Quantification of urinary exosomal PSA for the diagnosis of prostate cancer: a study protocol with emphasis on application of clinical laboratory-based techniques.

BACKGROUND: Prostate cancer is the most common cancer in men and represents a major problem of public health. The current method of diagnosing/screening for prostate cancer is invasive and costly. There have been renewed and innovative studies on searching urinary biomarker for prostate cancer diagnosis, especially with the technologies of urinary exosomes. However, the technologies of urine exosomes usually need expensive machines such as ultracentrifuge and they are difficult to standardization, which hinder their application in clinical laboratory. OBJECTIVE: In this study, our objective is to detect urinary exosomes from prostate cancer for the development of a test to aid in the diagnosis of prostate cancer. METHODS: The exosomes from a prostate cancer cell line LNCaP was used to set up the techniques. For the analysis of urine samples of patients, the methods include the collection of first-void urine by using Colli-Pee device, the isolation of urine exosome by optimized precipitation method and the detection of exosomal PSA by Elecsys® total PSA. We have modified and optimized the isolation of urinary exosomes with precipitation method. RESULTS: By using the exosomes from the prostate cancer cell line, we have found that the urinary exosomal PSA can be quantified by automatic technique of Elecsys® total PSA. It will be a 2-year study. We shall start to include the patients and controls in the summer of 2024. We expect the results to be published in the last quarter of 2026. CONCLUSIONS: This is the first study to detect the urinary exosomal PSA by the technique of Elecsys® total PSA for the diagnosis of prostate cancer. This study emphasizes on the techniques suitable for the implementation in clinical laboratory, which will facilitate application of urinary exosomes to simplify and improve the diagnosis/screening of prostate cancer.

Magnetically Steered Cell Therapy For Functional Restoration Of Intraocular Pressure Control In Open-Angle Glaucoma.

Trabecular meshwork (TM) cell therapy has been proposed as a next-generation treatment for elevated intraocular pressure (IOP) in glaucoma, the most common cause of irreversible blindness. Using a magnetic cell steering technique with excellent efficiency and tissue-specific targeting, we delivered two types of cells into a mouse model of glaucoma: either human adipose-derived mesenchymal stem cells (hAMSCs) or induced pluripotent cell derivatives (iPSC-TM cells). We observed a 4.5 [3.1, 6.0] mmHg or 27% reduction in intraocular pressure (IOP) for nine months after a single dose of only 1500 magnetically-steered hAMSCs, associated with restoration of function to the conventional outflow pathway, as judged by increased outflow facility and TM cellularity. iPSC-TM cells were also effective, but less so, showing only a 1.9 [0.4, 3.3] mmHg or 13% IOP reduction and increased risk of tumorigenicity. In both cases, injected cells remained detectable in the iridocorneal angle three weeks post-transplantation. Based on the locations of the delivered cells, the mechanism of IOP lowering is most likely paracrine signaling. We conclude that magnetically-steered hAMSC cell therapy has potential for long-term treatment of ocular hypertension in glaucoma. One Sentence Summary: A novel magnetic cell therapy provided effective intraocular pressure control in a mouse model of glaucoma, motivating future translational studies.

Characterization of extrachromosomal circular DNAs in plasma of patients with clear cell renal cell carcinoma.

BACKGROUND AND PURPOSE: Extrachromosomal circular DNAs (eccDNAs) have been recognized for their significant involvement in numerous biological processes. Nonetheless, the existence and molecular characteristics of eccDNA in the peripheral blood of patients diagnosed with clear cell renal cell carcinoma (ccRCC) have not yet been reported. Our aim was to identify potentially marked plasma eccDNAs in ccRCC patients. METHODS AND MATERIALS: The detection of plasma eccDNA in ccRCC patients and healthy controls was performed using the Tn5-tagmentation and next-generation sequencing (NGS) method. Comparisons were made between ccRCC patients and healthy controls regarding the distribution of length, gene annotation, pattern of junctional nucleotide motif, and expression pattern of plasma eccDNA. RESULTS: We found 8,568 and 8,150 plasma eccDNAs in ccRCC patients and healthy controls, respectively. There were no statistical differences in the length distribution, gene annotation, and motif signature of plasma eccDNAs between the two groups. A total of 701 differentially expressed plasma eccDNAs were identified, and 25 plasma eccDNAs with potential diagnostic value for ccRCC have been successfully screened. These up-regulated plasma eccDNAs also be indicated to originate from the genomic region of the tumor-associated genes. CONCLUSION: This work demonstrates the characterization of plasma eccDNAs in ccRCC and suggests that the up-regulated plasma eccDNAs could be considered as a promising non-invasive biomarker in ccRCC.

