Plasma Counterpart Research Articles

The Alzheimer's Association Global Biomarker Standardization Consortium (GBSC) plasma phospho-tau Round Robin study.

Phosphorylated tau (p-tau) is a specific blood biomarker for Alzheimer's disease (AD) pathology. Multiple p-tau biomarkers on several analytical platforms are poised for clinical use. The Alzheimer's Association Global Biomarker Standardisation Consortium plasma phospho-tau Round Robin study engaged assay developers in a blinded case-control study on plasma p-tau, aiming to learn which assays provide the largest fold-changes in AD compared to non-AD, have the strongest relationship between plasma and cerebrospinal fluid (CSF), and show the most consistent relationships between methods (commutability) in measuring both patient samples and candidate reference materials (CRM). Thirty-three different p-tau biomarker assays, built on eight different analytical platforms, were used to quantify paired plasma and CSF samples from 40 participants. AD biomarker status was categorised as "AD pathology" (n=25) and "non-AD pathology" (n=15) by CSF Aβ42/Aβ40 (US-FDA; CE-IVDR) and p-tau181 (CE-IVDR) methods. The commutability of four CRM, at three concentrations, was assessed across assays. Plasma p-tau217 consistently demonstrated higher fold-changes between AD and non-AD pathology groups, compared to other p-tau epitopes. Fujirebio LUMIPULSE G, UGOT IPMS, and Lilly MSD p-tau217 assays provided the highest median fold-changes. In CSF, p-tau217 assays also performed best, and exhibited substantially larger fold-changes than their plasma counterparts, despite similar diagnostic performance. P-tau217 showed the strongest correlations between plasma assays (rho=0.81 to 0.97). Plasma p-tau levels were weakly-to-moderately correlated with CSF p-tau, and correlations were non-significant within the AD group alone. The evaluated CRM were not commutable across assays. Plasma p-tau217 measures had larger fold-changes and discriminative accuracies for detecting AD pathology, and better agreement across platforms than other plasma p-tau variants. Plasma and CSF markers of p-tau, measured by immunoassays, are not substantially correlated, questioning the interchangeability of their continuous relationship. Further work is warranted to understand the pathophysiology underlying this dissociation, and to develop suitable reference materials facilitating cross-assay standardisation. Alzheimer's Association (#ADSF-24-1284328-C).

A Predictive Model for Initial Platinum-Based Chemotherapy Efficacy in Patients with Postoperative Epithelial Ovarian Cancer Using Tissue-Derived Small Extracellular Vesicles.

Epithelial ovarian cancer (EOC) is an often-fatal malignancy marked by the development of resistance to platinum-based chemotherapy. Thus, accurate prediction of platinum drug efficacy is crucial for strategically selecting postoperative interventions to mitigate the risks associated with suboptimal therapeutic outcomes and adverse effects. Tissue-derived extracellular vesicles (tsEVs), in contrast to their plasma counterparts, have emerged as a powerful tool for examining distinctive attributes of EOC tissues. In this study, 4D data-independent acquisition (DIA) proteomic sequencing was performed on tsEVs obtained from 58 platinum-sensitive and 30 platinum-resistant patients with EOC. The analysis revealed a notable enrichment of differentially expressed proteins that were predominantly associated with immune-related pathways. Moreover, pivotal immune-related proteins (IRPs) were identified by LASSO regression. These factors, combined with clinical parameters selected through univariate logistic regression, were used for the construction of a model employing multivariate logistic regression. This model integrated three tsEV IRPs, CCR1, IGHV_35 and CD72, with one clinical parameter, the presence of postoperative residual lesions. Thus, this model could predict the efficacy of initial platinum-based chemotherapy in patients with EOC post-surgery, providing prognostic insights even before the initiation of chemotherapy.

A review on the impact of single-stranded library preparation on plasma cell-free diversity for cancer detection.

In clinical oncology, cell-free DNA (cfDNA) has shown immense potential in its ability to noninvasively detect cancer at various stages and monitor the progression of therapy. Despite the rapid improvements in cfDNA liquid biopsy approaches, achieving the required sensitivity to detect rare tumor-derived cfDNA still remains a challenge. For next-generation sequencing, the perceived presentation of cfDNA is strongly linked to the extraction and library preparation protocols. Conventional double-stranded DNA library preparation (dsDNA-LP) focuses on assessing ~167bp double-stranded mononucleosomal (mncfDNA) and its other oligonucleosomal cell-free DNA counterparts in plasma. However, dsDNA-LP methods fail to include short, single-stranded, or nicked DNA in the final library preparation, biasing the representation of the actual cfDNA populations in plasma. The emergence of single-stranded library preparation (ssDNA-LP) strategies over the past decade has now allowed these other populations of cfDNA to be studied from plasma. With the use of ssDNA-LP, single-stranded, nicked, and ultrashort cfDNA can be comprehensively assessed for its molecular characteristics and clinical potential. In this review, we overview the current literature on applications of ssDNA-LP on plasma cfDNA from a potential cancer liquid biopsy perspective. To this end, we discuss the molecular principles of single-stranded DNA adapter ligation, how library preparation contributes to the understanding of native cfDNA characteristics, and the potential for ssDNA-LP to improve the sensitivity of circulating tumor DNA detection. Additionally, we review the current literature on the newly reported species of plasma ultrashort single-stranded cell-free DNA plasma, which appear biologically distinct from mncfDNA. We conclude with a discussion of future perspectives of ssDNA-LP for liquid biopsy endeavors.

