Noninvasive Biomarkers For Diagnosis Research Articles

1874P - A composite liquid biomarker for non-invasive diagnosis of resectable pancreatic ductal adenocarcinoma

1874P - A composite liquid biomarker for non-invasive diagnosis of resectable pancreatic ductal adenocarcinoma

Circulating microRNAs as predictive biomarkers for liver disease progression of chronic hepatitis C (genotype‐4) Egyptian patients

Egypt is one of the highest prevalence rates of hepatitis C virus (HCV) infection worldwide. HCV is among major reasons for chronic liver diseases. MicroRNA (miRNAs), small noncoding regulatory molecules play a key role in the pathogenesis of liver. Circulating miRNAs represent potential noninvasive biomarkers for diagnosis and monitoring patients with liver diseases progression. To investigate the potential role of circulating miRNAs for surveillance of liver disease progression, we assessed the expression of 20 liver-related miRNAs in sera of 47 chronic hepatitis C Egyptian patients compared with 25 controls using quantitative reverse-transcription polymerase chain reaction assay. The sensitivity and specificity were evaluated using receiver operating characteristic (ROC) curve. The correlations between their levels and the clinicopathological features were assessed. Fourteen miRNAs showed upregulation and six miRNAs showed downregulation. ROC curve analyses revealed that the explored miRNAs could serve as valuable biomarkers for chronic hepatitis with an area under the curve ranged from 0.708 (95% confidence interval [95% CI], 0.587 to 0.829; P = 0.004) for miR-199 up to 0.974 (95% CI, 0.943 to 1.00; P < 0.001) for miR-23b. The expression level of miR-21 demonstrated significant correlation with age, liver enzymes,ALT/AST, and α-fetoprotein level. AST level was directly correlated with miR-122, while an inversely correlated with miR-23b. Fibrosis and steatosis stages possessed positive correlation with miR-199 expression and negative correlation with miR-27a and miR-93. In conclusion, miR-23b and miR-106 might be a useful biomarker for chronic hepatitis C (CHC). MiR-27a, miR-93, and miR-199 might have a potential role in the progression of liver diseases. Unravel the role of these miRNAs in CHC patients might lead to precise prognosis and management.

Tenascin-C in brain injuries and edema after subarachnoid hemorrhage: Findings from basic and clinical studies.

Subarachnoid hemorrhage (SAH) by a rupture of cerebral aneurysms remains the most devastating cerebrovascular disease. Early brain injury (EBI) is increasingly recognized to be the primary determinant for poor outcomes, and also considered to cause delayed cerebral ischemia (DCI) after SAH. Both clinical and experimental literatures emphasize the impact of global cerebral edema in EBI as negative prognostic and direct pathological factors. The nature of the global cerebral edema is a mixture of cytotoxic and vasogenic edema, both of which may be caused by post-SAH induction of tenascin-C (TNC) that is an inducible, non-structural, secreted and multifunctional matricellular protein. Experimental SAH induces TNC in brain parenchyma in rats and mice. TNC knockout suppressed EBI in terms of brain edema, blood-brain barrier disruption, neuronal apoptosis and neuroinflammation, associated with the inhibition of post-SAH activation of mitogen-activated protein kinases and nuclear factor-kappa B in mice. In a clinical setting, more severe SAH increases more TNC in cerebrospinal fluid and peripheral blood, which could be a surrogate marker of EBI and predict DCI development and outcomes. In addition, cilostazol, a selective inhibitor of phosphodiesterase type III that is a clinically available anti-platelet agent and is known to suppress TNC induction, dose-dependently inhibited delayed cerebral infarction and improved outcomes in a pilot clinical study. Thus, further studies may facilitate application of TNC as biomarkers for non-invasive diagnosis or assessment of EBI and DCI, and lead to development of a molecular target drug against TNC, contributing to the improvement of post-SAH outcomes.

