNL-G-F: Tau double knock-in mouse brains in vivo and increased neuronal uptake and excitability in induced pluripotent stem cell-derived neurons (iNeurons) compared to E3/3 BDEVs in vitro. Notably, correlation analysis of BDEV-lipidome and proteome exhibited synergistic enrichment in unsaturated free fatty acid (FFA)18:2, a precursor of inflammatory w6 FFA, and neural cell adhesion molecule 1 (NCAM1). Treatment of iNeurons with FFA 18:2 induces NCAM1 expression, recruits tau into EVs and enhance their tau seeding activity, which is blocked by NCAM1 antibody in vitro . Finally, intracerebroventricular injection of NCAM1 antibody significantly alleviated pathological tau accumulation and glial inflammation in PS19 tauopathy mouse brains, which had previously been reported to exhibit increased level of 18:2 FFA. This highlights novel pathological mechanism in tau transfer mediated by E4/4 BDEVs and emphasizes strong therapeutic potential of targeting EV molecules in AD progression.

Docetaxel +/− ramucirumab remains the standard-of-care therapy for patients with metastatic non-small-cell lung cancer (NSCLC) after progression on platinum doublets and immune checkpoint inhibitors (ICIs). The aim of our study was to investigate whether the cancer gene mutation status was associated with clinical benefits from docetaxel +/− ramucirumab. We also investigated whether platinum/taxane-based regimens offered a better clinical benefit in this patient population. A total of 454 patients were analyzed (docetaxel +/− ramucirumab n=381; platinum/taxane-based regimens n=73). Progression-free survival (PFS) and overall survival (OS) were compared among different subpopulations with different cancer gene mutations and between patients who received docetaxel +/− ramucirumab versus platinum/taxane-based regimens. Among patients who received docetaxel +/− ramucirumab, the top mutated cancer genes included TP53 (n=167), KRAS (n=127), EGFR (n=65), STK11 (n=32), ERBB2 (HER2) (n=26), etc. None of these cancer gene mutations or PD-L1 expression was associated with PFS or OS. Platinum/taxane-based regimens were associated with a significantly longer mQS (13.00 m, 95% Cl: 11.20–14.80 m versus 8.40 m, 95% Cl: 7.12–9.68 m, LogRank P=0.019) than docetaxel +/− ramcirumab. Key prognostic factors including age, histology, and performance status were not different between these two groups. In conclusion, in patients with metastatic NSCLC who have progressed on platinum doublets and ICIs, the clinical benefit from docetaxel +/− ramucirumab is not associated with the cancer gene mutation status. Platinum/taxane-based regimens may offer a superior clinical benefit over docetaxel +/− ramucirumab in this patient population.

Abstract Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and promising biomarkers and therapeutics in the central nervous system (CNS). Human brain‐derived EVs (BDEVs) provide a comprehensive snapshot of physiological changes in the brain's environment, however, the isolation of BDEVs and the comparison of different methods for this purpose have not been fully investigated. In this study, we compared the yield, morphology, subtypes and protein cargo composition of EVs isolated from the temporal cortex of aged human brains using three established separation methods: size‐exclusion chromatography (SEC), phosphatidylserine affinity capture (MagE) and sucrose gradient ultracentrifugation (SG‐UC). Our results showed that SG‐UC method provided the highest yield and collected larger EVs compared to SEC and MagE methods as assessed by transmission electron microscopy and nanoparticle tracking analysis (NTA). Quantitative tandem mass‐tag (TMT) mass spectrometry analysis of EV samples from three different isolation methods identified a total of 1158 proteins, with SG‐UC showing the best enrichment of common EV proteins with less contamination of non‐EV proteins. In addition, SG‐UC samples were enriched in proteins associated with ATP activity and CNS maintenance, and were abundant in neuronal and oligodendrocytic molecules. In contrast, MagE samples were more enriched in molecules related to lipoproteins, cell‐substrate junction and microglia, whereas SEC samples were highly enriched in molecules related to extracellular matrix, Alzheimer's disease and astrocytes. Finally, we validated the proteomic results by performing single‐particle analysis using the super‐resolution microscopy and flow cytometry. Overall, our findings demonstrate the differences in yield, size, enrichment of EV cargo molecules and single EV assay by different isolation methods, suggesting that the choice of isolation method will have significant impact on the downstream analysis and protein discovery.

e21030 Background: Docetaxel +/- ramucirumab remains the standard of care therapy after progression on platinum doublet and immune checkpoint blockade (ICB) in patients with metastatic non-small cell lung cancer (NSCLC). However, the clinical benefit from docetaxel is short-lived with substantial toxicity. Additionally, whether cancer gene mutation status impacts the benefit from docetaxel has not been systemically studied. In this real-world study, we aim to investigate whether cancer gene mutation status is associated with benefit from docetaxel. We also explored whether IMPOWER150-based regimens (ABCP-atezolizumab + bevacizumab + carboplatin + paclitaxel; ACP-atezolizumab + carboplatin + paclitaxel; BCP-bevacizumab + carboplatin+paclitaxel) may serve as alternative options with better efficacy in this patient population. Methods: We retrospectively queried MD Anderson Cancer Center GEMINI database for patients with metastatic NSCLC who received docetaxel, docetaxel+ramucirumab, or one of the above-mentioned IMPOWER150-based regimens after progression on concomitant or sequential platinum-doublet and ICI. Primary endpoints include progression-free survival (PFS) and overall survival (OS). Results: 274 patients were analyzed (docetaxel n = 99; docetaxel +ramucirumab n = 142; ABCP n = 28; BCP n = 4, ACP n = 1). No significant difference was observed in mPFS (3.00m versus 3.93m, P = 0.156) or mOS (8.40m vs.8.67m, P = 0.737) between patients from docetaxel monotherapy group and docetaxel+ ramucirumab group. Cancer gene mutations in EGFR, KRAS, TP53 or PD-L1 expression (0-1%; 1-49%; ≥50%) were not associated with PFS or OS in the 241 patients treated with docetaxel +/- ramucirumab. Furthermore, there was no difference in mPFS (5.50m versus 3.47m, P = 0.516) or mOS (10.40m versus 8.83m, P = 0.737) in patients who received IMPOWER 150-based regimens compared to those who were treated with docetaxel+/- ramucirumab. The median treatment duration was 1.60m (0.70m-20.23m), 1.90m (0.70m-32.90m), 4.80m(0.70m-33.47m) and 11 patients (11.11%), 20 patients (14.08%), and 3 patients (9.09%) discontinued treatment due to adverse events in the patients treated with docetaxel monotherapy, docetaxel+ramucirumab combined therapy, and the IMPOWER150 regimens, respectively. Conclusions: These results from this real-world study suggested that unlike for ICI therapy, where onco-driver mutations have profound impact on the clinical benefit, the cancer gene mutation status does not impact the benefit from docetaxel +/- ramucirumab in NSCLC patients who progressed on platinum doublets and ICI. Furthermore, the clinical benefit from docetaxel +/- ramucirumab is short-lived. Novel treatment regimens are urgently needed in this clinical setting.