A Plasma Exosomal Metabolic Profiling of Nonalcoholic Fatty Liver Disease Patients Complicated with Impaired Fasting Glucose.

Nonalcoholic fatty liver disease is considered as the hepatic manifestation of metabolic syndrome. Detection of circulating exosomes together with metabolomic analysis of their cargo would provide early signals for metabolic derangements and complications associated with nonalcoholic fatty liver disease. Therefore, this study profiled exosomal metabolome of patients with nonalcoholic fatty liver disease and impaired fasting glucose. Plasma exosomes were extracted from nonalcoholic fatty liver disease patients with or without impaired fasting glucose through differential ultracentrifugation. Their metabolite profiles were examined by ultrahigh-performance liquid chrom atography-quadrupole time-of-flight mass spectrometry. Pathway analysis was carried out on platform MetaboAnalyst 4.0. Thirty-nine patients were enrolled, including nonalcoholic fatty liver disease-alone group (n = 26) and age-and gender-comparable nonalcoholic fatty liver disease plus impaired fasting glucose group (n = 13). Although less than and different from their plasma counterparts, a total of 10 significantly differential exosomal metabolites were identified. Nonalcoholic fatty liver disease plus impaired fasting glucose group had higher concentrations of linoleic acid, palmitamide, stearamide, and oleamide, as well as a lower concentration of phosphatidylethanolamine [20:5(5Z,8Z,11Z,14Z,17Z)/20:5(5Z,8Z,11Z,14Z,17Z)]. Pathway analysis showed an obviously changed metabolism of linoleic acid. Metabolomic analysis of plasma exosomes revealed a distinct change in fatty acids and related pathways in nonalcoholic fatty liver disease patients with impaired fasting glucose. These preliminary results provide a metabolomic snapshot and basis for further investigation of exosome biology for these patients.

CSF proteomics in autosomal dominant Alzheimer’s disease highlights parallels with sporadic disease

AbstractBackgroundAutosomal dominant Alzheimer’s disease (ADAD) offers a unique opportunity to study pathophysiological changes in a relatively young population with few comorbidities. A comprehensive investigation of proteome changes occurring in ADAD could provide valuable insights into AD‐related biological mechanisms and uncover novel biomarkers and therapeutic targets. Furthermore, ADAD might serve as a model for sporadic AD, but in‐depth proteome comparisons are lacking. We aimed to identify dysregulated CSF proteins in ADAD and determine the degree of overlap with sporadic AD.MethodUsing proximity extension‐based immunoassays (PEA) by Olink, we measured 1472 proteins in CSF of APP or PSEN1 mutation carriers (n = 22) and age‐ and sex‐matched controls (n = 20) from the Amsterdam Dementia Cohort. We compared protein abundance between groups using two‐sided t‐tests and identified enriched biological pathways. Using the same protein panels in paired plasma samples, we investigated correlations between CSF proteins and their plasma counterparts. Finally, we compared our results with recently published PEA data from an international cohort of sporadic AD (n = 230) and non‐AD dementias (n = 301). All analyses were false discovery rate‐corrected.ResultWe detected 66 differentially abundant CSF proteins (65 increased, one decreased) in ADAD compared to controls (q<0.05). The most strongly upregulated proteins (fold change >1.8) were related to immunity (CHIT1, ITGB2, SMOC2), cytoskeletal structure (MAPT, NEFL) and tissue remodeling (TMSB10, MMP‐10). Significant CSF‐plasma correlations were found for the upregulated proteins SMOC2 and LILR1B. 36 of the 66 differentially expressed proteins had been previously measured in the sporadic dementias cohort, 34 of which (94%) were also significantly upregulated in sporadic AD, with a strong correlation between the fold changes of these proteins in both cohorts (rs = 0.730, p<0.001). 29 of these 36 proteins (81%) were also upregulated among non‐AD patients with suspected AD co‐pathology.ConclusionThis CSF proteomics study demonstrates substantial biochemical similarities between ADAD and sporadic AD, suggesting involvement of the same biological processes. Besides known AD‐related proteins, we identified several relatively novel proteins, such as TMSB10, MMP‐10 and SMOC2, which have potential as novel biomarkers. With shared pathophysiological CSF changes, ADAD study findings might be translatable to sporadic AD, which could greatly expedite therapy development.

