Abstract Background: HPV16 (human papillomavirus) is a leading cause of oropharyngeal cancer (OPC) in the US. Antibodies to HPV16 early antigens are detectable in serum samples up to two decades before diagnosis and are potential biomarkers for early detection. In this study, we have developed and compared three custom HPV16 serology platforms for detection of OPC: MSD (Meso Scale Discovery) electrochemiluminescent detection, RAPID ELISA, and a novel semi-quantitative lateral flow assay (LFA) using a custom android application. Methods: For the MSD platform, recombinant HPV 16 antigens (E1, E2, E6, E7) were synthesized in mammalian cells and printed on multiplexed arrays. In RAPID ELISA, GST-tagged antigens (E1, E2, E6, E7) were expressed using mammalian in vitro transcription and translation and bound IgG detected. For the LFA, His-tagged recombinant proteins (HPV16 CE2 184-365aa and HPV16 E7) were synthesized, printed, and antibodies detected with protein G conjugated nanoparticles. Archived serum samples drawn from newly-diagnosed OPC patients (n = 60), with matching saliva samples (n=54, MSD assay only) were obtained from Mayo Clinic. Unmatched controls (n = 46) were obtained from AT Still. For the lateral flow assay, colorimetric image analysis was done using a titration curve of known concentration of monoclonal antibody. We optimized the biomarker panel (E2, E6, and E7 for MSD and ELISA, and for LFA E2, and E7) by using multiparametric analysis and plotting the receiver operating curves to the test performance, and assays were compared using the Cohen’s kappa statistic. Results: At 98% specificity, the MSD HPV16 serologic assay achieved 88% sensitivity, similar to RAPID ELISA, and the LFA assay at 85% sensitivity. The signal intensities for saliva samples were weaker at 24% sensitivity. The Cohen’s kappa value ranged from 0.74 to 0.78 for the comparison between all three assays. There was only one sample among all the cases that was negative by all three serology tests. Conclusion: All three assays have strong concordance for the detection of HPV16 early antigen serology. All the tests can facilitate large scale HPV serology research. Citation Format: Akshansh Kaushik, David Routman, Tej Patel, Joshua Eger, Padhmavathy Yuvaraj, Kathleen R. Bartemes, Marisa D. Griesel, Danielle E. Hunter, Katie Van Abel, Anu Mathew, Mingyue Wang, Leonid Dzantiev, Martin Stengelin, Jacob N. Wohlstadter, Ann Spolarich, Kristina R. Dahlstrom, Jennifer Blain Christen, Erich Sturgis, Karen Anderson. HPV serology for rapid detection of HPV positive oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5099.

Abstract Background: Early detection of lung cancer (LC) remains a critical unmet need. While computed tomography (CT) screening saves lives by detecting cancer at early stages, its utility is limited by high false positive rates and insufficient sensitivity. These limitations lead to unnecessary surgeries and missed malignancies, particularly in individuals presenting with indeterminate pulmonary nodules (IPNs). Objective: Our overarching goal is to develop circulating biochemical biomarkers that improve the specificity of CT screening for LC by differentiating malignant from benign IPNs. Methods: We applied a high-throughput systems immunoproteomics strategy to discover serum biomarkers able to discriminate between malignant and benign IPNs. This integrated approach profiles three classes of circulating biomarkers: autoantibodies, anti-microbial antibodies, and serum proteins. Results: In the discovery phase, we profiled IgG and IgA autoantibodies using Nucleic Acid Programmable Protein Array (NAPPA) against 13,330 full-length human proteins, along with microbial antibodies against 8,820 microbial antigens. These analyses were conducted using serum from 144 lung cancer cases and 143 benign controls from Vanderbilt University Medical Center. Antibodies with significant enrichment in cases (odds ratio p < 0.05 in the top decile) were prioritized, yielding 112 autoantibodies and 70 microbial antibodies associated with malignancy, as well as 50 autoantibodies and 230 microbial antibodies associated with benign disease. In the validation phase, we assessed the prioritized antibody candidates in 319 subjects from the Detection of Early Lung Cancer Among Military Personnel (DECAMP-1) cohort using our Multiplexed In-Solution Protein Array (MISPA). In parallel, we quantified 19 well-reported cancer-associated serum proteins across the same samples. A multimodal panel comprising 7 autoantibodies, 4 microbial antibodies, and 4 serum proteins achieved an area under the curve (AUC) of 0.81 in the discovery cohort and 0.74 in independent validation cohorts, showing improved discrimination of malignant versus benign nodules compared to clinical models alone. Additionally, to demonstrate clinical scalability, we confirmed performance of top markers in 46 cases and 230 controls from the German Lung Cancer Screening Intervention Study (LUSI) using the Meso Scale Discovery (MSD) electrochemiluminescence platform. Conclusions: These results demonstrate a fully integrated immunoproteomics pipeline for the discovery, validation, and potential clinical translation of multiplex antibody and protein biomarkers for lung cancer detection. Citation Format: Joshua LaBaer, Jin Park, Ji Qiu, Lusheng Song, Karen S. Anderson, Jennifer Molloy, Gomati Nandedkar, Daniel Woodley, Deborah Adams, Candyce McDaniel, Andruw Fierro, Renée Turzanski Fortner, Toria Trendler, Mingyue Wang, Leonid Dzantiev, Anu Mathew, Martin Stengelin, Wohlstadter Jacob. High throughput immunoproteomics for cancer biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2529.

