Non-invasive Early Biomarkers Research Articles

Abstract PR018: Plasma proteomic biomarkers reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade

Abstract Most patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses, stressing a critical need to identify early non-invasive biomarkers of response. Circulating biomarkers provide easy access for serial monitoring and can provide insight on the mechanisms of response to ICB. We performed plasma proteomics (2943 proteins) on 250 metastatic melanoma patients before and on ICB treatment and performed analysis to identify response- and time point-associated peripheral protein biomarkers. We next generated patient- matched peripheral blood lymphocyte (PBL) surface proteomic data to measure the proportions of immune cell subsets in circulation. We further obtained 13 tumor samples that were processed for single-cell RNA-sequencing (scRNA-seq). We fit linear mixed effects (LME) models to predict plasma protein abundances, using the sample time point, response status and interaction term between time point and response as covariates. We identified 397 proteins with a significant time point, response, or interaction effect. For proteins upregulated post-ICB, gene ontology analysis suggested these proteins were associated with inflammatory pathways involving the activation of effector immune functions. LME models fitted on the circulating immune cell proportions and parallel analysis of immune-related plasma proteins supported that post-ICB samples may be defined by increased immune activity, including T cell infiltration. Moreover, expression of genes corresponding to these post-ICB proteins in the tumor scRNA-seq data tended to be favored by immune subsets, namely those involved in immune cell recruitment and tumor reactivity, such as CXCL10+ macrophages and CXCL13+ T cells, respectively. On the other hand, expression of genes corresponding to plasma proteins upregulated in non-responders in the TME tended to be favored by suppressive myeloid subsets such as SPP1+ macrophages as well as malignant cells, suggesting the potential of immunosuppressive and pro-tumor biological processes to be involved in ICB resistance. Inference of cell-cell interactions in the TME further showed the involvement of these non-responder genes in potentially immunosuppressive and pro-tumor processes, including the PTPRC-MRC1 axis, involved in myeloid cell plasticity, the IGF1/2-IGF1R axis, involved in growth and survival of normal and neoplastic cells, and the GAS6-TYRO3 axis, associated with carcinogenetic mechanisms in multiple malignancies. We further identified a correlation between IL13 protein abundance in circulation and GAS6 expression in the tumor, suggesting that IL13 may reach the tumor from circulation to turn on the STAT6 pathway in receiving cells and upregulate GAS6 in turn to participate in this interaction with TYRO3. Together, these data represent one of the deepest peripheral biomarker studies using paired samples in melanoma patients treated with anti-PD1 therapy, and begin to elucidate the complex interplay between tumors and the systemic immune response within the host. Citation Format: Samuel J. Wright, Izabella Zamora, Milan Parikh, Deepika Yeramosu, Lynn Bi, Sarah Kang, Marijana Rucevic, Moshe Sade-Feldman, Baolin Liu, Ngan Nguyen, Jamey Guess, Alexis Schneider, David Lieb, Elliot Woods, William Michaud, Aleigha R. Lawless, Tatyana Sharova, Gyulnara Kasumova, Michelle S. Kim, Ryan J. Park, Russell W. Jenkins, Samuel J. Klempner, Ryan J. Sullivan, Keith T. Flaherty, Nir Hacohen, Genevieve M. Boland, Arnav Mehta. Plasma proteomic biomarkers reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR018.

MAPK signaling pathway induced LOX-1+ polymorphonuclear myeloid-derived suppressor cells in biliary atresia

Biliary atresia (BA) is a severe pediatric liver disease characterized by progressive bile duct destruction and fibrosis, leading to significant liver damage and frequently necessitating liver transplantation. This study elucidates the role of LOX-1+ polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in BA pathogenesis and assesses their potential as non-invasive early diagnostic biomarkers. Using flow cytometry, immunofluorescence, and molecular profiling, we analyzed the expression and activity of these cells in peripheral blood and liver tissues from BA patients and controls. Our findings reveal a significant increase in the frequencies and function of LOX-1+PMN-MDSCs in BA patients, along with MAPK signaling pathway upregulation, indicating their involvement in disease mechanisms. Additionally, the frequencies of LOX-1+PMN-MDSC in peripheral blood significantly positively correlate with liver function parameters in BA patients, demonstrating diagnostic performance comparable to traditional serum markers. These findings suggest that LOX-1+PMN-MDSCs contribute to the immunosuppressive environment in BA and could serve as potential diagnostic targets.

