Biomarker Assays Research Articles

Correlations of p‐tau217, p‐tau181 and tau levels between CSF and plasma that are measured by fully automated immunoassay platform

AbstractBackgroundCerebrospinal fluid (CSF)‐based ATN classification (A: β‐amyloid (Aβ) deposition, T: pathogenic tau accumulation, N: neurodegeneration) is being widely accepted for the characterization of Alzheimer’s disease (AD). However, for the implementation of ATN classification in clinical practice, blood‐based biomarker is preferable in terms of specimen collection ease. Therefore, we have developed the blood‐based biomarker assays that correlate well with CSF levels. We have previously demonstrated that the plasma tau and phosphorylated tau at threonine‐181 (p‐tau181) immunoassays can significantly differentiate between AD, mild cognitive impairment (MCI), and cognitively normal (CN) groups. Here, we evaluated the correlation of biomarker levels between CSF and plasma utilizing the biomarker panel with addition of phosphorylated tau at threonine‐217 (p‐tau217) immunoassay.MethodWe have measured p‐tau217, p‐tau181 and tau using commercially available specimens on a fully automated immunoassay platform, HISCLTM analyzer. The platform can achieve high degree of sensitivity, requires only 30 µl of sample and 17 minutes to complete the measurement per assay. The bio‐analytical performance for the immunoassays was evaluated by measuring lower limit of quantification (LLOQ). The developed immunoassays were used to measure the paired CSF and plasma specimens from AD (n = 10), MCI (n = 35) and CN (n = 9) groups.ResultThe quantitative sensitivity for p‐tau217, p‐tau181 and tau were sufficient to measure plasma specimens in all groups. The plasma biomarker levels were significantly different between the AD and CN groups for all the biomarkers. We have found good correlations of biomarker levels between CSF and plasma for p‐tau217 (rs = 0.594, p < 0.01), p‐tau181 (rs = 0.453, p < 0.01) and tau (rs = 0.326, p < 0.05).ConclusionOur results indicated the possibility of detecting plasma p‐tau217, p‐tau181 and tau levels which may reflect the CSF levels. In addition to these assays, we have also developed plasma Ab and neurofilament light chain assays that had demonstrated good correlation with CSF levels. Thus, all the plasma assays developed on the HISCL analyzer could assess ATN classification. The blood‐based biomarker panel using our fully automated assay will accelerate research in the dementia field and may help in appropriate diagnosis of patients with dementia in routine clinical practice.

Cross‐cohort correlation of plasma biomarkers for Alzheimer’s disease with cognitive measures in the Global Alzheimer’s Association Interactive Network

AbstractBackgroundRecent advances in assay development for Alzheimer’s disease (AD) biomarkers in the blood have led to increased sensitivity and specificity for detecting proteins such as the 42 amino acid amyloid‐beta (Aβ42), total and phosphorylated tau protein (t‐tau and p‐tau, respectively), and neurofilament light (NfL).MethodUsing data available in the Global Alzheimer’s Association Interactive Network (GAAIN), contributed by several studies such as the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL), the Health and Aging Brain Study: Health Disparities (HABS‐HD), and the Alzheimer’s Disease Neuroimaging Initiative (ADNI), we correlated AD plasma biomarkers with cognitive diagnosis outcomes in a preliminary analysis. A full study with additional data sets and analysis methods, including controlling for confounding variables such as age, sex, years of education, and co‐existing medical conditions, is planned.ResultUsing logistic regression in the GAAIN Interrogator data analysis tool, we preliminarily found that participants with Aβ42 levels in the upper quartile of all measurements were not associated with mild cognitive impairment (MCI) or dementia diagnoses, the upper quartile of t‐tau and p‐tau181 measurements were associated with dementia but not MCI diagnoses, and the upper quartile of NfL measurements were associated with both MCI and dementia diagnoses.ConclusionBlood‐based biomarker assays that detect proteins implicated in Alzheimer’s disease have the potential for early and accurate diagnosis and are less invasive and costly than lumbar punctures for cerebrospinal fluid (CSF) collection and positron emission tomography (PET) imaging. This meta‐analysis contributes to the growing knowledge base of the prognostic utility of blood‐based biomarker assays.

Abstract C016: Evaluating clinical biomarkers of FHD-286, a potent and selective inhibitor of BRG1/BRM, in metastatic uveal melanoma