A finger prick collection method for detecting blood biomarkers of neurodegeneration – a pilot study (DROP‐AD)

AbstractBackgroundPlasma biomarkers have proven to indicate developing cerebral pathologies and injury that underpin neurodegenerative disorders in a non‐invasive manner. However, the measurement of certain blood biomarkers requires on‐site sampling with strict, time‐limited and temperature dependant processing protocols. To further facilitate the usefulness of blood biomarkers, methods for remote and/or unsupervised sample collection would greatly increase the utility as a screening and monitoring tool in secondary care and therapeutic trials.This pilot study investigated a novel method to quantify neurofilament light (NfL), glial fibrillary acidic protein (GFAP) and phosphorylated tau (p‐tau) in capillary dry blood spots (DBScapillary) and venous dry blood spots (DBSvenous).MethodsWe included 77 memory clinic participants from ACE Alzheimer Center Barcelona. DBScapillary, DBSvenous, and EDTA plasma as well as neuropsychological measures were obtained from each participant. A subset (n = 23) had cerebrospinal fluid (CSF) biomarkers. Capillary whole blood was obtained by a macro lancet needle from the left index finger. A total of 65µL of capillary and venous blood were spotted and dried on Noviplex DBS cards, which were shipped overnight without temperature control or cooling to Gothenburg, Sweden. Samples were extracted from DBScapillary and DBSvenous cards, and NfL, GFAP, p‐tau181, and, in a subset (n = 11), p‐tau217 were measured by single molecular array (Simoa). Statistical analysis included Pearson correlation between DBS and EDTA plasma.ResultsDBScapillary GFAP (r = 0.7180, p<0.0001), and NfL (r = 0.6967, p<0.0001) correlated highly with their counterparts in EDTA plasma. We also demonstrated that DBScapillary p‐tau217 (r = 0.8955, p = 0.0002), but not DBScapillary p‐tau181 (r = 0.0132, p = 0.9447) correlated with the same measures in EDTA plasma, likewise DBSvenous GFAP (r = 0.7614, p<0.0001), NfL (r = 0.7701, p<0.0001), p‐tau217 (r = 0.9625, p<0.0001) and p‐tau181 (r = 0.6619, p<0.0001). Moreover, DBScapillary GFAP and DBSvenous GFAP, NfL and p‐tau181 correlated with MMSE, CDR and amyloid status (all p<0.05).ConclusionThis pilot study demonstrates the potential of remote collection and quantification of GFAP, NfL, p‐tau217 and p‐tau181 without low‐temperature storage/transportation or centrifugation (DBSvenous). Our data also suggests that this may be possible from a simple finger prick collection, including p‐tau217. We speculate that this simple, self‐executable method of blood collection would facilitate regular monitoring of patients with suspected neurological conditions.

A Plasma Exosomal Metabolic Profiling of Nonalcoholic Fatty Liver Disease Patients Complicated with Impaired Fasting Glucose.

Nonalcoholic fatty liver disease is considered as the hepatic manifestation of metabolic syndrome. Detection of circulating exosomes together with metabolomic analysis of their cargo would provide early signals for metabolic derangements and complications associated with nonalcoholic fatty liver disease. Therefore, this study profiled exosomal metabolome of patients with nonalcoholic fatty liver disease and impaired fasting glucose. Plasma exosomes were extracted from nonalcoholic fatty liver disease patients with or without impaired fasting glucose through differential ultracentrifugation. Their metabolite profiles were examined by ultrahigh-performance liquid chrom atography-quadrupole time-of-flight mass spectrometry. Pathway analysis was carried out on platform MetaboAnalyst 4.0. Thirty-nine patients were enrolled, including nonalcoholic fatty liver disease-alone group (n = 26) and age-and gender-comparable nonalcoholic fatty liver disease plus impaired fasting glucose group (n = 13). Although less than and different from their plasma counterparts, a total of 10 significantly differential exosomal metabolites were identified. Nonalcoholic fatty liver disease plus impaired fasting glucose group had higher concentrations of linoleic acid, palmitamide, stearamide, and oleamide, as well as a lower concentration of phosphatidylethanolamine [20:5(5Z,8Z,11Z,14Z,17Z)/20:5(5Z,8Z,11Z,14Z,17Z)]. Pathway analysis showed an obviously changed metabolism of linoleic acid. Metabolomic analysis of plasma exosomes revealed a distinct change in fatty acids and related pathways in nonalcoholic fatty liver disease patients with impaired fasting glucose. These preliminary results provide a metabolomic snapshot and basis for further investigation of exosome biology for these patients.

Extracellular vesicle biomarkers for complement dysfunction in schizophrenia.