Collagen-V and K-α-1 Tubulin Antibodies as Potential Markers of Unsuspected GERD-Related Lung Damage: Insights from a Cross-Sectional Analysis

PurposeOur group has proposed that aspiration of gastric contents leads to exposure of normally sequestered lung self-antigens (SAgs), specifically collagen-V (Col-V) and K-α-1-tubulin (Kα1T), which elicits an immune response characterized by increasing concentrations of self-antibodies (SAbs) anti-Col-V and anti-Kα1T. We sought to establish the point prevalence of abnormally elevated concentrations of SAbs among patients with pathological gastroesophageal reflux disease (GERD) and/or hiatal hernia undergoing antireflux surgery (ARS).MethodsFor this cross-sectional study, we retrieved a plasma aliquot from the Norton Thoracic Institute BioBank from blood samples that were taken preoperatively from patients who underwent ARS between November 2019 and August 2022. Enzyme-linked immunosorbent assays were employed to detect and quantify anti-Col-V and anti-Kα1T.ResultsSamples from 43 patients (females, n = 34 [79.1%]; mean age, 62 ± 12 years; and mean BMI, 30.5 ± 7 kg/m2) were analyzed. Before ARS, 28 (65.1%, CI95: 50.3–80.0%) patients had abnormally elevated concentrations of anti-Col-V and 19 (44.2%, CI95: 28.7–59.7%) had elevated concentrations of circulating anti-Kα1T. Overall, 13 patients (30.2%) had low (i.e., normal) concentrations of both SAbs, 13 (30.2%) were positive only for one, and 17 (39.5%) were positive for both SAbs.ConclusionA relative high point prevalence of abnormally elevated circulating SAbs (i.e., anti-Col-V and/or anti-Kα1T) before ARS was found. This result suggests clinically unsuspected pulmonary parenchymal injury secondary to GERD-related aspiration. Further studies are required to confirm this hypothesis and to identify alternative non-invasive early biomarkers of GERD-related lung damage.

Proteomic profiling of saliva exosomes for the identification of non-invasive early biomarker in patients with coronary artery disease

Proteomic profiling of saliva exosomes for the identification of non-invasive early biomarker in patients with coronary artery disease

#1186 Novel T-cell subsets as early non-invasive biomarkers of vascular damage in chronic kidney disease progression

Abstract Background and Aims The exploration of early biomarkers for diagnosing vascular alterations in CKD-MBD holds significant clinical potential. Previous evidence has demonstrated profound alterations in immune cell populations linked to vascular homeostasis in CKD dialysis patients. This study aimed to evaluate the utility of immune cell populations as early and progressive biomarkers for non-dialysis CKD patients, examining their correlation with vascular damage. Method A total of 43 non-dialysis CKD patients and 38 sex- and age-matched controls were included in the study. Patients were categorized into four groups based on their estimated glomerular filtration rate (eGFR): CKD-2/3a, CKD-3b, CKD-4, and CKD-5. The assessment of left carotid adventitial neovascularization and intima-media thickness (cIMT) was conducted using ultrasensitive ultrasonography without contrast, specifically employing Superb Microvascular Image (SMI) technology. Pulse wave velocity (PWV) was measured to determine aortic stiffness, utilizing the Complior Analyzer. Immunosescencent T-cells (CD4+CD28null), angiogenic T-cells (Tang) (CD3+CD31+CD184+) and their subsets (CD4+Tang, CD8+Tang and CD28nullTang) were quantified by flow cytometry in Peripheral Blood Mononuclear Cells. Results No significant differences in age and body mass index were observed between the control group and the four CKD stages. Notably, the values of estimated glomerular filtration rate (eGFR) exhibited a significant and progressive decrease, as indicated in the Table 1. Biochemical parameters related to CKD-MBD are also detailed in the same table. The carotid intima-media thickness (cIMT) was found to be significantly higher in CKD-5 compared to CKD 2-3a (CKD 2-3a = 0.66 ± 0.14 mm, CKD-5 = 0.80 ± 0.12 mm, p = 0.03). In patients at stage 4 of CKD, there was an increased number of adventitial vasa vasorum (control = 0.53 ± 1.33, CKD-4 = 1.67 ± 2.04, p = 0.0009) and a greater area of adventitial neovascularization in the carotid artery compared to the control group (control = 0.51 ± 1.31%, CKD-4 = 2.67 ± 2.27%, p = 0.0003). PWV was significantly higher in CKD-5 (control = 10.81 ± 2.01 m/s, CKD-5 = 12.91 ± 3.70 m/s, p = 0.045). Additionally, a significant correlation was observed between PWV and both the number of adventitial vasa vasorum at the carotid artery (r = 0.24, p = 0.028) and the area of adventitial neovascularization (r = 0.34, p = 0.002). CD4+CD28null T-cells were increased in CKD patients compared to control group (control = 24.99[5.52], CKD = 27.35[8.58], p = 0.004). Although no differences were found in total levels of Tang in CKD patients (control = 1.64 ± 0.89, CKD= 1.5 ± 1.02, p = 0.757), a strong negative correlation was observed across CKD stages (trend analysis for stages: r = −0.361, p = 0.017). Moreover, CKD patients had altered CD8+ and CD4+ Tang frequencies (CD4+Tang: control = 39.52[18.46], CKD = 33.60[12.84], p = 0.054; CD8+Tang: control = 40.03[14.38], CKD = 48.25[13.16], p = 0.012) and Tang CD28null were increased in CKD patients (control = 44.14[11.99], CKD = 48.62[12.08], p = 0.004). Finally, Tang frequency was significantly correlated with the number of adventitial vasa vasorum (r = 0.368, p = 0.032), area of adventitial neovascularization (r = −0.401, p = 0.019), PWV (r = −0.341, p = 0.048) and cIMT (r = −0.364, p = 0.034). Conclusion Carotid adventitial neovascularization, cIMT and PWV demonstrated an increase in advanced CKD stages, implying that they may possess limited clinical utility as early markers of vascular damage in the initial stages of CKD. Immunosenescence and vascular damage traits were found across the CKD spectrum. Altered Tang frequency may be considered a progressive biomarker of impaired vascular homeostasis in CKD.