Abstract Angiogenesis, the formation of new blood vessels from existing vasculature, is a hallmark of tumor progression, enabling malignant growth, invasion, and metastasis. Tumor-associated angiogenesis leads to the formation of leaky, poorly perfused vessels and localized hypoxia, fostering metabolic stress, immune evasion, and resistance to therapies. Vascular normalization through targeting of VEGF, Ang-2, and PDGF pathways can restore perfusion, enhance immune infiltration, and improve response. Although anti-angiogenic agents are widely used, no predictive biomarker identifies individuals that are likely to benefit. Previously, biomarker development for anti-angiogenic therapies focused on few vascular factors and did not capture the complexity of angiogenic signaling. To address this, we developed a 44-marker angiogenesis panel enabling high-throughput profiling of pro- and anti-angiogenic mediators relevant to oncology and vascular biology. Forty-four biomarkers encompassing established pro- and anti-angiogenic mediators (e.g., VEGF-A, Ang-2, Tie-2, IL-6) were literature-curated. Antibody pairs for each assay were optimized for the electrochemiluminescence detection-based MESO SCALE DISCOVERY (MSD) U-PLEX® platform. Double-spun platelet-poor EDTA plasma samples from Duke University was tested to identify a common diluent. Analytical characterization included LLOD/LLOQ, precision, spike recovery, and dilution linearity/parallelism determinations. The interclass correlation coefficient (ICC) and biological variance were evaluated using longitudinally collected plasma from 28 participants (27-79 y.o.), incorporating diurnal (morning and afternoon) draws over two months. Pharmacodynamic biomarker modulation was explored using retrospective plasma samples from two NCI studies —Cabozantinib + Panitumumab (NCT02008383) and Bevacizumab (NCT00416637). All assays met the predefined criteria, with >90% of assays achieving intra-/inter-assay precision of <15% CV. A single diluent supported multiplex compatibility. Dynamic ranges spanned 3-5 orders of magnitude, with analytical sensitivity for low and high abundance markers ranging from 0.06 pg/mL (IL-6) to 67.4 pg/mL (vWF), demonstrating sub-pg/mL detection. ICC analyses revealed good to excellent variable marker stability across the panel over two months. Retrospective analysis of both sets of NCI study samples demonstrated an association between (a) baseline markers and outcomes (OS, PFS) and (b) pharmacodynamic responses and expected treatment-induced changes, highlighting the value of the biomarker panel. This 44-plex panel demonstrates robust analytical performance, sensitivity, and reproducibility on the MSD® U-PLEX platform. The assay supports harmonized angiogenesis profiling, enabling standardized angiogenesis biomarker assessment in oncology research. Citation Format: Hans Layman, Mai Abdel-Ghani, Grace Galen, Rony Garcia-Vivas, Jenna Slezak, Anu Mathew, Mingyue Wang, Catherine Demos, Pankaj Oberoi, Andrew B. Nixon, Jacob N. Wohlstadter. Evaluation of the circulating angiome in cancer: Translational assessment of a 44-plex angiogenesis biomarker panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4802.

Research Use Only (RUO) sample-sparing MULTI-ARRAY serology immunoassays have been developed for human antibodies to several HPV-16 early antigens. These assays are intended to support research of oropharyngeal cancer (OPC) linked to the human papillomavirus (HPV).The HPV virus can infect the mouth and throat and cause cancers of the oropharynx, i.e. the back of the throat, including the base of the tongue and tonsils. HPV-related cancer incidence is increasing dramatically among men and is unlikely to decrease in the foreseeable future, despite vaccination efforts. As a result, this may become one of the most common cancers in middle-aged men in the United States by 2045. The HPV16 genome consists of six early genes (E1, E2, E4, E5, E6, and E7) and two late genes (L1 and L2) that constitute the viral capsid. Antibodies to early HPV-16 antigens are commonly present in those with OPC but are detected in only approximately 1% of the general population. These antibodies are present many years before cancer develops; thus, they are ideal biomarkers for early detection of OPC.Immunoassays for antibodies against HPV-16 early antigens E1, E2, E6, and E7 were developed in a high-throughput multiplexed format using electrochemiluminescence (ECL) detection, and MULTI-ARRAY 10-spot 96-well plates. Each well has an array presenting recombinant antigens manufactured by Meso Scale Diagnostics, LLC. The assay uses 25 uL of 2, 500x diluted serum or plasma. The assay format is simple: diluted sample is incubated in the well with the antigen array followed by a wash and detection of bound anti-HPV antibodies using an anti-human IgG antibody labeled with the SULFO-TAG ECL label. Assay performance was evaluated with approximately 200 commercially sourced samples from apparently healthy individuals and 14 samples from individuals known to be positive for at least one HPV-16 early antigen using an established reference method (Programmable Protein (RAPID) ELISA, Anderson 2015). There was excellent agreement between the two methods.This high-throughput MULTI-ARRAY assay may be useful in research applications requiring screening of a large number of samples for HPV-16 antibodies.For Research Use Only. Not for use in diagnostic procedures. Mingyue Wang, Jermaine Brown, John C. Smith, Leonid Dzantiev, Anu Mathew, Martin Stengelin, George Sigal, Padhma Yuvaraj, Karen S. Anderson, Jacob Wohlstadter. Serological assays for early detection of HPV-associated oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7086.