Schizophrenia, a complex neuropsychiatric disorder, frequently experiences a high rate of misdiagnosis due to subjective symptom assessment. Consequently, there is an urgent need for innovative and objective diagnostic tools. In this study, we used cutting-edge extracellular vesicles' (EVs) proteome profiling and XGBoost-based machine learning to develop new markers and personalized discrimination scores for schizophrenia diagnosis and prediction of treatment response. We analysed plasma and plasma-derived EVs from 343 participants, including 100 individuals with chronic schizophrenia, 34 first-episode and drug-naïve patients, 35 individuals with bipolar disorder, 25 individuals with major depressive disorder and 149 age- and sex-matched healthy controls. Our innovative approach uncovered EVs-based complement changes in patients, specific to their disease-type and status. The EV-based biomarkers outperformed their plasma counterparts, accurately distinguishing schizophrenia individuals from healthy controls with an area under curve (AUC) of 0.895, 83.5% accuracy, 85.3% sensitivity and 82.0% specificity. Moreover, they effectively differentiated schizophrenia from bipolar disorder and major depressive disorder, with AUCs of 0.966 and 0.893, respectively. The personalized discrimination scores provided a personalized diagnostic index for schizophrenia and exhibited a significant association with patients' antipsychotic treatment response in the follow-up cohort. Overall, our study represents a significant advancement in the field of neuropsychiatric disorders, demonstrating the potential of EV-based biomarkers in guiding personalized diagnosis and treatment of schizophrenia.

CSF proteomics in autosomal dominant Alzheimer's disease highlights parallels with sporadic disease.

Autosomal dominant Alzheimer's disease (ADAD) offers a unique opportunity to study pathophysiological changes in a relatively young population with few comorbidities. A comprehensive investigation of proteome changes occurring in ADAD could provide valuable insights into AD-related biological mechanisms and uncover novel biomarkers and therapeutic targets. Furthermore, ADAD might serve as a model for sporadic AD, but in-depth proteome comparisons are lacking. We aimed to identify dysregulated CSF proteins in ADAD and determine the degree of overlap with sporadic AD. We measured 1472 proteins in CSF of PSEN1 or APP mutation carriers (n = 22) and age- and sex-matched controls (n = 20) from the Amsterdam Dementia Cohort using proximity extension-based immunoassays (PEA). We compared protein abundance between groups with two-sided t-tests and identified enriched biological pathways. Using the same protein panels in paired plasma samples, we investigated correlations between CSF proteins and their plasma counterparts. Finally, we compared our results with recently published PEA data from an international cohort of sporadic AD (n = 230) and non-AD dementias (n = 301). All statistical analyses were false discovery rate-corrected. We detected 66 differentially abundant CSF proteins (65 increased, 1 decreased) in ADAD compared to controls (q < 0.05). The most strongly upregulated proteins (fold change >1.8) were related to immunity (CHIT1, ITGB2, SMOC2), cytoskeletal structure (MAPT, NEFL) and tissue remodelling (TMSB10, MMP-10). Significant CSF-plasma correlations were found for the upregulated proteins SMOC2 and LILR1B. Of the 66 differentially expressed proteins, 36 had been measured previously in the sporadic dementias cohort, 34 of which (94%) were also significantly upregulated in sporadic AD, with a strong correlation between the fold changes of these proteins in both cohorts (rs = 0.730, P < 0.001). Twenty-nine of the 36 proteins (81%) were also upregulated among non-AD patients with suspected AD co-pathology. This CSF proteomics study demonstrates substantial biochemical similarities between ADAD and sporadic AD, suggesting involvement of the same biological processes. Besides known AD-related proteins, we identified several relatively novel proteins, such as TMSB10, MMP-10 and SMOC2, which have potential as novel biomarkers. With shared pathophysiological CSF changes, ADAD study findings might be translatable to sporadic AD, which could greatly expedite therapy development.

OS08.3.A Distinct systemic and tumor microenvironment immune landscapes discriminate across sellar tumor types and controls through a methylation-based deconvolution method