#2908 Renal progenitor cells an early non-invasive biomarker of silent kidney injury in Fabry disease

Abstract Background and Aims Fabry's disease (FD), a hereditary condition, is linked to the X chromosome due to a mutation in the gene encoding alpha-galactosidase A (alpha-GalA). This mutation results in a defect in the metabolism of glycosphingolipids, leading to the deposition of globotriaosylceramide (GB3) primarily in organs such as the heart, kidneys, and nervous system. Renal injury can affect any level of the structure, with the podocytes being the most commonly affected due to their low regeneration and high contact with GB3. This leads to proteinuria, and if it exceeds 0.5 g, it is associated with a rapid deterioration of renal function. Renal assessment is carried out through blood analysis, proteinuria measurement, ultrasound, and the gold standard, renal biopsy. However, there is a growing trend towards less invasive biomarkers such as lysoGB3 titration in blood and urine, podocyturia, or renal progenitor cells (RPCs) in renal biopsy material or cell culture. RPCs possess self-renewal, clonogenicity, and multidifferentiation properties, contributing to cellular remodeling. They express specific markers like CD133/CD24 and CD106 in the parietal epithelium of Bowman's capsule. It is known that GB3 expresses CD77. The proliferative response of RPCs can become dysregulated, leading to detachment and elimination in the urine. The aim of the study was to demonstrate the presence of renal progenitor cells (RPCs) in urine as a non-invasive early marker for detecting renal damage in patients with Fabry's disease (FD) and to establish a correlation between RPC presence, GB3 deposition, and their potential association with the degree of renal injury. Method It is an open observational, case-control, single-center study on the urine of patients with Fabry's disease (FD) considered as cases, patients without a history considered as healthy controls, and patients with non-FD renal disease such as Gittelman syndrome or another biopsy-proven cause as positive controls. Renal progenitor cells (RPCs) were isolated using specific markers and flow cytometry, correlating these findings with renal function and albuminuria. Results A total of 75 recent urine samples were processed, corresponding to 59 patients: 16 with Fabry's disease (FD), 11 with Gittelman syndrome, 10 with chronic kidney disease (CKD), and 22 healthy controls. The samples were divided for sediment and microalbuminuria analysis, and separately for the isolation of renal progenitor cells (RPCs). The classification and quantification of the isolated cell types were performed, establishing positivity points using compensation panels with CD3 lymphocyte markers. This was followed by the identification of RPCs through positivity for CD133+/CD24+, CD106+, and CD106– markers. Once RPCs were identified, GB3 marking was carried out using CD77+. Differences were observed among the analyzed patient groups. In healthy controls, there was minimal or no presence of renal progenitor cells (RPCs), except in two patients where isolation was repeated in three different samples, maintaining positivity. Subsequent diagnoses revealed bilateral lithiasis and previously unknown hypertension in these cases. Patients with Gittelman syndrome showed a higher quantity of RPCs compared to healthy controls, but with P > .05. However, clear RPCs positivity was evident in patients with Fabry's disease (FD) and chronic kidney disease (CKD) confirmed by biopsy, with P < .05. Additionally, differences in the number of RPCs were noted based on the degree of renal disease when correlated with the presence of proteinuria < 0.5 g and > 0.5 g, with P < .05. Conclusion With these results, we can conclude that renal progenitor cells (RPCs) may serve as an early biomarker for silent renal injury of various etiologies. Furthermore, if we perform labeling with CD77, we can attribute it to the deposition of GB3 in Fabry's disease (FD).