Detection of exosomes by ZnO nanowires coated three-dimensional scaffold chip device

Exosomes as cell-derived vesicles have the potential to be novel biomarkers for noninvasive diagnosis of cancers. However, cost-effective detection of exosomes in routine clinical settings is still challenging. Herein, we present a ZnO nanowires coated three-dimensional (3D) scaffold chip device for effective immunocapture and classically visible and colorimetric detection of exosomes. The chip device is composed of 3D polydimethylsiloxane (PDMS) scaffold skeleton covered by free-standing ZnO nanowire array. The interconnected micropores of 3D scaffold induces the fluid flow with chaotic or vortex feature, and ZnO nanowire array provides large surface area for immobilization of exosome specific antibody as well as size exclusion-like effect for retaining exosomes. These synergistically and significantly enhance the capture of exosomes at a high flow rate. The captured exosomes are detected by horseradish peroxidase (HRP) labeled antibody which can initiate 3,3′,5,5′-tetramethylbenzidine (TMB)-based colorimetric sensing. The quantitative readout of exosomes is easily accomplished by UV–vis spectrometry or microplate reader with a linear range of 2.2 × 105 to 2.4 × 107 particles/μL and a minimal detectable concentration of 2.2 × 104 particles/μL. This chip device was applicable to clinical samples where cancer patients demonstrate statistically significant increase in exosomes compared with healthy individuals. Thus, our chip device is cost-effective and easy-to-use, facilitating visible and colorimetric assay with high sensitivity toward clinical applications.

Serum miR-29a and miR-122 as Potential Biomarkers for Non-Alcoholic Fatty Liver Disease (NAFLD).

Non-Alcoholic Fatty Liver Disease (NAFLD) is an over accumulation of triglyceride in the liver without alcohol consumption. Its major cause is insulin resistance. Patients with NAFLD can develop liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) are non-coding RNAs that regulate post-transcriptional gene silencing. Previous research reported that miR-29 family (a, b and c) and miR-122 have an important role in regulating insulin resistance related to NAFLD. The purpose of this study was to investigate that miR-29 and miR-122 can be possible biomarkers for non-invasive diagnosis of NAFLD. Serum samples were collected from 58 NAFLD patients and 34 healthy controls. MiRNAs were extracted from serum by using microRNA purification kit followed by polyuridylation, reverse transcription and quantitative real-time PCR. Also, we analyzed the correlation between miR-29 and miR-122 and level of liver inflammation in NAFLD patients. We found that the serum miR-29a levels in NAFLD patients were significantly lower (P = 0.006) than the control group, while miR-29c levels were unchanged, and miR-29b levels were undetectable. However, we found that serum miR-122 levels in NAFLD patients were significantly higher (P < 0.001) than those found in the control group. For miR-29a, the area under curve (AUC) was 0.679 (P = 0.0065) with 60.87% sensitivity and 82.35% specificity. For miR-122, the AUC was 0.831 (P < 0.0001) with 75.00% sensitivity and 82.35% specificity. Interestingly, the levels of serum miR- 122 were significantly different between patients without steatohepatitis (NAS < 4) and steatohepatitis (NAS ≥ 4), indicating that the levels of miR-122 were related to the severity of NAFLD. The levels of miR-29a and miR-122 might be beneficial and compelling as possible biomarkers for non-invasive diagnosis of NAFLD.

Analysis of the Metabolic Characteristics of Serum Samples in Patients With Multiple Myeloma.

Aims: This study aimed to identify potential, non-invasive biomarkers for diagnosis and monitoring of the progress in multiple myeloma (MM) patients.Methods: MM patients and age-matched healthy controls (HC) were recruited in Discovery phase and Validation phase, respectively. MM patients were segregated into active group (AG) and responding group (RG). Serum samples were collected were conducted to non-targeted metabolomics analyses. Metabolites which were significantly changed (SCMs) among groups were identified in Discovery phase and was validated in Validation phase. The signaling pathways of these SCMs were enriched. The ability of SCMs to discriminate among groups in Validation phase was analyzed through receiver operating characteristic curve. The correlations between SCMs and clinical features, between SCMs and survival period of MM patients were analyzed.Results: Total of 23 SCMs were identified in AG compared with HC both in Discovery phase and Validation phase. Those SCMs were significantly enriched in arginine and proline metabolism and glycerophospholipid metabolism. 4 SCMs had the discriminatory ability between MM patients and healthy controls in Validation phase. Moreover, 12 SCMs had the ability to discriminate between the AG patients and RG patients in Validation phase. 10 out of 12 SCMs correlated with advanced features of MM. Moreover, 8 out of 12 SCMs had the negative impact on the survival of MM. 5′-Methylthioadenosine may be the only independent prognostic factor in survival period of MM.Conclusion: 10 SCMs identified in our study, which correlated with advanced features of MM, could be potential, novel, non-invasive biomarkers for active disease in MM.