Abstract Background Systemic (Sys) and tumor microenvironment (TME) immune milieus play a pivotal role in tumor development, outcome and immunotherapy response predictions across a variety of central nervous system tumors. Genome-wide methylation profiling can reliably discriminate and estimate immune cell proportions present in the blood and within the tumor and has not been reported across sellar tumor types (STT). Material and Methods We estimated cell composition in liquid biopsy (LB, serum/plasma) and tissue specimens from 42 STT collections (i.e., pituitary neuroendocrine tumors [PitNETs; n=37] and craniopharyngiomas [CP; n=5]), and 26 nontumor controls (LB: 11; Tissue: 15) using MethylCIBERSORT, a methylation-based deconvolution algorithm and established immune cell signatures as reference. LB methylation was profiled with EPIC array. Correlations between estimated cell proportions across sample sources were explored (Spearman). Immune cell proportion hierarchical k-means clustering was performed across tissue and LB specimens. Similarly, mean comparisons between and across sample types and subgroups of interest were performed [Non-parametric Kruskal-Wallis, Wilcoxon rank-sum tests; p&amp;lt;0.05]. Results We identified three immune-clusters across tissue specimens which distinguished controls (k3-cluster) from sellar tumor specimens (k1- and k2- clusters), primarily attributable to differential B-cell and monocyte proportions. Interestingly, a subset of PitNET and CP, belonging to the k2-cluster, presented a distinct immune profile compared to their K1-sellar tumor counterparts. Analysis of plasma-derived immune clusters revealed that PitNETs were distributed across four distinct immune patterns and CP clustered together with controls and a PitNET subset. One of the PitNET clusters was enriched with patients that died during follow-up and presented an enrichment of CD4-(including the regulatory subtype), CD8 and CD56-T and depletion of natural killer cells. Differences across serum- and tissue-derived clusters were present but less prominent than their plasma counterparts. No correlation between immune cell proportions across other clinicopathological features within each tumor type (sex, age, histotypes, invasion etc) was observed. Conclusion Our results suggest that PitNETs are characterized by differential TME and systemic immune subtypes which also distinguish these tumors from CP and controls. Additionally, distinct systemic immune composition between tissue and LB sources, more readily observed in plasma, suggest that the systemic response to the presence of the tumor is distinct from the immune response noted in the TME. Tumor immune subtyping may allow the stratification of STT according to immunotherapy response vulnerabilities.

Cytokine changes in cerebrospinal fluid and plasma after emergency orthopaedic surgery

Neuroinflammation after surgery and its contribution to peri-operative neurocognitive disorders (PND) is not well understood. Studying the association between central and peripheral cytokines and neuroinflammation is a prelude to the development of treatments for PND. Here, we investigate the hypotheses that there is a greater cytokine response in cerebrospinal fluid (CSF) than plasma after orthopaedic surgery, and that plasma cytokine levels are directly related to CSF cytokine levels, indicating that plasma cytokine levels may have potential as biomarkers of neuroinflammation. Patients admitted with a fractured neck of femur were invited to participate in this study. Participants had a spinal catheter inserted just prior to induction of anaesthesia. Samples of blood and CSF were taken before, immediately after, and on the first day following emergency surgery. The catheter was then removed. Samples were analysed for the presence of ten cytokines by immunoassay. A spinal catheter was successfully inserted in 11 participants during the 18-month study period. Five plasma cytokines (IL-4, IL-6, IL-10, IL-12p70 and IL-13) rose significantly following surgery, whereas all ten CSF cytokines rose significantly (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IFN-γ and TNF-α) (adjusted-p < 0.05). Central (CSF) cytokine levels were consistently higher than their peripheral (plasma) counterparts after surgery, with some patients having a particularly marked neuroinflammatory response. The greatest increases occurred in IL-8 in CSF and IL-6 in plasma. There were significant, strong positive correlations between several of the measured cytokines in the CSF after surgery, but far fewer in plasma. There was no significant correlation between cytokine levels in the plasma and CSF at each of the three time points. To our knowledge, this is the first study to analyse paired samples of plasma and CSF for cytokine levels before and after emergency orthopaedic surgery. This study demonstrates that following surgery for a fractured neck of femur, there is a far greater rise in cytokines in the CSF compared to plasma. The lack of correlation between peripheral and central cytokines suggests measurement of peripheral cytokines are not necessarily related to which patients may have a large neuroinflammatory response.

Circulating Exosomal miRNAs as Novel Biomarkers Perform Superior Diagnostic Efficiency Compared With Plasma miRNAs for Large-Artery Atherosclerosis Stroke.

Recently, exosomal miRNAs have been reported to be associated with some diseases, and these miRNAs can be used for diagnosis and treatment. However, diagnostic biomarkers of exosomal miRNAs for ischemic stroke have rarely been studied. In the present study, we aimed to identify exosomal miRNAs that are associated with large-artery atherosclerosis (LAA) stroke, the most common subtype of ischemic stroke; to further verify their diagnostic efficiency; and to obtain promising biomarkers. High-throughput sequencing was performed on samples from 10 subjects. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on exosomes and plasma in the discovery phase (66 subjects in total) and the validation phase (520 subjects in total). We identified 5 candidate differentially expressed miRNAs (miR-369-3p, miR-493-3p, miR-379-5p, miR-1296-5p, and miR-1277-5p) in the discovery phase according to their biological functions, 4 of which (miR-369-3p, miR-493-3p, miR-379-5p, and miR-1296-5p) were confirmed in the validation phase. These four exosomal miRNAs could be used to distinguish LAA samples from small artery occlusion (SAO) samples, LAA samples from atherosclerosis (AS) samples, and LAA samples from control samples and were superior to plasma miRNAs. In addition, composite biomarkers achieved higher area under the curve (AUC) values than single biomarkers. According to our analysis, the expression levels of exosomal miR-493-3p and miR-1296-5p were negatively correlated with the National Institutes of Health Stroke Scale (NIHSS) score. The four identified exosomal miRNAs are promising biomarkers for the diagnosis of LAA stroke, and their diagnostic efficiency is superior to that of their counterparts in plasma.