OCT Intensity of the Region between Outer Retina Band 2 and Band 3 as a Biomarker for Retinal Degeneration and Therapy.

Optical coherence tomography (OCT) is widely used to probe retinal structure and function. This study investigated the outer retina band (ORB) pattern and reflective intensity for the region between bands 2 and 3 (Dip) in three mouse models of inherited retinal degeneration (Rs1KO, TTLL5KO, RPE65KO) and in human AMD patients from the A2A database. OCT images were manually graded, and reflectivity signals were used to calculate the Dip ratio. Qualitative analyses demonstrated the progressive merging band 2 and band 3 in all three mouse models, leading to a reduction in the Dip ratio compared to wildtype (WT) controls. Gene replacement therapy in Rs1KO mice reverted the ORB pattern to one resembling WT and increased the Dip ratio. The degree of anatomical rescue in these mice was highly correlated with level of transgenic RS1 expression and with the restoration of ERG b-wave amplitudes. While the inner retinal cavity was significantly enlarged in dark-adapted Rs1KO mice, the Dip ratio was not altered. A reduction of the Dip ratio was also detected in AMD patients compared with healthy controls and was also positively correlated with AMD severity on the AMD score. We propose that the ORB and Dip ratio can be used as non-invasive early biomarkers for retina health, which can be used to probe therapeutic gene expression and to evaluate the effectiveness of therapy.

Abstract 1854: Plasma proteomic biomarkers identify non-responders and reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade

Abstract Most patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses, stressing a critical need to identify early non-invasive biomarkers of response. Circulating biomarkers provide easy access for serial monitoring and can provide insight on the mechanisms of response to ICB. We performed plasma proteomics (~3000 proteins) on 250 metastatic melanoma patients before and on ICB treatment and performed analysis to identify response- and time point-associated peripheral protein biomarkers. We next generated patient-matched peripheral blood lymphocyte (PBL) surface proteomic data and tumor sample bulk transcriptomic data to build a multimodal dataset. We leveraged these data to train machine learning models for unsupervised analysis and to classify treatment responses and learned features predictive of survival and patient endotypes. Using univariate linear models to predict protein abundance based on time point, response and the interaction term for time point and response, we identified 343 time point-associated proteins, 141 response-associated proteins, and one interaction-associated protein. We further investigated response-related proteins by fitting multivariate logistic regression and cox proportional hazards regression models to further investigate the ability of these proteins to predict response, progression-free survival (PFS), and overall survival (OS) and identify those proteins that are most important in predicting response. We calculated covarying modules of proteins that were found to be either response-associated or time point-associated to discover underlying biological networks at different treatment time points and disease response states. Additionally, we leveraged these modules to endotype patients and identified trends in protein modules across patient subsets, including modules related to biological processes such as immune infiltration, apoptosis, metaplasia and cell adhesion. To understand the contribution of different compartments on plasma proteomic abundance, we studied correlations of plasma protein abundance with PBL surface protein abundance and tumor bulk mRNA-sequencing expression. Finally, we integrated our data modalities and fit various statistical models to evaluate the relative importance of plasma protein biomarkers in predicting response compared to PBL surface proteins and tumor bulk mRNA-sequencing expression. Together, these data represent one of the deepest peripheral biomarker studies using paired samples in melanoma patients treated with anti-PD1 therapy. Citation Format: Samuel J. Wright, Deepika Yeramosu, Milan Parikh, Marijana Rucevic, Ngan Nguyen, Russell W. Jenkins, Keith T. Flaherty, Nir Hacohen, Genevieve M. Boland, Arnav Mehta. Plasma proteomic biomarkers identify non-responders and reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1854.