Downregulated circulating microRNAs after surgery: potential noninvasive biomarkers for diagnosis and prognosis of early breast cancer

Success in curing breast cancer largely depends on the stage at diagnosis. Circulating microRNAs are becoming a promising noninvasive biomarker. We postulate that a postoperative decline in circulating microRNAs might have diagnostic and prognostic value. Applying high-throughput microarrays, we screened the dysregulated microRNAs in paired serum samples before and after surgery. The relative concentrations of putative markers between the early breast cancer and cancer-free groups were evaluated in the training set and verified in the validation set. Sensitivity, specificity, and receiver operating characteristic (ROC) curves were used to assess diagnostic value. Survival analysis was performed using Kaplan–Meier estimates and a Cox proportional hazards model. Five microRNAs significantly reduced after surgery were selected for the training set. We found that miR-130b-5p, miR-151a-5p, miR-206, and miR-222-3p were significantly higher in the breast cancer group. Each of the four microRNAs had potential diagnostic value. The combined four microRNAs (training set: area under the curve (AUC) 0.8457; validation set: AUC 0.9309) had better diagnostic value than each single microRNA. MiR-222-3p was an independent prognostic factor for disease-free survival (HR = 13.19; 95% CI, 1.06–163.59; P = 0.045). Patients with no fewer than three highly expressed miRNAs had shorter DFS than patients with 0–2 highly expressed miRNAs (HR = 2.293; 95% CI, 1.128–0.662; P = 0.022). Our findings indicate that postoperatively downregulated circulating miR-130b-5p, miR-151a-5p, miR-206, and miR-222-3p may be potential biomarkers for breast cancer diagnosis and prognosis.

Identification of beta-arrestin-1 as a diagnostic biomarker in lung cancer

BackgroundDistinguishing lung adenocarcinoma (ADC) from squamous cell carcinoma (SCC) has a tremendous therapeutic implication. Sometimes, the commonly used immunohistochemistry (IHC) markers fail to discriminate between them, urging for the identification of new diagnostic biomarkers.MethodsWe performed IHC on tissue microarrays from two cohorts of lung cancer patients to analyse the expression of beta-arrestin-1, beta-arrestin-2 and clinically used diagnostic markers in ADC and SCC samples. Logistic regression models were applied for tumour subtype prediction. Parallel reaction monitoring (PRM)-based mass spectrometry was used to quantify beta-arrestin-1 in plasma from cancer patients and healthy donors.ResultsBeta-arrestin-1 expression was significantly higher in ADC versus SCC samples. Beta-arrestin-1 displayed high sensitivity, specificity and negative predictive value. Its usefulness in an IHC panel was also shown. Plasma beta-arrestin-1 levels were considerably higher in lung cancer patients than in healthy donors and were higher in patients who later experienced a progressive disease than in patients showing complete/partial response following EGFR inhibitor therapy.ConclusionsOur data identify beta-arrestin-1 as a diagnostic marker to differentiate ADC from SCC and indicate its potential as a plasma biomarker for non-invasive diagnosis of lung cancer. Its utility to predict response to EGFR inhibitors is yet to be confirmed.

Human leucocyte antigen-G 14-bp InDel polymorphism and oral squamous cell carcinoma risk in Chinese Han population: A case-control study.