BACKGROUND:TAR DNA-binding protein-43 (TDP-43) has an important role in the pathogenesis of several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), and limbic predominant age-related TDP-43 encephalopathy (LATE). Improved immunoassays for detecting TDP-43 and its disease-associated modifications are needed, based on the hypothesis that proteins involved in disease pathology can serve as effective biomarkers. METHODS:Antibodies were screened against purified TDP-43 proteins and fragments, brain lysate, cerebrospinal fluid (CSF) and plasma from individuals with ALS (provided by MGH) and healthy controls (commercially obtained) to develop and preliminarily evaluate research use only (RUO) standard and ultrasensitive S-PLEX assays for TDP-43 and pTDP-43. RESULTS:The full length TDP-43 assay in standard format had a quantitative range of 10-275,000 pg/mL and sufficient sensitivity to quantitate 100% of the tested plasma samples. The assay displayed excellent dilution linearity. Whole blood, plasma and platelet-rich plasma show higher concentrations than red blood cells, serum and platelet-poor plasma, suggesting a need for good control over efficiency and timing of the separation of plasma from platelets and blood cells to avoid pre-analytical effects. To measure the lower TDP-43 levels in CSF samples, the more sensitive S-PLEX TDP-43 assay was used. It had a quantitative range of 5-34,000 pg/mL and was able to quantitate 48% of the tested CSF samples. The S-PLEX pTDP-43 assay was used to measure TDP-43 phosphorylated at S409/410 in plasma samples. It had a quantitative range of 1-50,000 pg/mL, and was able to quantitate 100% of the tested plasma samples. Specificity was confirmed by testing phosphorylated and non-phosphorylated purified protein. To preliminarily evaluate the assays, a small set of plasma and CSF samples from individuals with ALS was tested. Relative to the control samples, the ALS samples had higher median plasma TDP-43 levels (ratio = 4.8, p =  0.0018), lower median CSF TDP-43 levels (ratio = 0.49, p =  0.01), and non-significantly higher plasma pTDP-43 levels (ratio = 1.9, p =  0.11). CONCLUSIONS:Newly developed immunoassays for TDP-43 and pTDP-43 provide powerful tools for research on neurodegeneration biomarkers.

Research Use Only (RUO) sample-sparing MULTI-ARRAY immunoassays have been developed for several well-established cancer markers. The primary focus of this project is the development of assays to support research of suspicious lung nodules, but many of these selected markers are also relevant to other cancer types. These immunoassays include a serology panel, two biomarker panels, and a single-marker assay. The serology panel includes assays to detect autoantibodies to p53, CTAG-1, and CTAG-2. This panel requires 25 μL of 2, 500-fold diluted serum or plasma. A biomarker panel requiring 25 μL of 100-fold diluted serum or plasma detects the following biomarkers: CA15-3, SCFR/Kit, ErbB2, IGFBP-2, MIF, MMP-2, MMP-9 (total), REG4, S100A6, and TNF-RI. A second biomarker panel requiring 25 μLof 10-fold diluted serum or plasma detects the following biomarkers: CA125, Ca19-9, CEACAM-5 (CEA), EGFR, VEGFR-1/Flt-1, FLT3L, HE4, MMP-3 (total), and osteopontin. Finally, an assay for the core lung cancer biomarker cytokeratin-19 was developed. All assays had dynamic ranges that span 3-4 logs and are sufficiently sensitive to measure native levels in commercially sourced samples from apparently healthy individuals. The assays were preliminarily evaluated using more than 100 commercially sourced serum or plasma samples from apparently healthy individuals and more than 100 commercially sourced serum or plasma samples from patients with lung or other cancers. Many of the biomarkers had significantly different concentrations in several cancers compared to samples from apparently healthy individuals. These RUO assays may be useful in identifying biomarkers for a multi-marker panel to study early cancer. Mingyue Wang, Jermaine Brown, Daniel Lee, Salvia Misaghian, Taron Gorham, Leonid Dzantiev, Anu Mathew, Martin Stengelin, Ji Qiu, Karen Anderson, Joshua LaBaer, George Sigal, Jacob Wohlstadter. Sample-sparing immunoassays for early detection of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7087.

This study compares novel type 1 diabetes-related autoantibody assays developed to improve upon the standard radiobinding assay (RBA). Samples from 1505 individuals, followed for 5 years or to clinical type 1 diabetes, originally tested by RBA were aliquoted and sent blindly to 5 laboratories (BDC, IDR, DRI, MSD, Enable) to be tested by electrochemiluminescence (ECL) assays, Luciferase Immuno Precipitation System (LIPS) assays, multiplex antibody detection by agglutination-PCR (ADAP) assays, and N-terminally truncated GAD65 or IA2β autoantibody RBAs (tGADA/IA2βA). Findings: The fraction of samples that were concordant for negative/positive interpretations across all assays were 79.7% (GADA), 65.2% (IA-2A), 36.2% (IAA), and 67.5% (ZnT8A). The assays with the highest Youden index for predicting the previous RBA results differed by autoantibody: 0.65 LIPS(IDR) for IAA, 0.91 ECL(BDC) for ZnT8A, 0.82 tGADA RBA(IDR) for GADA, 0.91 ECL(MSD and BDC) for IA-2A. The Youden index for predicting 5-year type 1 diabetes varied significantly across assays and was highest for LIPS(DRI) for all autoantibody combinations, with little variation in the respective maximum Youden index. The discordance between assays makes it problematic to interpret positivity when comparing results from different assays. Longitudinal autoantibody assessments should be tested with the same assay.