Developing blood-brain barrier arterial spin labelling as a non-invasive early biomarker of Alzheimer’s disease (DEBBIE-AD): a prospective observational multicohort study protocol

IntroductionLoss of blood-brain barrier (BBB) integrity is hypothesised to be one of the earliest microvascular signs of Alzheimer’s disease (AD). Existing BBB integrity imaging methods involve contrast agents or ionising...

Exosomal UMOD gene expression and urinary uromodulin level as early noninvasive diagnostic biomarkers for diabetic nephropathyin type 2 diabetic patients.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Exosomes are promising biomarkers for disease diagnosis and uromodulin is a kidney-specific protein. So, this study was designed to investigate the change in the gene expression of urinary exosomal uromodulin mRNA and urinary uromodulin level and determine the diagnostic potential of these noninvasive biomarkers in the early stage of diabetic nephropathy in type 2 diabetic patients. This study included 100 participants; urinary exosomes were isolated using polyethylene glycol (PEG). Gene expression of exosomal uromodulin mRNA was determined by quantitative real-time polymerase chain reaction (q-RT-PCR). The urinary uromodulin levels were determined by an enzyme-linked immunosorbent assay (ELISA). In this study, the gene expression of exosomal uromodulin (UMOD) mRNA and the level of urinary uromodulin showed a significant increase in all diabetic groups with and without nephropathy compared to the control group. The exosomal UMOD mRNA showed a significant positive correlation with urinary uromodulin in all groups. Multiple logistic regression showed that urinary uromodulin was an independent determinant for DN. A diagnostic model of two indicators, exosomal UMOD mRNA and urinary uromodulin, can significantly predict DN. The area under the curve is 0.095, with a 95% confidence interval of 0.98-1, and 0.81, with a 95% confidence interval of 0.69-0.92, for the exosomal UMOD mRNA and urinary uromodulin, respectively. Urinary exosomal mRNA of UMOD and urinary uromodulin levels are progressively elevated in an early stage of DN, even before the microalbuminuria stage, so they could be used as early predictors for DN.

The Utility of Urinary Titin to Diagnose and Predict the Prognosis of Acute Myocardial Infarction.

Early detection and management are crucial for better prognosis in acute myocardial infarction (AMI). Serum titin, a component of the sarcomere in cardiac and skeletal muscle, was associated with AMI. Thus, we hypothesized that urinary N-fragment titin may be a biomarker for its diagnosis and prognosis. Between January 2021 and November 2021, we prospectively enrolled 83 patients with suspected AMI. Their urinary N-fragment titin, serum high-sensitivity troponin I (hsTnI), creatine kinase (CK), and creatine kinase-MB (CK-MB) were measured on admission. Then, urinary titin was assessed as diagnostic and prognostic biomarker in AMI. Among 83 enrolled patients, 51 patients were diagnosed as AMI. In AMI patients who were admitted as early as 3 h or longer after symptom onset, their urinary titin levels were significantly higher than non-AMI patients who are also admitted 3 h or longer after symptom onset (12.76 [IQR 5.87-16.68] pmol/mgCr (creatinine) and 5.13 [IQR 3.93-11.25] pmol/mgCr, p = 0.045, respectively). Moreover, the urinary titin levels in patients who died during hospitalization were incredibly higher than in those who were discharged (15.90 [IQR 13.46-22.61] pmol/mgCr and 4.90 [IQR 3.55-11.95] pmol/mgCr, p = 0.023). Urinary N-fragment titin can be used as non-invasive early diagnostic biomarker in AMI. Furthermore, it associates with hospital discharge disposition, providing prognostic utility.