Abstract The BRG/Brahma-associated factors (BAF) family of chromatin remodeling complexes (also referred to as the mSWI/SNF complex) regulates chromatin accessibility and gene expression through its ATP-dependent remodeling activity. FHD-286, a first-in-class compound that potently and selectively inhibits the ATPase components of the BAF complex, BRG1 and BRM, was evaluated in a Phase I dose escalation in subjects with metastatic uveal melanoma (mUM) (FHD-286-001). The pharmacodynamics of FHD-286 were assessed to demonstrate proof-of-mechanism and understand the downstream molecular and clinical impact of BRG1/BRM inhibition. In addition, circulating tumor DNA (ctDNA) was evaluated as an early predictor of overall survival benefit. Seventy-three subjects were on a daily dosing regimen ranging from 2.5 to 10 mg or an intermittent regimen of 1-week on/1-week-off ranging from 10 to 22.5 mg. Tumor biopsies were collected at screening and either Cycle 3, or end of treatment. Biomarker changes in the tumor were observed by histological assessment, by IHC/IF biomarker assays, and RNA sequencing. Observations were suggestive of a differentiation effect by FHD-286 on the tumor cells, including a decrease in stemness genes and an increase in mature melanocytic markers. Evidence of necrosis and a trend of decreasing tumor cell density were also seen. In addition, ctDNA was measured in serial plasma samples using a targeted NGS panel. A reduction in ctDNA was shown in approximately 50% of subjects, which correlated with an increased overall survival benefit. Lastly, serial blood samples collected in Paxgene RNA tubes were analyzed by RNA sequencing. A robust dose-dependent gene signature was identified as a peripheral readout of target engagement at steady-state in both dosing regimens and across dose cohorts. In summary, we observed changes in the tumor cells at the biopsy site and ctDNA that suggest FHD-286 has a biological impact on uveal melanoma that may lead to clinical benefit. Given the limitations and challenges to obtain tumor biopsies from every subject and at a frequent rate, the identified PD gene signature in the blood is a valuable tool to measure target engagement in this study, and other potential solid tumor indications. Citation Format: Jessica Wan, Kim Horrigan, GiNell Elliott, Liv Johannessen, Mike Collins, Alexander J. Lazar, Dillon Corrigan, Caitlin Patriquin, Sam Agresta, Jessica Piel, Martin Hentemann. Evaluating clinical biomarkers of FHD-286, a potent and selective inhibitor of BRG1/BRM, in metastatic uveal melanoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C016.

Abstract B008: Machine-learning enabled quantification of colocalized multiplex IHC signals with spectral overlap

Abstract Background: Immunohistochemistry (IHC) is the gold standard for detecting protein biomarkers in cancer. In breast cancer, several nuclear biomarkers are relevant for guiding treatment decisions, including estrogen receptor (ER) and progesterone receptor (PR), indicative of response to endocrine therapy, and the proliferation marker Ki67, indicative of response to CDK inhibition1. However, these nuclear markers are detected using chromogens that colocalize and spectrally overlap with hematoxylin (HTX), the standard nuclear counterstain for IHC. Thus, the accurate detection and quantification of nuclear antigens is challenging. To overcome this limitation of IHC-based biomarker assays, we developed a method to identify colocalized signals after staining with chromophores with spectral overlap. Methods: Multiplex IHC (mIHC) using primary antibodies against Ki67, PR, and ER was performed on a breast cancer tissue microarray (N=65 cores). Antigens were detected by dabsyl-, tamra-, and Cy5-conjugated secondary antibodies, respectively, and slides were counterstained with HTX. The cores were scanned with brightfield imaging (Aperio AT2 and 3DHistech microscopes). The same cores were imaged on a custom brightfield microscope using an LED light source and 10 nm wide band-pass filters at 405, 450, 520, 550, 589, 632 and 660 nm. Images were spectrally unmixed using absorption spectra of the different chromophores and the background estimated based on the images themselves and aligned with the brightfield images. Pathologist-trained individuals collected exhaustive manual annotations in regions of interest for each marker and HTX using unmixed channels. A convolutional neural network (CNN) was trained using the annotations for each marker and HTX and applied to the WSI. Predicted cell locations were aggregated across the four channels to obtain an exhaustive “super-annotation” co-expression map of the WSI. Super-annotations were then used to train a CNN to detect ER/PR/Ki67/HTX colocalization in the aligned RBG image of the same slide. Predictions from the super-annotation model were compared to ER/PR/Ki67/HTX detection done via HALO (Indica Labs). Results: Our multi-spectral imaging pipeline allowed for comprehensive annotations of all nuclei positive for at least one of the three markers or HTX. Using our super-annotation model, the count error of nuclei that are positive for HTX and one, two, or three markers was reduced by 7.8%, 6.4%, and 0.03%, respectively, compared to HALO. Conclusions: We have developed a method to identify colocalized antigens after mIHC using chromogens with spectral overlap. Related work to extend this approach to membranous biomarkers is ongoing and will enable additional novel analyses (e.g., describing the precise spatial organization of PD-L1+ tumor cells relative to CD8+ tumor-infiltrating lymphocytes). The approach that we describe here has great potential to facilitate the utilization of mIHC for the clinical detection of biomarkers in oncology.

Plasma pTau217: single vs multiple phospho‐site assays.