Three blood-based biomarkers of neurological injury—glial fibrillary acidic protein (GFAP), neurofilament light (Nf-L), and Tau—have emerged as promising biomarkers of neurological disorders and injuries such as hypoxic-ischemic encephalopathy (HIE), traumatic brain injury, and Alzheimer’s disease (AD). The low levels of GFAP, Nf-L, and Tau in serum and plasma require highly sensitive assays to detect them. Here, we report the analytical validation of an ultrasensitive, electrochemiluminescence-based, multiplexed immunoassay for neurological biomarker assessment. The MSD S-PLEX Neurology Panel 1 kit uses ultrasensitive S-PLEX assay technology to simultaneously measure GFAP, Nf-L, and Tau (total) in a 96-well plate format. Analytical validation was performed in a series of studies based in part on Clinical and Laboratory Standards Institute guidelines to evaluate precision, dilution linearity, interference screening, detection capability, stability, spike recovery, and cross reactivity. Reproducibility was performed to assess precision across three different laboratories at MSD, Johns Hopkins University, and All Children’s Hospital using three independent reagent lots and an identical sample set. In this study, the assay used 5 μL of a sample per replicate. This differs from the commercial protocol for this panel because of the extremely high concentrations observed in HIE samples. The quantifiable range of the assay was 2.4–4,200 pg/mL for GFAP, 7.6–10,000 pg/mL for Nf-L, and 0.34–740 pg/mL for Tau. A set of 15 common interfering substances was screened, and none showed interference exceeding 18%. Dilution of spiked samples up to 256-fold recovered at 80-120% of the expected value for all analytes. Precision error was calculated using identical samples across 3 independent laboratories and 3 production lots of the panel (180 measurements) demonstrating excellent reproducibility. The samples included adult and umbilical cord serum and plasma, samples containing human anti-mouse antibodies, anti-nuclear antibodies, and clinical samples, including those from subjects with AD. The MSD S-PLEX Neurology Panel 1 kit provides a new, analytically validated tool for assessing human GFAP, Nf-L, and Tau. The kit is sufficiently sensitive to detect these analytes in normal serum and plasma and its dynamic range is capable of quantifying elevated levels found in target neurological disorders.

2044 Background: Diffuse gliomas are aggressive malignant tumors with poor prognosis. The current standard of care includes measurement of molecular biomarkers in biopsy samples. One unmet clinical need is to identify non-invasive biomarkers that may be used for differential diagnosis of gliomas from other brain tumors. Pre-clinical and clinical validation of such biomarkers could eliminate the need for biopsy, and support the implementation of more personalized and/or emerging treatments and the earlier enrolment of patients into clinical trials. Our objective is to use multidimensional proteomics to identify and validate potential plasma biomarkers for glioma management. Methods: We used the proximity extension assay from Olink Proteomics to analyze 3,000 proteins in plasma of patients with diffuse gliomas and meningiomas (as controls). By data visualization, we identified several plasma proteins that were increased or decreased in gliomas in comparison to meningiomas. Several candidate markers were selected for validation with an independent set of retrospectively collected samples by using quantitative research-use-only electrochemiluminescence assays available from Meso Scale Discovery. In the validation set, which included longitudinal data from patients, patient information included biopsy-requiring molecular tumor abnormalities such as IDH1 status, ATRX expression, MGMT promoter methylation, CDKN2A/B/p16 status, V1p 19q co-deletion and NF1 status. In the validation stage, we focused on diffuse gliomas. Results: In the discovery phase, associations between proteins were plotted to determine potential predictive ability for discriminating diffuse gliomas vs. meningiomas. A partitioning algorithm was fit to determine the optimal combination of GFAP (the strongest biochemical marker), age and sex, as well as with other candidate proteins. Differential expression was seen for a few other proteins such as NEFL, PROK1, FABP4, MMP3 and LMOD1. In the cross-sectional validation phase, we verified strong associations between GFAP and FABP4 plasma concentration and GBM, astrocytomas, oligodendrogliomas and meningiomas, where these markers could differentiate between the groups. Within diffuse gliomas, NEFL, GFAP, FABP4 and IL13 were significantly different. Conclusions: This study highlights the potential of plasma biomarkers to revolutionize glioma patient management through liquid biopsy applications. The strong associations observed between plasma protein concentrations and glioma subtypes support a diagnostic power that addresses a critical unmet need in neuro-oncology. More specifically, these biomarkers can help with patient differential diagnosis at initial presentation, with future aims to investigate the prognostic value and the possibility of acting as surrogates of molecular changes that are currently used for optimizing therapy.