DEveloping BBB-ASL as non-Invasive Early biomarker of Alzheimer's Disease (DEBBIE-AD): Study design

DEveloping BBB-ASL as non-Invasive Early biomarker of Alzheimer's Disease (DEBBIE-AD): Study design

A comprehensive proteomic profiling of urinary exosomes and the identification of early non-invasive biomarker in patients with coronary artery disease

Urinary small extracellular vesicles or exosomes (uEVs) source could be an emerging trove of biomarkers in coronary artery disease (CAD). It is a chronic inflammatory disease having a long asymptomatic phase of fatty-fibrous development in arteries leading to angina, myocardial infarction, and death. Our study was aimed at identifying differential protein expression profiling of uEVs in CAD. We collected urine samples of CAD patients (n = 41) age 18–65 years and gender matched healthy controls (n = 41). We isolated uEVs using differential ultracentrifugation. Further, uEV samples were characterized by western blotting exosome markers (Flotillin, TSG, CD63, and CD9), nano tracking analysis, and transmission and scanning electron microscopy. A total of 508 proteins were identified by iTRAQ-based mass spectrometry. We observed protein expression levels of AZGP1, SEMG1/2, ORM1, IGL, SERPINA5, HSPG2, prosaposin, gelsolin, and CD59 were upregulated, and UMOD, KNG1, AMBP, prothrombin, and TF were downregulated. Protein-protein interactions, gene ontology and pathway analysis were performed to functionally annotate identified uEVs proteins. A novel uEVs differential protein signature is shown. On validating UMOD protein by ELISA in two clinically different CAD, stable-CAD patients had lower levels than healthy controls whereas recent myocardial infarction patients had lowest. Our findings suggest UMOD importance as early diagnostic biomarker. SignificanceCoronary artery disease is a chronic inflammatory disease caused by gradual deposition of cholesterol and fat along with other proteins to develop plaque inside arteries. This further leads to blockage of artery, heart attack and death. There are no identifiable early biomarkers to diagnose this. For the first time, we have identified the differentially expressed proteins isolated from non-invasive uEV of CAD patients compared to healthy controls by using MS Orbitrap and iTRAQ labelling of peptides. We have identified decreased levels of UMOD protein in CAD. These findings have been confirmed by ELISA. Furthermore, the levels of UMOD were observed as more highly decreased in recent myocardial infarction CAD patients, indicating the importance of this protein as an early diagnostic biomarker. Conclusively, our study represents a non-invasive urinary EVs trove of differentially expressed proteins in CAD. This will form a groundwork for understanding the pathophysiology of CAD and will help in future translational research utilizing uEVs.

Salivary Oxidative Stress Assessment in Tobacco Users with and without Potentially Malignant Disorders and Micronuclei Estimation using Fluorescent Microscopy

Abstract Background: Consumption of tobacco in either smoking or nonsmoking form produces free radicals, contributing to an increased risk of oral cancer as a result of oxidative stress. Biochemical changes in antioxidant enzymes are responsible for tobacco-associated oral carcinogenesis. Due to the production of reactive oxygen species and oxidative stress, the promotion of carcinogenesis occurs in the cells. Oral potentially malignant disorders (OPMDs) is an assembly of benign lesions with morphologically altered clinical or histopathological tissue that has a greater than normal risk of transforming into malignant lesions after diagnosis at a later date. Micronuclei are the goal site for early genotoxic events and are involved in carcinogenic events. Micronuclei in exfoliated cells are useful biomarkers in assessing cytogenetic damage. Early detection will help to devise appropriate treatment plans, thereby improving patient survival rates. Aim and Objectives: To estimate the salivary superoxide dismutase (SOD) levels and the expression of micronuclei in exfoliated buccal cells of tobacco users before the onset of tobacco-induced oral lesions and correlating the same with patients with OPMDs to determine if this can be useful in predicting early malignant potential in subjects with habitual tobacco usage. Materials and Methods: The study included three groups. Group I – Tobacco users with OPMD (n = 50), Group II – Tobacco users without OPMD (n = 50), and Group III – Healthy controls (n = 50). Sandwich enzyme-linked immunosorbent assay was done for saliva samples for quantification of salivary SOD. Exfoliated buccal cells were smeared and stained with acridine orange stain and viewed under a fluorescent microscope for micronuclei estimation. Results: The study showed a significant difference in salivary SOD levels and increased micronuclei expression among tobacco users than healthy controls. Conclusion: Our study results revealed a gradual and steady decrease of SOD levels and increased micronuclei expression from healthy control to tobacco users without lesions and tobacco users with lesion, and this can be used as an effective noninvasive early diagnostic biomarker in assessing malignant progression in tobacco users. Furthermore, the evaluation of micronuclei estimation can serve as a simple, quick, and noninvasive chair-side procedure for regular oral cancer screening in patients with tobacco usage.