AbstractBackgroundBlood biomarkers have gained much attention in recent years given their increasing ability to indicate amyloid pathology and excellent performance in distinguishing diagnostic groups in the context of Alzheimer’s disease (AD). Plasma pTau217 has been considered the best candidate biomarker to serve as diagnostic and prognostic tool for clinical trials. However, biomarker assays targeting tau phosphorylation on Thr217 may differ because of their composition (e.g., targeting multiple or single phosphorylation sites) which may lead to distinct associations with pathology. Thus, we aimed to compare two plasma pTau217 immunoassays that differ in regard to their target specificity.MethodParticipants from the TRIAD cohort with available cross‐sectional plasma and imaging data, incorporating within the AD spectrum, were included in this study (Young = 25;CU‐ = 107;CU+ = 32;MCI+ = 42;AD+ = 47;MCI‐ = 19). Plasma pTau was quantified using the assays from Janssen (pTau217+; which exhibits enhanced signal with co‐phosphorylation of pTau212) and from ALZpath (single phosphorylation on pTau217), both performed on the Simoa platform. Amyloid and tau pathologies were indexed by PET using [18F]AZD4694 and [18F]MK6240, respectively. ANCOVA compared biomarker values across groups, Spearman rank tested the correlations between them. Linear regression models (LM) evaluated the association between plasma and PET biomarkers globally and at the voxel level.ResultBiomarker levels (fold mean of CU‐) were comparable across diagnostic groups, showing the expected increases in amyloid positive groups. Both plasma assays were highly correlated with amyloid (Fig.1), which was also observed on the LM adjusted by age and sex, and on the voxel‐wise analysis (Fig.2). Associations with tau PET SUVR (medial temporal) were also significant and showed similar distribution between the two pTau assays. When neocortical tau was evaluated, however, pTau217+ had a better association as well as higher correlation coefficients within amyloid+ (Fig.3) and neocortical tau+ groups than did ALZpath pTau217.ConclusionIn general, pTau217 assays with different antibody specificity showed very similar associations with PET. However, pTau217+ achieved a better association with neocortical tau as compared to ALZpath pTau217. A biomarker targeting multiple phosphorylation sites might be a better proxy of advanced tau, therefore may offer improved ability to detect and exclude participants with advanced AD pathology for clinical trials.

Abstract C013: Clinical biomarkers based on PK/PD modeling to guide the development for a first-in-class, highly selective SMARCA2 (BRM) degrader, PRT3789

Abstract Background: The SWI/SNF family of chromatin-remodeling complexes is frequently dysregulated in multiple tumor types, resulting in aberrant expression of genes. Damaging mutations resulting in loss of function of one of its core catalytic subunits SMARCA4 (BRG1) occur in multiple tumor types, including in 5-10% of non-small cell lung cancer (NSCLC). SMARCA4-deleted cells become highly dependent on the other catalytic subunit, SMARCA2 (BRM), for their survival, therefore selective degradation of SMARCA2 has therapeutic potential in these cancer types. PRT3789 is a first-in-class, potent and selective SMARCA2 degrader that is currently under evaluation in a Phase 1 study in patients with advanced solid tumors with loss of SMARCA4 (NCT05639751). We deeply characterized the degradation kinetic parameters of PRT3789 based on preclinical PK/PD studies, which enabled dose-selection. Based on this modeling, we developed a suite of biomarker assays that can be used to assess target engagement in this Phase 1 study. Methods: A sensitive and quantitative plate-based immunoassay utilizing the MSD® platform was developed for the measurement of SMARCA2 protein in human PBMCs following treatment with PRT3789. A secondary, qPCR-based assay was developed for assessment of SMARCA2 transcriptional targets in peripheral blood that may be associated with response to SMARCA2 degradation by PRT3789. Additionally, an immunohistochemistry (IHC) assay for the assessment of SMARCA2 and SMARCA4 protein expression in tumor tissue was developed on the Leica BOND III platform using commercially available antibodies. Results: Detailed in vitro enzymology characterized the degradation kinetic parameters of PRT3789 and was successfully used to predict the PRT3789 effects observed in PK/PD and xenograft efficacy studies in mice. The MSD® assay demonstrated a wide dynamic range of detection for SMARCA2 with a lower level of quantification of 23 pg/mL, as well as exhibiting acceptable spike recovery and minimal cross-reactivity to SMARCA4. Based on the qPCR assay, we developed a differential gene expression (DGE) score based on expression of an 8-gene panel that correlates with SMARCA2 protein degradation in PBMCs. The IHC assay demonstrated discrete and robust nuclear tumor staining with a dynamic intensity range and limited cytoplasmic background for both SMARCA2 and SMARCA4. A panel of NSCLC tumor samples with known SMARCA4 mutations were further evaluated in this assay. Loss of SMARCA4 protein expression in tumor cells by IHC was associated with nonsense and frameshift mutations in the SMARCA4 gene. Conclusions: We demonstrate the sensitive assessment of SMARAC2 protein degradation and effects on downstream gene expression in PBMCs, which will be used to assess target engagement following treatment with PRT3789 in a Phase 1 clinical study. Citation Format: Min Wang, Andrew Moore, Alexander Grego, Miles Cowart, Joseph Rager, Gina Paris, Koichi Ito, Naveen Babbar, Sandy Geeganage, Peegy Scherle, Neha Bhagwat. Clinical biomarkers based on PK/PD modeling to guide the development for a first-in-class, highly selective SMARCA2 (BRM) degrader, PRT3789 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C013.