Adult-type diffuse gliomas, including glioblastoma (GBM), astrocytoma, and oligodendroglioma, are among the most aggressive brain tumors, often exhibiting poor prognosis. Diagnosis currently relies on MRI and biopsy, but for individuals ineligible for resection, non-invasive diagnostic tools are lacking. Additionally, distinguishing primary from recurrent disease and monitoring therapy response remain critical unmet needs. We evaluated seven candidate proteomic markers in plasma from individuals with GBM (n=143), astrocytoma (n=50), oligodendroglioma (n=32), and non-tumor controls (n=30) using research-use-only electrochemiluminescence assays (Meso Scale Discovery). Key questions included: (1) Do protein levels change between primary and recurrent GBM? (2) Do protein concentrations correlate with survival in primary vs. recurrent disease? (3) Can markers predict GBM tumor burden? (4) Can they distinguish GBM from other gliomas and controls? The results showed that neurofilament light chain (NEFL) increased significantly from primary to recurrent GBM (unadjusted p = 0.008). In primary GBM, low fatty acid binding protein 4 (FABP4) correlated with survival (adjusted p = 0.028), but no significance was observed in recurrent disease after adjustment. Machine learning (Lasso regression) showed poor performance in predicting tumor volume (cross-validated R² = 0.192). However, dimensionality reduction (PCA-UMAP) revealed distinct clustering of GBM samples versus astrocytomas, oligodendrogliomas, and controls. A random forest classifier trained on a 70:30 split achieved strong diagnostic performance (test AUC = 0.96, sensitivity = 0.95, specificity = 0.83). To conclude, plasma proteomic markers, particularly NEFL and FABP4, show promise for monitoring recurrence and prognostic stratification in GBM. While tumor volume prediction was unreliable, machine learning models excelled in differentiating GBM from other gliomas and controls, suggesting clinical utility for non-invasive diagnosis. Further validation is warranted to refine biomarker panels for precision oncology applications.

BACKGROUND:Alzheimer's disease (AD) is a heterogeneous neurodegenerative disease with a decades-long prodromal period. Monitoring of sequential pathological changes in neurodegeneration, inflammation, neurovascular dysfunction, oxidative stress and metabolic stress may provide the opportunity for intervention before symptom onset. Assessment with multiple biomarkers may also inform more tailored therapeutic intervention. METHODS:Using MULTI-ARRAY technology, 54 biomarkers were measured using less than 200 μL of CSF from individuals with AD dementia (n = 100), mild cognitive impairment (MCI) with progression to dementia during the following 3-year follow-up (n = 100), MCI non-progressors (n = 100), and subjective cognitive decline (SCD) (n = 93), collected by ACE Alzheimer Center Barcelona. Biomarkers were selected to cover multiple putative disease mechanisms such as neurovascular dysfunction, inflammation, neurodegeneration, tissue injury, and metabolic stress. One-way ANOVA with Bonferroni correction was applied to determine groupwise differences. Area under the curve (AUC) for receiver operating characteristic curves was calculated to assess biomarker utility for predicting dementia progression. RESULTS:For 43 assays, more than 80% of samples provided concentrations within the dynamic range of the assay. We found concentration differences of 30 CSF biomarkers to be statistically significant across cognitive groups, with the most significant groupwise comparisons between the dementia groups (AD and MCI progressors) and the non-dementia groups (MCI non-progressors and SCD). There were 17 analytes for which mean comparisons were statistically different between MCI progressor and non-progressor groups. Ten proteins, pTau217, total tau, neurofilament light, GFAP, MIF, MMP-10, YKL-40, neurofilament heavy, MIP-1α, and IL-15, demonstrated an AUC > 0.7 for differentiation of MCI progressors and non-progressors, showing promise for differentiating MCI individuals at risk of progressing to dementia, with ptau217 being the most significant (AUC > 0.99). CONCLUSIONS:Here we present an exploratory study with quantitative immunoassays, where we identified several CSF biomarkers indicative of dementia or progression to dementia covering multiple pathological mechanisms. Further successful integration into a biomarker panel could help personalize treatment, stratify individuals for therapeutic studies and provide a better understanding of how these early pathologies impact disease progression.

2045 Background: Diffuse gliomas were recently reclassified based on the 2021 WHO Classification criteria. Several molecular changes which carry diagnostic and prognostic power have been added to the classification parameters, including IDH1 mutation and MGMT promotor methylation. To characterize these molecular changes, however, an invasive biopsy is required. Our goal was to examine the relationship between seven plasma biomarkers for diffuse glioma and the established molecular changes and delineate if these markers can be used as surrogates of these molecular changes. Methods: Seven candidate markers, namely glial fibrillary acidic protein (GFAP), neurofilament light (NEFL), matrix metalloproteinase 1, 3, 9 (MMP1, MMP3, MMP9), total Tau (tTau) and fatty acid binding protein 4 (FABP4) were evaluated by quantitative research-use-only electrochemiluminescence assays available from Meso Scale Discovery by comparing the protein concentration distribution with non-parametric Wilcoxon rank sum tests and multiple testing adjustment. The molecular markers tested were IDH1, MGMT promotor and ATRX. The discovery cohort consisted of 49 IDH1 mutant (39%) and 77 IDH1 wildtype (61%) gliomas. Among this retrospective cohort were 103 primary samples (collected at diagnosis) and 23 recurrent samples (collected at time of recurrence). The retrospective validation cohort consisted of 36 IDH1 mutant (22%) and 129 IDH1 wildtype (78%), with 64 primary samples and 76 recurrent samples. Results: Several of the proteomic markers showed significant associations with genetic markers at an adjusted significance level of P < 0.05. For IDH1 status, the strongest association was with NEFL, with IDH1 wildtype samples showing higher levels of the protein. For ATRX expression, high FABP4 was correlated with ATRX retention. As expected, survival analysis based on molecular markers yielded that IDH1 status was most predictive of survival both in primary tumors and recurrent tumors. MGMT promotor methylation was predictive of survival in primary cases but not recurrent cases. When combining the genetic markers with protein concentrations, we were able to see some improvement in survival prediction. Conclusions: We demonstrate that some plasma biomarkers, particularly NEFL and FABP4, show significant associations with key molecular changes in diffuse gliomas, including IDH1 status and ATRX retention/loss. Future research will determine whether these proteomic markers can serve as surrogates for molecular alterations and assist in potentially improved diagnosis and monitoring of diffuse gliomas.