DNA methylation profiles in urothelial bladder cancer tissues and children with schistosomiasis from Eggua, Ogun State, Nigeria

BackgroundSquamous cell carcinoma has been attributed to chronic schistosomiasis and is the predominant type of bladder cancer in schistosomiasis endemic areas. The aim of this study was to assess early promoter DNA methylation in selected genes implicated in schistosomiasis-associated bladder cancer (SABC).MethodsA total of 159 urine samples were collected from school-aged children in Eggua Community of Ogun State and examined by microscopy for Schistosoma haematobium eggs. From this sample, a subset of 34 (21.1%) urine samples positive for S. haematobium, age and sex-matched with negative urine control samples, and 16 formalin-fixed paraffin-embedded bladder cancer tissues obtained from the University College Hospital were subjected to DNA isolation and bisulphite DNA conversion. Quantitative methylation-specific PCR was used to determine the methylation status of APC, RARβ2, RASSF1A, and TIMP3 in the samples.ResultsHigh degrees of methylation of RARβ2(67.7%), RASSF1A (38.2%), and TIMP3(52.9%) was more common in urogenital schistosomiasis (UGS)-positive urine samples than negative urine (control) samples and in bladder cancer tissues. Promoter DNA methylation in the positive urine samples was 1.4-fold, 13.3-fold, 3.4-fold, and 3.8-fold higher in APC, RARβ2, RASSF1A, and TIMP3, respectively, than in the matched controls. The odds of promoter methylation were likely to increase with age group for APC (OR: 1.615) and TIMP3(OR: 2.000); sex for TIMP3(OR: 2.644); and haematuria for RARβ2(OR: 1.094), RASSF1A (OR: 1.143), and TIMP3(OR: 1.842), although there were no significant associations. Conclusions: Gene promoter DNA methylation in tumour suppressor genes was observed in schistosomiasis cases. Hence, promoter DNA methylation may occur during active schistosomiasis in children. This result may serve as an early non-invasive biomarker to detect and hint at the risk of developing SABC later in life.

Pathogenic roles of microRNAs and competing endogenous RNAs in Hirschsprung disease (HSCR): Potential diagnostic markers for HSCR

AbstractMicroRNAs (miRNAs) are endogenous small non‐coding single‐stranded RNAs. They can bind to the 3′‐untranslated region (3′UTR) of mRNAs and regulate the expression of their target genes by inducing degradation or translation inhibition of the corresponding mRNAs. Competing endogenous RNAs (ceRNAs) can disable miRNAs by combining miRNAs response elements (MREs) with miRNAs. Hirschsprung disease (HSCR) is a common pediatric surgical disease in which cells derived from the enteric neural crest fail to colonize the distal colon, but its pathogenesis is not very clear. In recent years, with more and more studies on miRNAs in HSCR, miRNAs seem to be involved in the pathogenesis of HSCR. miRNAs in HSCR affect the proliferation and migration of enteric neural crest cells mainly through target genes, and ceRNAs inhibit miRNAs, thus participating in the pathogenesis of HSCR. It was reported that some miRNAs in the serum of children with HSCR were significantly higher than those in the control group. Therefore, miRNAs are expected to be a new noninvasive early screening biomarker and targeted therapy point for HSCR. Here, we provide a summary of the understanding of miRNAs and ceRNAs in regulating enteric nervous system proliferation and migration and their roles in the pathogenesis of HSCR.

Changes in urinary exosomal protein CALM1 may serve as an early noninvasive biomarker for diagnosing diabetic kidney disease