Head‐to‐head comparison of CSF AD biomarker assays: fully‐automated high‐throughput platforms (ECLIA and CLEIA) vs ELISA

AbstractBackgroundBiomarkers in cerebrospinal fluid (CSF) can help to diagnose Alzheimer’s disease (AD) and classify patients according to the amyloid/tau/neurodegeneration (ATN) research framework.MethodWe directly compared the performance of three different testing systems, i..e. traditional solid‐phase sandwich ELISA assays (Innotest®, Fujirebio Europe N.V., Gent, Belgium, and IBL, Minneapolis, USA), a fully automated electrochemiluminescence immunoassays (ECLIA) platform (Elecsys® CSF assays measured on the cobas e 411 analyzer, Roche Diagnostics International Ltd, Rotkreuz, Switzerland), and a fully automated chemiluminescent enzyme immunoassays (CLEIA) platform (Lumipulse® G600II, Fujirebio Europe N.V., Gent, Belgium), in 50 early AD patients (64±8.1y) and 25 cognitively unimpaired (CU) controls (65±9.4y)] from the same cohort. Individual CSF biomarkers Aβ42, Aβ40, pTau181 and tTau were measured using all systems. Ratios for pTau181/Aβ42, tTau/Aβ42, Aβ42/Aβ40, pTau181/Aβ42/Aβ40, tTau/Aβ42/Aβ40, pTau181/Aβ40, and tTau/Aβ40 were calculated.ResultAcross all systems, biomarkers and their ratios accurately distinguished AD patients from CU in a comparable manner: ROC: AUCs >0.96 (upper bound of CI:1.0)], except for ELISA Aβ42/Aβ40 [AUC = 0.948 (CI: 0.901‐0.995)]; according to Youden Index (YI)‐derived optimal cut‐offs, sensitivity and specificity were comparably high. YI cut‐offs and manufacturer (MA) cut‐offs were highly consistent. However, for the ECLIA platform (Elecsys®), the YI cut‐off led to significantly higher sensitivity for tTau (97.8% [0.88‐1.00%]/ 80.9%) and pTau181 (97.8% [0.88‐1.00%]/ 83.0%), and higher specificity for Aβ42 (96.0% [79‐100%]/ 76.0%), than MA cut‐off, maybe due to high prevalence of AD. The two fully automated platforms showed markedly reduced maximal coefficients of variation (CV) than ELISA.ConclusionIn addition to increased time efficiency, fully automated platforms showed more consistent results than ELISA assays which favor single analyte measurements. Improved accuracy using YI cut‐offs suggests that in‐house cut‐offs might be important for optimal performance. The two fully automated platforms had comparable accuracy. Thus, the platform of choice might depend on the user’s needs; for instance, the pTau181/Aβ42 ratio (Elecsys®; (AUC: 1.0 [1.0‐1.0], sensitivity 100%, specificity 100%)) combines the two AD hallmarks (A and T) in one test. For ATN classification, the Aβ42/Aβ40 ratio (Lumipulse®, AUC: 1.0 [1.0‐1.0], sensitivity 100%, specificity 100%) might be a more accurate marker for amyloid positivity (A+) than Aβ42 alone.

Racial and Ethnic Differences in Plasma Biomarker Eligibility in a Preclinical Alzheimer’s Disease Trial

AbstractBackgroundIn Alzheimer’s disease (AD) trials, differential screen failure due to cognitive and biomarker requirements may contribute to underrepresentation of racially and ethnically minoritized groups. The AHEAD 3‐45 Study (NCT04468659) is an ongoing program testing lecanemab at the stage of preclinical AD that utilizes plasma biomarker prescreening, acquired before cognitive, clinical, and medical history eligibility assessments, to enrich for participants likely to qualify based on amyloid PET eligibility criteria.MethodsWe examined the frequency of plasma amyloid biomarker eligibility among racial and ethnic groups in the AHEAD Study. We assigned participants ages 55‐80 to mutually exclusive groups: Hispanic Black (HB), Hispanic White (HW), Non‐Hispanic Asian (NHA), Non‐Hispanic Black (NHB), and Non‐Hispanic White (NHW). We used univariate logistic regression models to explore group differences in screen failure rates as determined by an algorithm that includes the plasma Aβ 42/40 ratio, age, and APOE status. The algorithm indicates an adequately high probability of elevated brain amyloid (>20 centiloids). We further explored whether APOE ε4 status (carrier vs non‐carrier) contributed to group differences.ResultsAmong 4274 participants undergoing plasma screening, 59 (1.4%) were HB, 622 (14.6%) were HW, 74 (1.73%) were NHA, 329 (7.7%) were NHB, and 3190 (74.6%) were NHW. Screen failure rates were 86% for HB, 76% for HW; 80% for NHA; 77% for NHB, and 62% for NHW. Using NHW participants as a reference group, we observed increased odds of screen failure among all other groups (HB OR = 4.0 95% CI 2.0, 9.1; HW OR = 2.0 95% CI 1.6, 2.4; NHA OR = 2.5 95% CI 1.4, 4.5; NHB OR = 2.1, 95% CI 1.6, 2.7). Observed differences were consistent across APOE ε4 carriers and non‐carriers.ConclusionDifferential rates of amyloid eligibility were observed despite the lack of systematic sample bias due to clinical or cognitive requirements observed in previous studies. Potential explanations for these observations include differences in clinical trial access, incidences of elevated amyloid, needed cutoffs for biomarker assays, and confounding due to comorbidities or other unmeasured covariates.This work is supported by a public‐private partnership between Alzheimer’s Clinical Trial Consortium (U24 AG057437) and Eisai.