TAR DNA-binding protein-43 (TDP-43) has an important role in the pathogenesis of several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), and limbic predominant age-related TDP-43 encephalopathy (LATE). Improved immunoassays for detecting TDP-43 and its disease-associated modifications are needed, based on the hypothesis that proteins involved in disease pathology can serve as effective biomarkers. Antibodies were screened against purified TDP-43 proteins and fragments, brain lysate, cerebrospinal fluid (CSF) and plasma from individuals with ALS (provided by MGH) and healthy controls (commercially obtained) to develop and preliminarily evaluate research use only (RUO) standard and ultrasensitive S-PLEX assays for TDP-43 and pTDP-43. The full length TDP-43 assay in standard format had a quantitative range of 10-275,000 pg/mL and sufficient sensitivity to quantitate 100% of the tested plasma samples. The assay displayed excellent dilution linearity. Whole blood, plasma and platelet-rich plasma show higher concentrations than red blood cells, serum and platelet-poor plasma, suggesting a need for good control over efficiency and timing of the separation of plasma from platelets and blood cells to avoid pre-analytical effects. To measure the lower TDP-43 levels in CSF samples, the more sensitive S-PLEX TDP-43 assay was used. It had a quantitative range of 5-34,000 pg/mL and was able to quantitate 48% of the tested CSF samples. The S-PLEX pTDP-43 assay was used to measure TDP-43 phosphorylated at S409/410 in plasma samples. It had a quantitative range of 1-50,000 pg/mL, and was able to quantitate 100% of the tested plasma samples. Specificity was confirmed by testing phosphorylated and non-phosphorylated purified protein. To preliminarily evaluate the assays, a small set of plasma and CSF samples from individuals with ALS was tested. Relative to the control samples, the ALS samples had higher median plasma TDP-43 levels (ratio = 4.8, p  =  0.0018), lower median CSF TDP-43 levels (ratio = 0.49, p  =  0.01), and non-significantly higher plasma pTDP-43 levels (ratio = 1.9, p  =  0.11). Newly developed immunoassays for TDP-43 and pTDP-43 provide powerful tools for research on neurodegeneration biomarkers.

e14017 Background: Adult-type diffuse gliomas are common malignant tumors known for their high recurrence rates, regardless of grade. High recurrence is due to incomplete resection of the tumors and the infiltrative nature of the tumor cells. Unfortunately, prediction of recurrence relies solely on MRI, a method that is expensive and susceptible to errors, treatment and pseudo-progression effects. Capturing recurrence prior to MRI is an unmet clinical need and could allow for earlier intervention or enrolment of patients into clinical trials. Liquid biopsy has emerged as a promising approach. Methods: Previously collected patient plasma was retrieved from three sites: Northwestern University Tumor Biobank, Penn State Neuroscience Biorepository and University Health Network Biobank (Cross-sectional samples(all primary vs all recurrent) n = 264, patients with multiple samples (longitudinal) n = 44; Groups = Glioblastoma (GBM), astrocytoma, oligodendroglioma) and analyzed retrospectively for seven proteomic markers: GFAP, NEFL, FABP4, MMP1, MMP3, MMP9 and total tau (tTau) using research-use-only electrochemiluminescence assays available from Meso Scale Discovery. For the cross-sectional analysis (independent samples per patient), Wilcoxon rank sum test with post hoc Holm’s correction was used to compare biomarker values in samples from individuals with primary and recurrent tumors, after adjusting for sex differences. For the longitudinal analysis of paired primary and recurrence samples, Wilcoxon signed-rank test was used. Survival probability was tested through Kaplan-Meier survival curves. Results: In the cross-sectional analysis of the diffuse gliomas (GBM = 77+129 [Batch 1 + Batch 2], Oligodendroglioma = 23 +11, Astrocytoma = 26 + 25), we found that in the GBM group, the median values of MMP9 and GFAP were lower during recurrence. The median NEFL value was higher during GBM recurrence but did not achieve statistical significance, although the same effect was observed in the longitudinal analysis. In survival analyses, higher MMP3 and MMP9 in primary case samples across all diffuse gliomas were significantly associated with poorer survival, but this significance was lost during recurrence, indicating potentially important differences between a primary and a recurrent state. Conclusions: This cross-sectional and longitudinal retrospective pilot study evaluated seven plasma markers for the potential capability of predicting tumor recurrence in adult-type diffuse gliomas. While we found no significant differences across diffuse gliomas overall, subgroup analyses revealed recurrence-associated patterns. These findings suggest that certain markers could complement imaging for recurrence detection and perhaps prediction. Larger and more comprehensive studies are warranted.