Human leucocyte antigen-G (HLA-G) is a nonclassical HLA class I molecule involved in tumour immune escape. The purpose of this study was to investigate the association between the 14-bp insertion/deletion (InDel) polymorphism in the 3' untranslated region (3'-UTR) of HLA-G gene and oral squamous cell carcinoma (OSCC) risk in Chinese Han population (216 cases and 193 healthy controls), and furthermore, to evaluate serum soluble HLA-G (sHLA-G) levels in the OSCC patients. Our results demonstrated that the Ins allele was significantly less frequent in the OSCC patients than that in the healthy controls (odds ratio [OR]=0.75; 95% confidence interval [CI]: 0.57-0.99; p=0.040). Distribution of the 14-bp genotypes in the OSCC patients and the healthy controls revealed that the Ins/Ins genotype was associated with decreased OSCC risk in both the codominant model (Ins/Ins versus Del/Del; OR=0.57; 95% CI=0.33-0.99; p=0.044) and the log-additive model (OR=0.76; 95% CI: 0.58-0.99; p=0.044). The serum sHLA-G level was significantly higher in the OSCC patients than those in the healthy controls (p<0.001). Receiver operating characteristic (ROC) curve revealed the valuable diagnostic value of sHLA-G for OSCC detection, with an area under the ROC curve (AUC) of 0.891 (95% CI: 0.856-0.925, p<0.001). The OSCC patients with Ins/Ins genotype had lower serum sHLA-G levels than those with Ins/Del and Del/Del genotypes (p=0.015). Furthermore, serum sHLA-G levels were significantly increased with the increasing TNM stages of the OSCC patients (p=0.017). Our findings revealed that the HLA-G 14-bp InDel polymorphism might be a genetic risk factor for OSCC susceptibility, and the serum sHLA-G may act as a promising biomarker for noninvasive diagnosis of OSCC.

Hemodynamic assessments of venous pulsatile tinnitus using 4D-flow MRI.

To use 4D-flow MRI to characterize hemodynamics of transverse and sigmoid sinus in venous pulsatile tinnitus (PT) patients and to investigate their differences vs healthy controls. A total of 21 patients with venous PT and 11 healthy controls were included in the retrospective study. All participants underwent 4D-flow and magnetic resonance venography scan in a 3.0T magnetic resonance scanner. All visualization, quantification, and analysis of 4D-flow data were performed using dedicated software. Two independent reviewers evaluated the existence of vortex or turbulence. Covariance analysis adjusted for age was used to compare average through-plane velocity (Vtp_avg), maximum through-plane velocity (Vtp_max), average velocity (Vavg), maximum velocity (Vmax), average blood flow (Flowavg), and pulsatility index (PI) between PT and control group. There were hemodynamic differences between PT patients and healthy controls. Compared with the control group, the PT group showed significantly higher Vtp_avg, Vtp_max, Vavg, Vmax, and Flowavg, and slightly higher PI. For the assessment of flow pattern, inter-reader reproducibility was excellent (κ = 1.00). Vortex or turbulence was observed in PT patients with good sensitivity (86.4%) and specificity (90.9%). Drainage dominance was more frequently observed in patients (15/21, 71.4%) than healthy controls (4/11, 36.4%). Significant hemodynamic differences were found between venous PT patients and healthy controls with 4D-flow MRI. Hemodynamic conditions could serve as noninvasive biomarkers for diagnosis and treatment evaluation of venous PT. This study provides Class III evidence that 4D-flow MRI accurately identifies patients with venous PT.

Abstract 5408: Elevated level of serum miR-1290 is correlated with high-grade serous ovarian epithelial ovarian cancer and might be a potential biomarker

Abstract Objective: There is a critical need for improved diagnostic markers to detect ovarian high grade serous ovarian cancer (HGSOC). MicroRNAs (miRNAs) stably exist in circulating blood, reflecting tissue or organ conditions and present in circulating microvesicles such as exosomes. Recent studies have confirmed the potential use of miRNAs profiling as a novel non-invasive biomarker for diagnosis of HGSOCs. The aim of this study is to identify which miRNAs are highly produced from HGSOCs and analyze whether serum miRNA can discriminate patients with HGSOC from healthy controls. Methods: Secreted exosomes from ovarian cancer cell lines were collected and exosomal miRNAs extracted. miRNA microarray was performed and several elevated miRNAs specific to HGSOCs were picked up. Among these, we focused on miR-1290. Serum from 71 pre-operative, 46 post-operative ovarian cancer patients and 13 healthy controls were gathered and its expression levels were detected by quantitative Real Time PCR. Results: In HGSOC patients, miR-1290 emerged overexpressed compared to healthy controls (3.52-fold). ROC analysis showed that at the cut-off of 1.61(healthy controls ; 1), the sensitivity and specificity were 63 % and 85 % respectively for detecting HGSOC (AUC = 0.71). The AUC of CA125 + miR-1290 combination was 1.00. Its expression singnificantly decreased after operation(5.87 → 1.17 ; P &amp;lt; 0.01). In advanced stage HGSOC patients, moreover, it expressed marginally higher than early stage ones(4.23 VS 1.58 ; P = 0.23). Conclusions: Serum miR-1290 can be a potential diagnostic biomarker for HGSOC. Citation Format: Masaki Kobayashi, Sawada Kenjiro, Yoshimura Akihiko, Miyamoto Mayuko, Nakatsuka Erika, Kodama Michiko, Hashimoto Kae, Mabuchi Seiji, Kimura Tadashi. Elevated level of serum miR-1290 is correlated with high-grade serous ovarian epithelial ovarian cancer and might be a potential biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5408.