Remarkable plasma-resistance performance by nanocrystalline Y2O3\xb7MgO composite ceramics for semiconductor industry applications

Motivated by recent finding of crystallographic-orientation-dependent etching behavior of sintered ceramics, the plasma resistance of nanocrystalline Y2O3-MgO composite ceramics (YM) was evaluated for the first time. We report a remarkably high plasma etching resistance of nanostructure YM surpassing the plasma resistance of commercially used transparent Y2O3 and MgAl2O4 ceramics. The pore-free YM ceramic with grain sizes of several hundred nm was fabricated by hot press sintering, enabling theoretical maximum densification at low temperature. The insoluble two components effectively suppressed the grain growth by mutual pinning. The engineering implication of the developed YM nanocomposite imparts enhanced mechanical reliability, better cost effectiveness with excellent plasma resistance property over their counterparts in plasma using semiconductor applications.

Characterization of carp seminal plasma Wap65-2 and its participation in the testicular immune response and temperature acclimation

Two functionally distinct isoforms of warm-temperature acclimation related 65-kDa protein (Wap65-1 and Wap65-2) with a role in the immune response are present in fish. To our knowledge, contrary to Wap65-1, Wap65-2 has neither been isolated nor functionally characterized in carp especially in reproductive system. The aim of this study was to characterize Wap65-2 and ascertain its functions in immune response and temperature acclimation within reproductive system. Wap65-2 corresponded to one of the most abundant proteins in carp seminal plasma, with a high immunologic similarity to their counterparts in seminal plasma of other fish species and a wide tissue distribution, with predominant expression in the liver. The immunohistochemical localization of Wap65-2 to spermatogonia, Leydig cells, and the epithelium of blood vessels within the testis suggests its role in iron metabolism during spermatogenesis and maintenance of blood-testis barrier integrity. Wap65-2 secretion by the epithelial cells of the spermatic duct and its presence around spermatozoa suggests its involvement in the protection of spermatozoa against damage caused by heme released from erythrocytes following hemorrhage and inflammation. Our results revealed an isoform-specific response of Wap65 to temperature acclimation and Aeromonas salmonicida infection which alters blood-testis barrier integrity. Wap65-2 seems to be related to the immune response against bacteria, while Wap65-1 seems to be involved in temperature acclimation. This study expands the understanding of the mechanism of carp testicular immunity against bacterial challenge and temperature changes, in which Wap65-2 seems to be involved and highlights their potential usefulness as biomarkers of inflammation and temperature acclimation.

Expression of Matrix Metalloproteinases and Their Tissue Inhibitors in Peripheral Blood Leukocytes and Plasma of Children with Nonalcoholic Fatty Liver Disease.

Gene expression profiles of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) were evaluated in peripheral blood leukocytes of children with nonalcoholic fatty liver disease (NAFLD). Gene expression patterns were correlated with their plasma protein counterparts, systemic parameters of liver injury, and selected markers of inflammation. The MMP-2, MMP-9, MMP-12, MMP-14, TIMP-1, TIMP-2, TGF-β, and IL-6 transcripts levels were tested by the real-time PCR. Plasma concentrations of MMP-9, TIMP-1, MMP-9/TIMP-1 ratio, MMP-2/TIMP-2 ratio, sCD14, leptin, resistin, IL-1 beta, and IL-6 and serum markers of liver injury were estimated by ELISA. The MMP-9, TIMP-2 expression levels, plasma amounts of MMP-9, TIMP-1, and the MMP-9/TIMP-1 ratio were increased in children with NAFLD. Concentrations of AST, ALT, GGT, and leptin were elevated in serum patients with NAFLD, while concentration of other inflammatory or liver injury markers was unchanged. The MMP-2 and MMP-9 levels correlated with serum liver injury parameters (ALT and GGT concentrations, respectively); there were no other correlations between MMP/TIMP gene expression profiles, their plasma counterparts, and serum inflammatory markers. Association of MMP-2 and MMP-9 expression with serum liver injury parameters (ALT, GGT) may suggest leukocyte engagement in the early stages of NAFLD development which possibly precedes subsequent systemic inflammatory responses.