The association of neighborhood socioeconomic status, cognition, cardiovascular risk factors, and vascular risk biomarkers in the HABS‐HD study

AbstractBackgroundNeighborhood socioeconomic status (NSES) is linked to poorer cognition and physical health, influencing the risk of cardiovascular disease and mortality. Adverse neighborhood conditions have been also associated with higher cardiovascular biomarker levels. This study goal is to examine whether NSES is associated with cognitive function and whether this relationship is explained by associations between NSES and cardiovascular risk and biomarker factors in a sample of non‐Hispanic Whites (NHW) and Mexican American (MA) elders.MethodWe studied 1391 subjects from the Health and Aging Brain Study (HABS‐HD) with signed consent, fasting blood samples, 3T MRI, clinical evaluation, and cognitive testing. Area deprivation index (ADI) was used as a measure NSES. Cardiovascular risk factors (CVRF) were ascertained by self‐ report and medical history, and biomarkers were assayed via a multiple‐plex biomarker assay platform using electrochemiluminescence. Three linear regression models examined the association of ADI with cognitive performance, adjusting for age, sex, and education, CVRF, and vascular risk biomarkers.ResultSixty percent of the sample were female. MA were significantly younger, less educated, and had higher ADI scores than NHW. They also had lower scores in global cognition and executive function measures. MA had a higher prevalence of hypertension, diabetes and obesity (p = <0.0001). After controlling for demographic variables, ADI was associated with SEVLT [NHW (B = ‐0.003, t = ‐2.18, p = 0.02); MA (B = ‐0.005, t = ‐2.96, p = 0.003)] and Trails B [NHW (B = ‐0.006, t = ‐2.55, p = 0.01); MA (B = ‐0.01, t = ‐3.76, p = <0.001)] scores in both ethnic groups. Dyslipidemia influenced the association of ADI with SEVLT in MA, while obesity influenced the association of ADI and trails B in NHW. The association of ADI and trails B was partially influenced by IL6 in MA, and this association was partially influenced by TNF, CRP, and VCAM values in NHW.ConclusionNSES may influence cognitive functioning. This relationship is only partially explained by CVRF and vascular blood biomarkers. Future research is needed on the longitudinal relationships of NSES, cognitive impairment, and vascular factors.

Diagnostic of Novel Plasma Biomarkers in Controls, SCD and MCI Subjects

AbstractBackgroundAlzheimer’s disease (AD) is the most common cause of dementia, and a challenging disease for the development of plasma biomarkers. Blood samples are the most easily collectable samples for medical diagnosis. This study evaluates the potential of plasma biomarkers in identifying early stages of AD.Method189 participants were recruited at the NYU Alzheimer Disease Research Center. This cohort includes n = 75 with normal cognition (NL), n = 77 with subjective cognitive decline (SCD), n = 37 with mild cognitive impairment (MCI). Comprehensive neuropsychological and magnetic resonance imaging evaluations were conducted for all patients. Plasma biomarkers assays (total tau [t‐tau], neurofilament light [NfL], glial fibrillary acid protein [GFAP], ubiquitin carboxyl‐terminal hydrolase L1 [UCH‐L1], Aβ1‐42, Aβ1‐40 and pTau181) were measured using the SIMOA SR‐X, a novel technology that employs highly sensitive immunoassays with a limit of detection (LOD) under 100 fg/ml.ResultThe levels of NfL, t‐Tau, GFAP and UCH‐L1 were measured using the Neurology 4‐plex A. NfL levels showed a significant difference between NL and SCD (one tailed t‐ test p = 0.0459). GFAP showed statistically significant differences between NL and MCI, (one tailed t‐test p = 0.0369). Aβ1‐42 showed a significant difference between SCD and MCI (one tailed t‐test p = 0.0493). pTau181 levels showed a significant difference between NL, SCD and NL, MCI (p = 0.0447 and p = 0.0439, respectively). The ratio of pTau181/Aβ1‐42 shows a significant difference between NL and MCI (one tailed t‐test p = 0.0187). Correlation of these biomarkers with brain imaging and cognitive measures is underway.ConclusionBiomarker levels of NfL, GFAP, Aβ1‐42, pTau181 and Ratio pTau181/Aβ1‐42 in plasma samples showed several significant differences between the three groups of subjects. Plasma biomarkers can be useful for the diagnosis of AD related pathology at early stages.

Role of specific nutritional biomarkers in predicting post-operative complications among patients undergoing elective abdominal surgery.

Nutritional biomarkers like serum prealbumin, transferrin, retinol-binding protein (RBP), C-reactive protein (CRP), leptin, and insulin-like growth factor 1 (IGF1) have the inherent ability to diagnose undernutrition objectively before it is clinically manifested. The primary objective of the study was to evaluate the diagnostic efficacy of the specific nutritional biomarkers in predicting post-operative complications. A prospective cohort study was conducted in the department of surgery and included all patients aged 18 years and above who underwent elective abdominal surgery. Demographic details and clinical and surgical details were documented from the case records. Nutritional biomarker assay was done at admission. The post-operative complications occurring until discharge were graded using the Clavien-Dindo classification. The diagnostic accuracy of the specific nutritional biomarkers in predicting post-operative complications was assessed. A total of 320 patients were included in the study. Of these, 126 (39.38%) developed post-operative complications. Major complications accounted for 19.05% of the complications, while 80.95% were minor complications. Patients with blood prealbumin level less than 17.287 mg/dL had a higher incidence of complications (p < 0.001). Serum transferrin levels less than 168.04 mg/dL and IGF1 levels less than < 44.51 ng/ml showed increased incidence of complications (p < 0.001). The AUC was found to be the highest for serum IGF1 with 0.7782. Sensitivity was equally high for IGF1 and serum transferrin, with 76.98% for the former and 76.19% for the latter. Specific nutritional biomarkers, like serum prealbumin and transferrin, were efficient in predicting postoperative complications of patients before undergoing elective abdominal surgeries even after adjusting for confounders. This can facilitate preoperative corrective measures to lower the overall postoperative complications.