Evaluation of Serum Paired MicroRNA Ratios for Differential Diagnosis of Non-Small Cell Lung Cancer and Benign Pulmonary Diseases.

To clarify whether there are different expressions between lung cancer and benign pulmonary diseases, we studied seven microRNAs (miRNAs) in serum from patients with non-small cell lung cancer (NSCLC), benign pulmonary nodules and four pulmonary inflammation diseases. We detected the expression of miRNAs using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). We found that five miRNA ratios-miR-15b-5p/miR-146b-3p, miR-20a-5p/miR-146b-3p, miR-19a-3p/miR-146b-3p, miR-92a-3p/miR-146b-3p, and miR-16-5p/miR-146b-3p-show higher expression in the NSCLC group than the benign pulmonary nodule group, and 13 ratios of miRNAs were significantly upregulated in the NSCLC group compared with the pulmonary inflammation diseases group. Receiver operating characteristic (ROC) curve analysis was performed. For NSCLC and benign pulmonary nodules, the sensitivity and specificity were 0.70 and 0.90, respectively. For NSCLC and pulmonary inflammation diseases, the sensitivity and specificity were 0.81 and 0.71, respectively. The ratios of miRNAs can be used as potential non-invasive biomarkers for diagnosis of early-stage NSCLC and benign pulmonary diseases.

Plasma miRNAs in diagnosis and prognosis of pancreatic cancer: A miRNA expression analysis

The differential expression of microRNAs (miRNAs) in plasma of pancreatic cancer (PC) patients may act as a diagnostic biomarker. A four-stage study was performed to identify plasma miRNAs with potential in detecting PC. Exiqon panels (20 PC vs. 10 normal controls (NCs)) were applied in the screening phase to obtain miRNA profiling. The identified miRNAs were further assessed in the training (40 PC vs. 40 NCs) and testing stages (112 PC vs. 116 NCs) with qRT-PCR assays. A six-miRNA signature including up-regulated miR-122-5p, miR-125b-5p, miR-192-5p, miR-193b-3p, miR-221-3p and miR-27b-3p was identified. The signature could accurately discriminate PC patients from NCs with areas under the receiver operating characteristic curve of 0.848, 0.833 and 0.937 for the training, testing and the external validation stage (41 PC vs. 50 NCs), respectively. The multivariate Cox regression analyses showed that down-regulated plasma miR-125b-5p could predict worse OS independent from late tumor stage and high CA19-9. All the six miRNAs except miR-122-5p showed high expression levels in PC tissues than those in matched normal tissues. MiR-122-5p and miR-193b-3p were up-regulated, while miR-221-3p was down-regulated in plasma exosomes from PC patients. Bioinformatics analysis demonstrated that the miRNAs might involve in several molecular pathways closely related with PC such as p53 signaling pathway, pancreatic cancer, TGF-beta signaling pathway and so on. In conclusion, we identified a six-miRNA signature in plasma which could act as a non-invasive biomarker in diagnosis and prognosis of PC. Plasma miR-125b-5p might act as an independent biomarker in predicting OS of PC patients.

Plasma long non-coding RNA HOTAIR as a potential biomarker for gastric cancer.