Alzheimer's disease (AD) is a heterogeneous neurodegenerative disease with a decades-long prodromal period. Monitoring of sequential pathological changes in neurodegeneration, inflammation, neurovascular dysfunction, oxidative stress and metabolic stress may provide the opportunity for intervention before symptom onset. Assessment with multiple biomarkers may also inform more tailored therapeutic intervention. Using MULTI-ARRAY technology, 54 biomarkers were measured using less than 200 μL of CSF from individuals with AD dementia ( n  = 100), mild cognitive impairment (MCI) with progression to dementia during the following 3-year follow-up ( n  = 100), MCI non-progressors ( n  = 100), and subjective cognitive decline (SCD) ( n  = 93), collected by ACE Alzheimer Center Barcelona. Biomarkers were selected to cover multiple putative disease mechanisms such as neurovascular dysfunction, inflammation, neurodegeneration, tissue injury, and metabolic stress. One-way ANOVA with Bonferroni correction was applied to determine groupwise differences. Area under the curve (AUC) for receiver operating characteristic curves was calculated to assess biomarker utility for predicting dementia progression. For 43 assays, more than 80% of samples provided concentrations within the dynamic range of the assay. We found concentration differences of 30 CSF biomarkers to be statistically significant across cognitive groups, with the most significant groupwise comparisons between the dementia groups (AD and MCI progressors) and the non-dementia groups (MCI non-progressors and SCD). There were 17 analytes for which mean comparisons were statistically different between MCI progressor and non-progressor groups. Ten proteins, pTau217, total tau, neurofilament light, GFAP, MIF, MMP-10, YKL-40, neurofilament heavy, MIP-1α, and IL-15, demonstrated an AUC > 0.7 for differentiation of MCI progressors and non-progressors, showing promise for differentiating MCI individuals at risk of progressing to dementia, with ptau217 being the most significant (AUC > 0.99). Here we present an exploratory study with quantitative immunoassays, where we identified several CSF biomarkers indicative of dementia or progression to dementia covering multiple pathological mechanisms. Further successful integration into a biomarker panel could help personalize treatment, stratify individuals for therapeutic studies and provide a better understanding of how these early pathologies impact disease progression.

BACKGROUND:To investigate evidence of residual viral infection, intrathecal immune activation, central nervous system (CNS) injury, and humoral responses in cerebrospinal fluid (CSF) and plasma in patients recovering from coronavirus disease 2019 (COVID-19), with or without neurocognitive post-COVID condition (PCC). METHODS:Thirty-one participants (25 with neurocognitive PCC) underwent clinical examination, lumbar puncture, and venipuncture ≥3 months after COVID-19 symptom onset. Healthy volunteers were included. CSF and plasma severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and spike antigen (N-Ag, S-Ag), and CSF biomarkers of immune activation and neuronal injury were analyzed. RESULTS:SARS-CoV-2 N-Ag or S-Ag were undetectable in all samples and no participant had pleocytosis. We detected no significant differences in CSF and plasma cytokine concentrations, albumin ratio, IgG index, neopterin, β2M, or in CSF biomarkers of neuronal injury and astrocytic damage. Furthermore, principal component analysis (PCA1) analysis did not indicate any significant differences between the study groups in the marker sets cytokines, neuronal markers, or anti-cytokine autoantibodies. CONCLUSIONS:We found no evidence of ongoing viral replication, immune activation, or CNS injury in plasma or CSF in patients with neurocognitive PCC compared with COVID-19 controls or healthy volunteers, suggesting that neurocognitive PCC is a consequence of events suffered during acute COVID-19 rather than persistent viral CNS infection or residual CNS inflammation.

Background:Certain demyelinating disorders, such as neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) exhibit serum autoantibodies against aquaporin-4 (αAQP4) and myelin oligodendrocyte glycoprotein (αMOG). The variability of the autoantibody presentation warrants further research into subtyping each case.Methods:To elucidate the relationship between astroglial and neuronal protein concentrations in the peripheral circulation with occurrence of these autoantibodies, 86 serum samples were analyzed using immunoassays. The protein concentration of glial fibrillary acidic protein (GFAP), neurofilament light chain (NFL) and tau protein was measured in 3 groups of subcategories of suspected NMOSD: αAQP4 positive (n = 20), αMOG positive (n = 32) and αMOG/αAQP4 seronegative (n = 34). Kruskal-Wallis analysis, univariate predictor analysis, and multivariate logistic regression with ROC curves were performed.Results:GFAP and NFL concentrations were significantly elevated in the αAQP4 positive group (p = 0.003; p = 0.042, respectively), and tau was elevated in the αMOG/αAQP4 seronegative group (p < 0.001). A logistic regression model to classify serostatus was able to separate αAQP4 seropositivity using GFAP + tau, and αMOG seropositivity using tau. The areas under the ROC curves (AUCs) were 0.77 and 0.72, respectively. Finally, a combined seropositivity versus negative status logistic regression model was generated, with AUC = 0.80.Conclusion:The 3 markers can univariately and multivariately classify with moderate accuracy the samples with seropositivity and seronegativity for αAQP4 and αMOG.