Real-time quantification of proteins secreted of conditioned media from mesenchymal stromal cells (MSC) in co-culture with hematopoietic progenitor cells

The interaction of mesenchymal stromal cells (MSC) in co-cultures with hematopoietic progenitor cells (HPC) in ex-vivo models induce the secretion of pro-inflammatory protein factors and influences HPC cell proliferation and differentiation processes. Different studies have shown the quantification of these biomarkers in other conditioned media using an ELISA and Luminex™ type immunoassay format. In this paper, we developed real-time quantification of biomarkers in conditioned media of these cells in co-cultures from bone marrow, umbilical cord blood, and Wharton's jelly sources using surface plasmon resonance imaging (SPRi). We show the detection and quantification of CCL2, CCL5, bFGF, IL-6, IL-8 and VEGF proteins with high sensitivity and specificity. SPRi can be used with a real-time analysis methodology and could be considered a tool for biomarker assays.

Evaluation of novel biomarkers for early pregnancy outcome prediction†.

To assess performance and discriminatory capacity of commercially available enzyme-linked immunosorbent assays of biomarkers for predicting first trimester pregnancy outcome in a multi-center cohort. In a case-control study at three academic centers of women with pain and bleeding in early pregnancy, enzyme-linked immunosorbent assays of biomarkers were screened for assay performance. Performance was assessed via functional sensitivity, assay reportable range, recovery/linearity, and intra-assay precision (%Coefficient of Variation). Top candidates were analyzed for discriminatory capacity for viability and location among 210 women with tubal ectopic pregnancy, viable intrauterine pregnancy, or miscarriage. Assay discrimination was assessed by visual plots, area under the curve with 95% confidence intervals, and measures of central tendency with two-sample t-tests. Of 25 biomarkers evaluated, 22 demonstrated good or acceptable assay performance. Transgelin-2, oviductal glycoprotein, and integrin-linked kinase were rejected due to poor performance. The best biomarkers for discrimination of pregnancy location were pregnancy-specific beta-1-glycoprotein 9, pregnancy-specific beta-1-glycoprotein 1, insulin-like growth factor binding protein 1, kisspeptin (KISS1), pregnancy-specific beta-1-glycoprotein 3, and beta parvin (PARVB). The best biomarkers for discrimination of pregnancy viability were pregnancy-specific beta-1-glycoprotein 9, pregnancy-specific beta-1-glycoprotein 3, EH domain-containing protein 3, KISS1, WAP four-disulfide core domain protein 2 (HE4), quiescin sulfhydryl oxidase 2, and pregnancy-specific beta-1-glycoprotein 1. Performance of commercially available enzyme-linked immunosorbent assays was acceptable for a panel of novel biomarkers to predict early pregnancy outcome. Of these, six and seven candidates demonstrated good discriminatory capacity of pregnancy location and viability, respectively, when validated in a distinct external population. Four markers demonstrated good discrimination for both location and viability.

Animal-free assessment of developmental toxicity: Combining PBPK modeling with the ReproTracker assay

in vitro screening platforms to assess teratogenic potential of compounds are emerging rapidly. ReproTracker is a human induced pluripotent stem cells (hiPSCs)-based biomarker assay that is shown to identify the teratogenicity potential of new pharmaceuticals and chemicals reliably. In its current state, the assay is limited to identifying the potential teratogenic effects and does not immediately quantify a clinical dose relevant to the exposure of chemicals or drugs observable in mothers or fetuses. The goal of this study was to evaluate whether the ReproTracker assay can be extrapolated in vivo and quantitatively predict developmental toxicity exposure levels of two known human teratogens, thalidomide, and carbamazepine. Here, we utilized Physiologically Based Pharmacokinetic (PBPK) modeling to describe the pharmacokinetic behavior of these compounds and conducted an in vitro to in vivo extrapolation (IVIVE) approach to predict human equivalent effect doses (HEDs) that correspond with in vitro concentrations potentially associated with adverse outcomes in ReproTracker. The HEDs derived from the ReproTracker concentration predicted to cause developmental toxicity were close to the reported teratogenic human clinical doses and the HED derived from the rat or rabbit developmental toxicity study. The ReproTracker derived-HED revealed to be sensitive and protective of humans. Overall, this pilot study demonstrated the importance of integrating PBPK model in extrapolating and assessing developmental toxicity in vitro. The combination of these tools demonstrated that they could improve the safety assessment of drugs and chemicals without animal testing.