Abstract B31: Endocervical microRNA profiling for detection of ovarian cancer

Abstract Though the necessity for ovarian cancer detection at earlier stages has long been acknowledged, biomarker-based discoveries have yielded underwhelming outcomes. The failures of CA-125 and other approaches have raised concern over the sensitivity and robustness of serum biomarkers. However, the discovery that most ovarian cancers originate in the fallopian tube may offer a unique opportunity; since the natural washdown of fallopian tube cells and debris travels through the uterus and into the cervix and vagina, we hypothesized that biomarkers may be concentrated in these proximal fluids. Though we previously demonstrated that tumor DNA is present in the vaginal tract of ovarian cancer patients, the deep sequencing required and low sensitivity (60%) render it insufficient for clinical use. Therefore, we examined other potential biomarkers with high stability that are detectable with methods that are cost effective and noninvasive, such as endocervical sampling. We performed mass spectroscopy on 41 endocervical cytobrush (ECC) samples and found the greatest discriminating pathway between ovarian cancer and benign patients was exosomal composition and trafficking (p=1.3 × 10−21). MicroRNAs (miRNAs) are common exosomal cargo, boast high extracellular stability in bodily fluids, and govern a multitude of cellular processes that can influence ovarian cancer initiation and progression. We therefore used NanoString to perform comprehensive miRNA profiling in matched ECC and plasma samples from high-grade serous ovarian cancer and normal patients. We discovered 13 significant differentially expressed ECC miRNAs and 7 plasma miRNAs individually capable of distinguishing cancer from normal. Not only did ECC samples yield a higher number of significant hits than plasma samples, but these hits also showed better sensitivity and specificity as assessed through their concordance index (c-index) scores generated by area under receiver operator curve analysis (c-index averages of 0.9141 in ECC vs. 0.8796 in plasma). 3 miRNAs overlapped between ECC and plasma, and in each case higher c-index scores were present in ECC samples than in plasma. ECC samples also showed more dramatic differences in expression between cancer and benign (1.4 log2 fold change in ECC vs. 0.8 log2 fold change in plasma), suggesting ECC may yield more consistent and detectable differences in miRNA expression than its plasma counterpart. The miRNAs discovered in our ECC samples showed individual sensitivities and specificities that lie within 95% confidence intervals, indicating potential for a viable positive predictive value for a clinical screening test. Crucial next steps are to validate these data in a larger cohort and determine if a similar distinguishing signature is present in the earliest events in fallopian tube transformation. Together, these data support further investigation of proximal tissue sampling for detection of ovarian cancer. Citation Format: Alexandra R. Harris, Danielle C. Llaneza, Mark R. Conaway, Thomas P. Conrads, Katherine Croft, John R. Cornelison, Charles N. Landen. Endocervical microRNA profiling for detection of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B31.

Abstract B49: Substantial performance differences among RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control study—important considerations for liquid biopsies

Abstract Cancer biomarker studies require procedures that provide accurate and precise test results with high analytical sensitivity. Consequently, the growing use of extracellular RNA from human biofluids as clinically relevant biomarker requires the implementation of benchmarked methods for sample collection, processing, and profiling. While several small-scale studies have pointed at the impact of individual preanalytical factors, no comprehensive study has addressed the many preanalytical variables affecting downstream sequencing of blood-derived exRNAs. In the Extracellular RNA Quality Control study, we have systematically evaluated the type of blood collection tube (n=10, including 5 so-called preservation tubes), the time between blood draw and plasma preparation (n=3), different plasma types (n=3, i.e., platelet-free, -poor, and -rich plasma), and RNA purification methods using the supplier-specified minimum and maximum plasma input volumes (n=15). The impact of these preanalytical factors is assessed by deep transcriptome profiling of all small and messenger RNAs from healthy donors’ plasma, using TruSeq Small RNA sequencing and TruSeq RNA Exome sequencing, respectively. All experiments are conducted in triplicate (for a total of 270 transcriptomes) using 191 synthetic RNA spike-in molecules as processing controls over a relevant dynamic range. When comparing blood collection tubes, serum mRNA seems very similar to EDTA plasma mRNA, but serum-derived small RNAs are markedly different in biotype composition compared to their plasma counterparts. Furthermore, several plasma tubes with preservation reagents do not stabilize RNA very well, as is reflected by increasing RNA concentrations and number of detected genes over time. Also, their reproducibility is generally compromised. In addition, we demonstrate large differences in RNA purification kit performance in terms of reproducibility, sensitivity, and observed transcriptome complexity. Among others, we note a 50-fold difference in mRNA yield and a 5-fold difference in the number of detected mRNAs. We summarized the results in 12 performance parameters that enable an informed selection of the most optimal sample processing workflow. In conclusion, using a systematic approach, we put forward robust quality control metrics for exRNA quantification methods with validated SOPs for sample collection, processing, and profiling. Our results are crucially important for all future RNA-based liquid biopsy-guided precision oncology applications. Authors in random order; abstract submitted on behalf of the exRNAQC Consortium. Citation Format: Anneleen Decock, Jasper Anckaert, Celine Everaert, Justine Nuytens, Scott Kuersten, An Hendrix, Jo Vandesompele, Pieter Mestdagh, Bert Dhondt, Ruben Van Paemel, Kimberly Verniers, Gary Schroth, Carolina Fierro, Nurten Yigit, Kathleen Schoofs, Annelien Morlion, Jill Deleu, Eva Hulstaert, Francisco Avila Cobos, Nele Nijs, Eveline Vanden Eynde, Hetty Hilde Helsmoortel, Olivier De Wever, Annouck Philippron. Substantial performance differences among RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control study—important considerations for liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B49.