BackgroundThe risk of the development and progression of diabetic kidney disease (DKD) was increased by abnormal calcium release. However, it is still unknown whether calcium signal pathway-related proteins are changed in urinary exosomes. This study aims to explore the changes in urinary exosomal proteins, which may provide novel biomarkers for diagnosing DKD. MethodsUrinary exosomes were isolated from 132 participants by size exclusion chromatography method and 72 participants were tested by LC-MS/MS (Discovery phase). Correlation and multivariate logistics analysis were applied to evaluate selected urinary proteins. Western blot and ELISA were used to validate the selected protein (Validation phase: n = 60). The diagnostic performance of the selected biomarker was evaluated by receiver operating characteristic curve analyses between the discovery and validation phases. ResultsSixteen calcium signal pathway-related proteins were identified, however, only Calmodulin-1(CALM1) was continuously increased. Different expression of CALM1 was found in patients with different level of estimated glomerular filtration rate (eGFR) in two cohorts. The level of CALM1 was correlated with eGFR and serum creatinine levels in two cohorts. Multivariate analysis revealed that serum albumin (ALB) levels and CALM1 were independent risk factors for DKD. A diagnostic model based on CALM1 and serum ALB levels that could significantly distinguish DKD was established and validated. ConclusionsSignificant changes in calcium signal pathway-related urinary exosomal proteins were observed. The CALM1 may serve as an early noninvasive biomarker for diagnosing DKD.

Circulating miR-146b and miR-27b are efficient biomarkers for early diagnosis of Equidae osteoarthritis

One of the most orthopedic problems seen in the equine is osteoarthritis (OA). The present study tracks some biochemical, epigenetic, and transcriptomic factors along different stages of monoiodoacetate (MIA) induced OA in donkeys in serum and synovial fluid. The aim of the study was the detection of sensitive noninvasive early biomarkers. OA was induced by a single intra-articular injection of 25 mg of MIA into the left radiocarpal joint of nine donkeys. Serum and synovial samples were taken at zero-day and different intervals for assessment of total GAGs and CS levels as well as miR-146b, miR-27b, TRAF-6, and COL10A1 gene expression. The results showed that the total GAGs and CS levels increased in different stages of OA. The level of expression of both miR-146b and miR-27b were upregulated as OA progressed and then downregulated at late stages. TRAF-6 gene was upregulated at the late stage while synovial fluid COL10A1 was over-expressed at the early stage of OA and then decreased at the late stages (P < 0.05). In conclusion, both miR-146b and miR-27b together with COL10A1 could be used as promising noninvasive biomarkers for the very early diagnosis of OA.

Genome-wide mRNA profiling in urinary extracellular vesicles reveals stress gene signature for diabetic kidney disease

Urinary extracellular vesicles (uEV) are a largely unexplored source of kidney-derived mRNAs with potential to serve as a liquid kidney biopsy. We assessed ∼200 uEV mRNA samples from clinical studies by genome-wide sequencing to discover mechanisms and candidate biomarkers of diabetic kidney disease (DKD) in Type 1 diabetes (T1D) with replication in Type 1 and 2 diabetes. Sequencing reproducibly showed >10,000 mRNAs with similarity to kidney transcriptome. T1D DKD groups showed 13 upregulated genes prevalently expressed in proximal tubules, correlated with hyperglycemia and involved in cellular/oxidative stress homeostasis. We used six of them (GPX3, NOX4, MSRB, MSRA, HRSP12, and CRYAB) to construct a transcriptional "stress score" that reflected long-term decline of kidney function and could even identify normoalbuminuric individuals showing early decline. We thus provide workflow and web resource for studying uEV transcriptomes in clinical urine samples and stress-linked DKD markers as potential early non-invasive biomarkers or drug targets.

Salivary and serum expression of TNF-α and Ki-67 in oral potentially malignant lesions

BackgroundOral potentially malignant lesions (OPML) are suspicious lesions that may turn into malignancy according to the degree of dysplasia. Early diagnosis of these lesions allows their treatment before malignant transformation. Saliva is a liquid biopsy that can be obtained easily from the patient and assessed for biomarkers expression. Our study aimed to investigate the possibility of using salivary and serum TNF-α and Ki-67 in early detection of OPML. Patients with OPML were selected according to histopathological evaluation of the suspected lesions. The lesions were stained immunohistochemically by tumor necrosis factor alpha (TNF-α) and Ki-67. Saliva and blood samples were collected from the patients for detecting the expression of the markers by ELISA.ResultsThe level of TNF-α and Ki-67 was higher in patients with OPML than healthy individuals regarding both saliva and serum. TNF-α was significantly higher in saliva than in serum unlike Ki-67 which was insignificantly higher in saliva than in serum. The OPML show negative expression of TNF-α in OPML immunohistochemically unlike Ki-67 which show intense positive expression in OPML immunohistochemically.ConclusionsSaliva can be used as an early noninvasive biomarker detector depending on selecting the appropriate salivary biomarker. Salivary TNF-α can be used in early diagnosis of OPML.