Real World Experience and Clinical Utility of Esoguard® - Interim Data from the Lucid Registry

Background: Barrett’s Esophagus (BE) is the only known precursor to esophageal adenocarcinoma (EAC), but historically, screening rates have been low, likely influenced by limitations of upper endoscopy (UE) as the traditional screening modality. EsoGuard® (EG) is a DNA biomarker assay, and EsoCheck® (EC) a noninvasive, swallowable capsule device designed to collect cells from a targeted region of the esophagus. EG and EC in combination offers a well-tolerated, in-office triage test to facilitate BE detection in patients with multiple risk factors. The Lucid Registry captures real-world data from commercial use of EC with EG; we present an interim review of clinical utility data from the first 517 subjects. Methods: Multicenter, prospective, registry capturing data from patients undergoing EC/EG in the commercial setting. This snapshot includes subjects enrolled from registry initiation (April 14, 2023) through August 16, 2023. The primary measure of clinical utility was provider decision impact, namely agreement between EG results and physician decision on whether to refer for subsequent UE. The relationship between BE/EAC risk factors and EG positivity rates was also assessed. Results: Average age was 47.9±14.3 years, 47.2% had history of gastroesophageal reflux disease (GERD), and 63.8% had ≥3 traditional BE/EAC risk factors. 58.8% of subjects were firefighters; when firefighting was treated as an additional BE/EAC risk factor, 81.2% of the study population had ≥3. EG positivity was 14.1%. 437 subjects were followed to the clinical utility endpoint: agreement between EG (+) results and referral for UE was 100%; agreement between EG (-) results and non-referral for UE was 99.4%; concordance between EG results and UE referral was 97.9%. Conclusions: Experience from the Lucid Registry demonstrates that physicians who have adopted EC/EG in the commercial setting are reliably utilizing EG as a triage test to inform decisions on which patients to refer for further endoscopic evaluation of BE.

Combination of verteporfin-photodynamic therapy with 5-aza-2'-deoxycytidine enhances the anti-tumour immune response in triple negative breast cancer.

Triple negative breast cancer (TNBC) is a subtype of breast cancer characterised by its high tumourigenic, invasive, and immunosuppressive nature. Photodynamic therapy (PDT) is a focal therapy that uses light to activate a photosensitizing agent and induce a cytotoxic effect. 5-aza-2'-deoxycytidine (5-ADC) is a clinically approved immunomodulatory chemotherapy agent. The mechanism of the combination therapy using PDT and 5-ADC in evoking an anti-tumour response is not fully understood. The present study examined whether a single dose of 5-ADC enhances the cytotoxic and anti-tumour immune effect of low dose PDT with verteporfin as the photosensitiser in a TNBC orthotopic syngeneic murine model, using the triple negative murine mammary tumour cell line 4T1. Histopathology analysis, digital pathology and immunohistochemistry of treated tumours and distant sites were assessed. Flow cytometry of splenic and breast tissue was used to identify T cell populations. Bioinformatics were used to identify tumour immune microenvironments related to TNBC patients. Functional experiments showed that PDT was most effective when used in combination with 5-ADC to optimize its efficacy. 5-ADC/PDT combination therapy elicited a synergistic effect in vitro and was significantly more cytotoxic than monotherapies on 4T1 tumour cells. For tumour therapy, all types of treatments demonstrated histopathologically defined margins of necrosis, increased T cell expression in the spleen with absence of metastases or distant tissue destruction. Flow cytometry and digital pathology results showed significant increases in CD8 expressing cells with all treatments, whereas only the 5-ADC/PDT combination therapy showed increase in CD4 expression. Bioinformatics analysis of in silico publicly available TNBC data identified BCL3 and BCL2 as well as the following anti-tumour immune response biomarkers as significantly altered in TNBC compared to other breast cancer subtypes: GZMA, PRF1, CXCL1, CCL2, CCL4, and CCL5. Interestingly, molecular biomarker assays showed increase in anti-tumour response genes after treatment. The results showed concomitant increase in BCL3, with decrease in BCL2 expression in TNBC treatment. In addition, the treatments showed decrease in PRF1, CCL2, CCL4, and CCL5 genes with 5-ADC and 5-ADC/PDT treatment in both spleen and breast tissue, with the latter showing the most decrease. To our knowledge, this is the first study that shows which of the innate and adaptive immune biomarkers are activated during PDT related treatment of the TNBC 4T1 mouse models. The results also indicate that some of the immune response biomarkers can be used to monitor the effectiveness of PDT treatment in TNBC murine model warranting further investigation in human subjects.

In Vivo Assessment of Laboratory-Grown Kidney Tissue Grafts.

Directed differentiation of stem cells is an attractive approach to generate kidney tissue for regenerative therapies. Currently, the most informative platform to test the regenerative potential of this tissue is engraftment into kidneys of immunocompromised rodents. Stem cell-derived kidney tissue is vascularized following engraftment, but the connection between epithelial tubules that is critical for urine to pass from the graft to the host collecting system has not yet been demonstrated. We show that one significant obstacle to tubule fusion is the accumulation of fibrillar collagens at the interface between the graft and the host. As a screening strategy to identify factors that can prevent this collagen accumulation, we propose encapsulating laboratory-grown kidney tissue in fibrin hydrogels supplemented with candidate compounds such as recombinant proteins, small molecules, feeder cells, and gene therapy vectors to condition the local graft environment. We demonstrate that the AAV-DJ serotype is an efficient gene therapy vector for the subcapsular region and that it is specific for interstitial cells in this compartment. In addition to the histological evaluation of epithelial tubule fusion, we demonstrate the specificity of two urine biomarker assays that can be used to detect human-specific markers of the proximal nephron (CD59) and the distal nephron (uromodulin), and we demonstrate the deposition of human graft-derived urine into the mouse collecting system. Using the testing platform described in this report, it will be possible to systematically screen factors for their potential to promote epithelial fusion of graft and host tissue with a functional intravital read-out.