Long non-coding RNAs (lncRNAs) Hox transcript antisense intergenic RNA ( HOTAIR) has been suggested to be implicated in gastric cancer tumorigenesis and progression; however, little is known about the role of the plasma HOTAIR in gastric cancer diagnosis and prognosis. The current study was aimed at investigating the clinical relevance of plasma long non-coding HOTAIR as a non-invasive diagnostic biomarker in gastric cancer. Plasma HOTAIR expression was measured in 50 patients with newly diagnosed gastric cancer and 50 age- and sex-matched healthy controls using quantitative reverse transcription polymerase chain reaction. Plasma level of HOTAIR was significantly higher in gastric cancer patients compared with healthy controls ( P < 0.001). By using receiver operating characteristic curve analysis, it was found that plasma HOTAIR could diagnose gastric cancer with 88% sensitivity and 84% specificity. Furthermore, increased HOTAIR expression was associated with advanced tumor stages, higher grades, and metastasis. Plasma HOTAIR might serve as a potential non-invasive biomarker for diagnosis of gastric cancer.

Adiponectin, Leptin, IGF-1, and Tumor Necrosis Factor Alpha As Potential Serum Biomarkers for Non-Invasive Diagnosis of Colorectal Adenoma in African Americans.

The potential role of adiponectin, leptin, IGF-1, and tumor necrosis factor alpha (TNF-α) as biomarkers in colorectal adenoma is not clear. Therefore, we aimed to investigate the blood serum levels of these biomarkers in colorectal adenoma. The case–control study consisted of serum from 180 African American patients with colon adenoma (cases) and 198 healthy African Americans (controls) at Howard University Hospital. We used ELISA for adiponectin, leptin, IGF-1, and TNF-α detection and quantification. Statistical analysis was performed by t-test and multivariate logistic regression. The respective differences in median leptin, adiponectin, IGF-1, and TNF-α levels between control and case groups (13.9 vs. 16.4), (11.3 vs. 46.0), (4.5 vs. 12.9), and (71.4 vs. 130.8) were statistically significant (P < 0.05). In a multivariate model, the odds ratio for adiponectin, TNF-α, and IGF-1 were 2.0 (95% CI = 1.6–2.5; P < 0.001), 1.5 (95% CI = 1.5(1.1–2.0); P = 0.004), and 1.6 (95% CI = 1.3–2.0; P < 0.001), respectively. There was a positive correlation between serum adiponectin and IGF-1 concentrations with age (r = 0.17, P < 0.001 and r = 0.13, P = 0.009), TNF-α, IGF-1, and leptin concentration with body mass index (BMI) (r = 0.44, P < 0.001; r = 0.11, P = 0.03; and r = 0.48, P < 0.001), respectively. Also, there was a negative correlation between adiponectin and leptin concentrations with BMI (r = −0.40, P < 0.001), respectively. These data support the hypothesis that adiponectin, IGF-1, and TNF-α high levels correlate with higher risk of colon adenoma and can thus be used for colorectal adenomas risk assessment.

Exhaled nitric oxide as a potential marker for detecting non-ulcer dyspepsia and peptic ulcer disease

Nitric oxide (NO) plays a key role in the development of peptic ulcer disease (PUD). Conversely, the gastric pathogen Helicobacter pylori colonizes the human stomach and contributes to the development of non-ulcer dyspepsia (NUD) and PUD. However, the underlying relation between molecular NO in exhaled breath and H. pylori-associated NUD and PUD remains largely unknown. Here, we found that the excretion kinetics of NO profiles in exhaled breath are altered markedly in H. pylori-infected NUD and PUD subjects. In our observations, PUD led to considerably higher enrichments of NO in exhaled breath compared to NUD, thus revealing a potential link between exhaled NO and ulcer and non-ulcer complications. Our findings therefore suggest that molecular NO in exhaled breath could be used as a potential biomarker for non-invasive diagnosis and selective differentiation of NUD from PUD. Our observations also highlight that alterations of NO in the gastric environment can play an important role in the pathogenesis of peptic ulcers and thus may provide a new strategy for precise evolution of the actual disease state without the need for endoscopic biopsy, even after the eradication of H. pylori infection.