Comparison of plasma and urinary microRNA-483-5p for the diagnosis of adrenocortical malignancy

IntroductionMinimally invasive circulating microRNAs might be used for the preoperative differentiation of adrenocortical carcinoma (ACC) and adrenocortical adenoma (ACA). So far, the best blood-borne microRNA biomarker of ACC is circulating hsa-miR-483-5p. The expression of urinary hsa-miR-483-5p as a non-invasive marker of malignancy and its correlation with plasma hsa-miR-483-5p, has not been investigated, yet. AimOur aim was to investigate the expression of urinary hsa-miR-483-5p and its correlation with its plasma counterpart. MethodsPlasma and urinary samples from 23 ACC and 23 ACA patients were analysed using real-time RT-qPCR. To evaluate the diagnostic applicability of hsa-miR-483-5p, ROC-analysis was performed. ResultsSignificant overexpression of hsa-miR-483-5p was observed in carcinoma patients’ plasma samples compared to adenoma patients’ (p < 0.0001, sensitivity: 87%, specificity: 78.3%). In urinary samples, however, no significant difference could be detected between ACC and ACA patients. ConclusionsPlasma hsa-miR-483-5p has been confirmed as significantly overexpressed in adrenocortical cancer patients and thus might be exploited as a minimally invasive preoperative marker of malignancy. The applicability of urinary hsa-miR-483-5p for the diagnosis of adrenocortical malignancy could not be confirmed.

Aplysia allatotropin-related peptide and its newly identified d-amino acid–containing epimer both activate a receptor and a neuronal target

l- to d-residue isomerization is a post-translational modification (PTM) present in neuropeptides, peptide hormones, and peptide toxins from several animals. In most cases, the d-residue is critical for the biological function of the resulting d-amino acid-containing peptide (DAACP). Here, we provide an example in native neuropeptides in which the DAACP and its all-l-amino acid epimer are both active at their newly identified receptor in vitro and at a neuronal target associated with feeding behavior. On the basis of sequence similarity to a known DAACP from cone snail venom, we hypothesized that allatotropin-related peptide (ATRP), a neuropeptide from the neuroscience model organism Aplysia californica, may form multiple diastereomers in the Aplysia central nervous system. We determined that ATRP exists as a d-amino acid-containing peptide (d2-ATRP) and identified a specific G protein-coupled receptor as an ATRP receptor. Interestingly, unlike many previously reported DAACPs and their all-l-residue analogs, both l-ATRP and d2-ATRP were potent agonists of this receptor and active in electrophysiological experiments. Finally, d2-ATRP was much more stable than its all-l-residue counterpart in Aplysia plasma, suggesting that in the case of ATRP, the primary role of the l- to d-residue isomerization may be to protect this peptide from aminopeptidase activity in the extracellular space. Our results indicate that l- to d-residue isomerization can occur even in an all-l-residue peptide with a known biological activity and that in some cases, this PTM may help modulate peptide signal lifetime in the extracellular space rather than activity at the cognate receptor.

Tissue acylcarnitine status in a mouse model of mitochondrial β-oxidation deficiency during metabolic decompensation due to influenza virus infection

Despite judicious monitoring and care, patients with fatty acid oxidation disorders may experience metabolic decompensation due to infection which may result in rhabdomyolysis, cardiomyopathy, hypoglycemia and liver dysfunction and failure. Since clinical studies on metabolic decompensation are dangerous, we employed a preclinical model of metabolic decompensation due to infection. By infecting mice with mouse adapted influenza and using a pair-feeding strategy in a mouse model of long-chain fatty acid oxidation (Acadvl−/−), our goals were to isolate the effects of infection on tissue acylcarnitines and determine how they relate to their plasma counterparts. Applying statistical data reduction techniques (Partial Least Squares-Discriminant Analysis), we were able to identify critical acylcarnitines that were driving differentiation of our experimental groups for all the tissues studied. While plasma displayed increases in metabolites directly related to mouse VLCAD deficiency (e.g. C16 and C18), organs like the heart, muscle and liver also showed involvement of alternative pathways (e.g. medium-chain FAO and ketogenesis), suggesting adaptive measures. Matched correlation analyses showed strong correlations (r > 0.7) between plasma and tissue levels for a small number of metabolites. Overall, our results demonstrate that infection as a stress produces perturbations in metabolism in Acadvl−/− that differ greatly from WT infected and Acadvl−/− pair-fed controls. This model system will be useful for studying the effects of infection on tissue metabolism as well as evaluating interventions aimed at modulating the effects of metabolic decompensation.