Serum Beta-Defensin-2 is a biomarker for psoriasis but not subclinical atherosclerosis: Role of IL17a, PI-3 kinase and Rac1.

Beta-defensins (BDs) are antimicrobial peptides secreted upon epithelial injury. Both chemotactic and antimicrobial properties of BDs function as initial steps in host defense and prime the adaptive immune system in the body. Psoriasis, a chronic immune-mediated inflammatory disease, has both visible cutaneous manifestations as well as known associations with higher incidence of cardiometabolic complications and vascular inflammation. We aimed to investigate the circulating expression of beta-defensin-2 (BD2) in psoriasis at baseline compared to control subjects, along with changes in BD2 levels following biologic treatment at one-year. The contribution of BD2 to subclinical atherosclerosis is also assessed. In addition, we have sought to unravel signaling mechanisms linking inflammation with BD2 expression. Multimodality imaging as well inflammatory biomarker assays were performed in biologic naïve psoriasis (n=71) and non-psoriasis (n=53) subjects. A subset of psoriasis patients were followed for one-year after biological intervention (anti-Tumor Necrosis Factor-α (TNFα), n=30; anti-Interleukin17A (IL17A), n=21). Measurements of circulating BD2 were completed by Enzyme-Linked Immunosorbent Assay (ELISA). Using HaCaT transformed keratinocytes, expression of BD2 upon cytokine treatment was assessed by quantitative polymerase chain reaction (qPCR) and ELISA. Herein, we confirm that human circulating BD2 levels associate with psoriasis, which attenuate upon biologic interventions (anti-TNFα, anti-IL-17A). A link between circulating BD2 and sub-clinical atherosclerosis markers was not observed. Furthermore, we demonstrate that IL-17A-driven BD2 expression occurs in a Phosphatidylinositol 3-kinase (PI3-kinase) and Rac1 GTPase-dependent manner. Our findings expand on the potential role of BD2 as a tractable biomarker in psoriasis patients and describes the role of an IL-17A-PI3-kinase/Rac signaling axis in regulating BD2 levels in keratinocytes.

Do Global Dietary Nutrient Datasets Associate with Human Biomarker Assessments? A Regression Analysis

BackgroundTo assess the state and trends of global nutrition, our best tools are nationally representative assessments using human biomarker assays, yet these are expensive and logistically challenging. We instead often rely on more easily produced global nutrient datasets—measures of nutrients provided by the diet—as a proxy for nutritional intake and deficiency, due to their greater geographic and temporal coverage. However, the accuracy of global nutrient datasets is questionable. ObjectiveWe aimed to test whether estimates of inadequate dietary intake derived from existing global nutrient datasets reliably associate with biophysical deficiency. DesignWe performed linear regressions of estimates of inadequate dietary nutrient intake derived from three global nutrient datasets—Global Dietary Database, Global Nutrient Database, and Global Expanded Nutrient Supply (GENuS) model—against the existing suite of nationally representative biomarker survey data for three key nutrients of global concern in two vulnerable demographic groups: zinc, folate, and vitamin A in females of childbearing age; and zinc and vitamin A in children younger than 5 y. ResultsWe found significant associations (P < 0.1) for only 3 of 22 regressions between global nutrition datasets and biophysical deficiency: zinc for females of childbearing age from GENuS and Global Dietary Database, and zinc for children under 5 y from GENuS. Folate and vitamin A show no reliable relationship between nutrient datasets and independent biomarker surveys. Applying the successful models for zinc to the accompanying full datasets yield estimates of global zinc deficiency of 31%–37% for these demographic groups. ConclusionsWe found that few estimates of nutritional inadequacy from global dietary datasets are associated with more direct measures of biophysical deficiency from biomarker studies. Researchers and policymakers must be cautious when applying global nutrient datasets to questions of global health and use them for limited applications.

Behavioral and Neuropathological Phenotyping of the Tau58/2 and Tau58/4 Transgenic Mouse Models for FTDP-17.

The Tau58/2 and Tau58/4 mouse lines expressing 0N4R tau with a P301S mutation mimic aspects of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). In a side-by-side comparison, we report the age-dependent development of cognitive, motor, and behavioral deficits in comparison with the spatial-temporal evolution of cellular tau pathology in both models. We applied the SHIRPA primary screen and specific neuromotor, behavioral, and cognitive paradigms. The spatiotemporal development of tau pathology was investigated immunohistochemically. Levels of sarkosyl-insoluble paired helical filaments were determined via a MesoScale Discovery biomarker assay. Neuromotor impairments developed from age 3 months in both models. On electron microscopy, spinal cord neurofibrillary pathology was visible in mice aged 3 months; however, AT8 immunoreactivity was not yet observed in Tau58/4 mice. Behavioral abnormalities and memory deficits occurred at a later stage (>9 months) when tau pathology was fully disseminated throughout the brain. Spatiotemporally, tau pathology spread from the spinal cord via the midbrain to the frontal cortex, while the hippocampus was relatively spared, thus explaining the late onset of cognitive deficits. Our findings indicate the face and construct validity of both Tau58 models, which may provide new, valuable insights into the pathologic effects of tau species in vivo and may consequently facilitate the development of new therapeutic targets to delay or halt neurodegenerative processes occurring in tauopathies.