Abstract LB-A06: Pipeline for High Throughput Analysis of Exosomes in Clinical Biofluids

Abstract Purpose/Objective(s): Because Extracellular Vesicles (EVs) carry surface receptors that are characteristic of their cells of origin, EVs have tremendous potential as non-invasive biomarkers for diagnosis, risk-stratification, treatment selection, and treatment monitoring. We developed a first-in-class pipeline to characterize EV heterogeneity and provide high-sensitivity quantification of informative EVs in biofluids before, during, and after treatment. By combining multiplex assays with high-resolution, single EV flow cytometric methods together into a Mutiplex-to-Single EV Analysis (Mt-SEA) pipeline, we are able to characterize a broad range of relevant EV subsets, while also accurately measuring the concentration of specific EV populations. Exploratory studies presented here validate the performance of Mt-SEA method by confirming strong correlations of liquid biopsy EV repertoires with tumor burden and responses to treatment. Furthermore, EV analysis with Mt-SEA may identify previously unrecognized prognostic epitopes or EVs subsets. Materials/Methods: To evaluate the use of this pipeline in an exploratory clinical cohort, we evaluated EVs from plasma samples of Adult T Cell Leukemia/Lymphoma patients receiving palliative radiation. Plasma was obtained before and after treatment (n = 5 treatment courses). A customized multiplex array was used with detection antibodies to identify more than 40 major EV subsets. General exosome and EV detection epitopes included CD63, CD9, and CD81. Tumor-specific epitopes for each patient included CD4, CD5, and CD25, based on available histo-/cyto-pathology results. Next, high-resolution single EV analyses were performed with nanoFACS sorting and a prototype nanoFCM analyzer. Results: The Mt-SEA pipeline provided a broad survey of EV populations, followed by individual EV analysis ATLL-derived EVs were detected in each pre-treatment sample, with reduced specific ATLL-derived EV subsets concentrations at the end of treatment. Furthermore, ATLL-specific EVs from patients with progressive systemic disease prior to treatment were found to carry CD44 and CD133 epitopes, consistent with increasing tumor aggressiveness. Responses to treatment that were clinically evident mirrored changes in the Mt-SEA EV profiles, and Mt-SEA identified new candidate prognostic EV profiles associated with clinical outcomes that would not have been predicted. Conclusions: Optimal leverage the information carried by tumor- and immune cell-derived EVs (to use EVs as clinical biomarkers) will require a combination of methods to 1) broadly characterize EVs and 2) rigorously enumerate specific selected EV subsets using single molecule detection. The Mt-SEA pipeline achieves both of these goals. Our exploratory study with these ATLL cases demonstrates that Mt-SEA provides unexpected insights into tumor biology, along with robust estimations of concentrations of EV subsets of interest. Detection of tumor-associated EVs and detection of EV repertoire changes during treatment paves the way to future evaluation of the Mt-SEA pipeline for personalized, adaptive therapies in a wider range of tumor types. Citation Format: Jennifer Jones, Joshua Welsh, Jason Savage, Jenn Marte, James Gulley, Kevin Conlon, Thomas Waldmann, Kevin Camphausen, Jay Berzofsky. Pipeline for High Throughput Analysis of Exosomes in Clinical Biofluids [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A06.

Cell-Free Circulating Methylated SEPT9 for Noninvasive Diagnosis and Monitoring of Colorectal Cancer.

Identification of early-stage tumor and monitoring therapeutic efficacy and recurrence or metastasis of colorectal cancer (CRC) are urgently warranted for improving the outcome of CRC patients and reducing the disease-related mortality. In this study, we evaluated the diagnostic value of cell-free circulating methylated SEPT9 (mSEPT9) for CRC and beyond CRC and examined the potentiality of mSEPT9 in assessing therapeutic efficacy and monitoring recurrence of CRC. Our results confirmed the favorable diagnostic value of plasma mSEPT9 for CRC, with a sensitivity of 61.22% (95% confidence interval (CI): 51.33%–70.27%) and specificity of 93.7% (95% CI: 91.09%–95.57%) using 2/3 algorithm. The positive rate of mSEPT9 in CRC was correlated with tumor size, histological grade, and general histological type (P < 0.05). Beyond CRC, gastric cancer patients also presented a high positive rate of plasma mSEPT9 (70%). The conversions between preoperative and postoperative plasma mSEPT9 reflected the therapeutic efficacy of curatively intended surgery for CRC patients. The persistent positivity of plasma mSEPT9 after surgery (within 7–14 days) was highly associated with impending recurrences or metastases (within one year), with a sensitivity of 100%. Postoperative mSEPT9 status during follow-up also provided valuable indication for CRC recurrence or metastases, with a good consistency (kappa = 0.818, P = 0.001). Our results verified the reliability of plasma mSEPT9 as a biomarker for noninvasive diagnosis of CRC. More significantly, we revealed its valuable role in appraising CRC therapeutic efficacy and monitoring CRC recurrences or metastases. Further studies with larger sample sizes are needed to verify and elucidate the clinical utility of the promising findings.