Gliomas are aggressive malignant tumors, with poor prognosis. There is an unmet need for the discovery of new, non-invasive biomarkers for differential diagnosis, prognosis, and management of brain tumors. Our objective is to validate four plasma biomarkers – glial fibrillary acidic protein (GFAP), neurofilament light (NEFL), matrix metalloprotease 3 (MMP3) and fatty acid binding protein 4 (FABP4) – and compare them with established brain tumor molecular markers and survival. Our cohort consisted of patients with benign and malignant brain tumors (GBM = 77, Astrocytomas = 26, Oligodendrogliomas = 23, Secondary tumors = 35, Meningiomas = 70, Schwannomas = 15, Pituitary adenomas = 15, Normal individuals = 30). For measurements, we used ultrasensitive electrochemiluminescence multiplexed immunoassays. High plasma GFAP concentration was associated with GBM, low GFAP and high FABP4 were associated with meningiomas, and low GFAP and low FABP4 were associated with astrocytomas and oligodendrogliomas. NEFL was associated with progression of disease. Several prognostic genetic alterations were significantly associated with all plasma biomarker levels. We found no independent associations between plasma GFAP, NEFL, FABP4 and MMP3, and overall survival. The candidate biomarkers could not reliably discriminate GBM from primary or secondary CNS lymphomas. GFAP, NEFL, FABP4 and MMP3 are useful for differential diagnosis and prognosis, and are associated with molecular changes in gliomas.

Abstract Background There are numerous benefits to performing salivary serology measurements for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen for coronavirus disease 2019 (COVID-19). Here, we used a sensitive multiplex serology assay to quantitate salivary IgG against 4 SARS-CoV-2 antigens: nucleocapsid, receptor-binding domain, spike, and N-terminal domain. Methods We used single samples from 90 individuals with COVID-19 diagnosis collected at 0 to 42 days postsymptom onset (PSO) and from 15 uninfected control subjects. The infected individuals were segmented in 4 groups (0–7 days, 8–14 days, 15–21 days, and >21 days) based on days PSO, and values were compared to controls. Results Compared to controls, infected individuals showed higher levels of antibodies against all antigens starting from 8 days PSO. When applying cut-offs with at least 93.3% specificity at every time interval segment, nucleocapsid protein serology had the best sensitivity at 0 to 7 days PSO (60% sensitivity [35.75% to 80.18%], ROC area under the curve [AUC] = 0.73, P = 0.034). Receptor-binding domain serology had the best sensitivity at 8 to 14 days PSO (83.33% sensitivity [66.44%–92.66%], ROC AUC = 0.90, P < 0.0001), and all assays except for N-terminal domain had 92% sensitivity (75.03%–98.58%) at >14 days PSO. Conclusions This study shows that our multiplexed immunoassay can distinguish infected from uninfected individuals and reliably (93.3% specificity) detect seroconversion (in 60% of infected individuals) as early as the first week PSO, using easy-to-collect saliva samples.

BACKGROUND:Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in blood has high sensitivity in adults with acute coronavirus disease 2019 (COVID-19), but sensitivity in pediatric patients is unclear. Recent data suggest that persistent SARS-CoV-2 spike antigenemia may contribute to multisystem inflammatory syndrome in children (MIS-C). We quantified SARS-CoV-2 nucleocapsid (N) and spike (S) antigens in blood of pediatric patients with either acute COVID-19 or MIS-C using ultrasensitive immunoassays (Meso Scale Discovery). METHODS:Plasma was collected from inpatients (<21 years) enrolled across 15 hospitals in 15 US states. Acute COVID-19 patients (n = 36) had a range of disease severity and positive nasopharyngeal SARS-CoV-2 RT-PCR within 24 hours of blood collection. Patients with MIS-C (n = 53) met CDC criteria and tested positive for SARS-CoV-2 (RT-PCR or serology). Controls were patients pre-COVID-19 (n = 67) or within 24 hours of negative RT-PCR (n = 43). RESULTS:Specificities of N and S assays were 95-97% and 100%, respectively. In acute COVID-19 patients, N/S plasma assays had 89%/64% sensitivity; sensitivities in patients with concurrent nasopharyngeal swab cycle threshold (Ct) ≤35 were 93%/63%. Antigen concentrations ranged from 1.28-3844 pg/mL (N) and 1.65-1071 pg/mL (S) and correlated with disease severity. In MIS-C, antigens were detected in 3/53 (5.7%) samples (3 N-positive: 1.7, 1.9, 121.1 pg/mL; 1 S-positive: 2.3 pg/mL); the patient with highest N had positive nasopharyngeal RT-PCR (Ct 22.3) concurrent with blood draw. CONCLUSIONS:Ultrasensitive blood SARS-CoV-2 antigen measurement has high diagnostic yield in children with acute COVID-19. Antigens were undetectable in most MIS-C patients, suggesting that persistent antigenemia is not a common contributor to MIS-C pathogenesis.