Cell-free plasma hypermethylated CASZ1, CDH13 and ING2 are promising biomarkers of esophageal cancer.

Identifying sensitive and specific biomarkers for early detection of cancer is immensely imperative for early diagnosis and treatment and better clinical outcome of cancer patients. This study aimed to construct a specific DNA methylation pattern of cancer suppressor genes and explore the feasibility of applying cell-free DNA based methylation as a biomarker for early diagnosis of esophageal squamous cell carcinoma (ESCC). We recruited early stage ESCC patients from Yangzhong County, China. The Illumina Infinium 450K Methylation BeadChip was used to construct a genome-wide DNA methylation profile. Then, differentiated genes were selected for the validation study using the Sequenom MassARRAY platform. The frequency of methylation was compared between cancer tissues, matched cell-free DNAs and normal controls. The specific methylation profiles were constructed, and the sensitivity and specificity were calculated. Seven CG sites in three genes CASZ1, CDH13 and ING2 were significantly hypermethylated in ESCC as compared with normal controls. A significant correlation was found between the methylation of DNA extracted from cancer tissues and matched plasma cell-free DNA, either for individual CG site or for cumulative methylation analysis. The sensitivity and specificity reached 100% at an appropriate cut-point using these specific methylation biomarkers. This study revealed that aberrant DNA methylation is a promising biomarker for molecular diagnosis of esophageal cancer. Hypermethylation of CASZ1, CDH13 and ING2 detected in plasma cell-free DNA can be applied as a potential noninvasive biomarker for diagnosis of esophageal cancer.

Serum Cytokine Profiling in Cats with Acute Idiopathic Cystitis.

BackgroundFeline idiopathic cystitis (FIC) is a common lower urinary tract disorder of domestic cats that resembles interstitial cystitis/painful bladder syndrome (IC/PBS) in humans. Diagnosis of FIC is based on clinical signs and exclusion of other disorders because of a lack of specific pathologic findings or other objective biomarkers. Cytokines are potential noninvasive biomarkers to define the presence, severity, and progression of disease, and response to treatment.ObjectivesThe objective of this pilot study was to determine concentrations of selected cytokines in serum from healthy cats and cats with acute FIC.AnimalsSerum samples from 13 healthy cats and from 12 cats with nonobstructive acute FIC were utilized.MethodsMultiplex analysis of 19 cytokines (CCL2, CCL5, CXCL1, CXCL12, CXCL8, Flt3L, GM‐CSF, IFN‐γ, IL‐12 (p40), IL‐13, IL‐18, IL‐1β, IL‐2, IL‐4, IL‐6, PDGF‐BB, SCF, sFas, and TNF‐α) was performed with a commercially available feline‐specific multiplex bead‐based assay.ResultsMean serum concentrations of IL‐12 (p40; P < 0.0001), CXCL12 (P = 0.002), IL‐18 (P = 0.032), and Flt3L (P = 0.0024) were significantly increased in FIC cats compared to healthy cats. GM‐CSF, IL‐1b, IL‐2, and PDGF‐BB were undetectable or detected in an insufficient number of cats to allow meaningful comparisons.Conclusions and Clinical ImportanceWe have identified increased serum concentrations of pro‐inflammatory cytokines and chemokines CXCL12, IL‐12, IL‐18, and Flt3L in FIC‐affected cats. These findings suggest potential candidates for noninvasive biomarkers for diagnosis, staging, and therapeutic outcome monitoring of affected cats and provide additional insight into the etiopathogenesis of FIC.