Detection of HPV in urine for cervical cancer screening: Feasibilty of an assay system.

The detection of urine HPV is considered as a promosing alternative to increase the screening coverage of cervical cancer. However, the validated assay of urine HPV is still scarse. We described a nouvel assay syetem for the urine-based detection of HPV in the framework of HPV screening. This sytsem consisted of Automate Nimbus extraction of DNA and Anyplex™ II HPV HR Detection PCR of HPV DNA. We validated this system by spiking HPV-infected cervical cancer cell line HeLa cells into normal urine and compared the prelimary results of cervical samples and urine samples. We found that this system could detect as few as 5 HeLa cells in normal urine model. Some discordances of HPV results between cervical samples and urine samples were observed. We concluded that this assay system could be applied for the detection of HPV in urine. A large scale study is necessary to evaluate the clinical significance of this assay system.

Transmission electron microscopy assessment of a novel method for isolating pure exosomes from serum.

Serum exosomes frequently are used for liquid biopsy. Serum exosomes normally are isolated using ultracentrifugation; however, ultracentrifugation is time-consuming, labor intensive and requires a high-speed centrifuge. Many commercial kits use a precipitation-based method; however, this process can result in substantial contamination. We developed a new method to isolate pure serum exosomes. We isolated serum exosomes using precipitation, extracted them using acetone, then isolated them again by precipitation. We used transmission electron microscopy (TEM) to examine the morphology of serum exosomes. TEM indicated that our isolated exosomes were pure with typical morphology and with a size ranging from 40 to 150 nm. Flow cytometry revealed expression of exosome markers, CD63, CA81 and CD9. Our double precipitation method enables ready extraction of pure exosomes from serum. Our double precipitation method simplifies detection of serum exosomal biomarkers for diagnosis and prognosis of disease.

Urine-based detection of HPV for cervical cancer screening: Time for standardized tests.

Cervical cancer is preventable because it has an established etiology, mainly attributed to a detectable pathogen, human papillomavirus (HPV). In 2018, the world health organization issued an unprecedented call for global action to eliminate cervical cancer by 2030. The adaptation of regular screening programs is fundamental to achieve the goal of cervical cancer elimination. However, it is still difficult to achieve satisfactory coverage rates of screening in developing countries as well as in developed countries because many women are reluctant to participate in gynecologic examination. HPV detection in urine is a convenient, widely acceptable by women and relatively affordable without the necessity for clinical visits to improve the coverage rates of cervical cancer screening. Unfortunately, the clinical implementation of urine-based tests for HPV detection has been hindered by the lack of standardized tests. Further optimization of protocols and standardization of urinary HPV detection are expected to be realized. With the advantages of urine sampling to overcome cost, personal, and cultural barriers, time has come for the standardized tests to facilitate a wide clinical implementation of urinary HPV detection that will significantly contribute to the WHO's goal, that is, to eliminate the cervical cancer globally.

Development of the Thymus and Kidney and Effects of Resveratrol on Their Aging in a Short-Lived Fish.

Annual fishes of the genus Nothobranchius have been widely used in cognitive, behavioral, and genetic studies, and have become an excellent animal model for studying aging. However, the development and degeneration of immune organs in annual fishes and the antagonistic effects of resveratrol remain unclear. In the present study, the development of thymus and kidney was investigated systematically using Nothobranchius guentheri from larvae, juveniles, and young and old fish with hematoxylin and eosin staining. We found that thymus primordium was observed first in the larvae at 2 days after hatching (dah). After the lymphoid cells became evident at 5 dah, the thymus acquired an irregular shape at 7 dah. Then it formed a wedge shape at 15 dah. Thymus looked as homogeneous distribution of lymphocytes at 1 month old, and it differentiated into cortex and medulla approximately in 2-month-old fish. Combined with TUNEL and senescence-associated ß-galactosidase (SA-ß-gal) staining, it showed the degeneration of the thymus appeared in 4-month-old fish. Kidney primordium appeared on 1 dah, and the glomerulus was visible at 7 dah. The nephrogenic activity was most apparent in 1-month-old fish. A large hematopoietic tissue was arranged in the renal interstitium in 2- and 3-month-old fish. In 6-month-old fish, the kidney structure became less dense. By 12 months, the kidney exhibited the most pronounced histological characteristics of aging. Feeding resveratrol ameliorated renal fibrosis and SA-ß-gal staining with age, increased SIRT1 and SIRT3 expression, and decreased the levels of NF-κB and inflammatory factors in thymus and kidney of the fish. We provided basic data for the development and degeneration of immune organs and resveratrol's anti-aging effects in short-lived fish.

A specific technique of immunolabelling of urinary small extracellular vesicle biomarkers for the diagnostic of renal cancer.

Small extracellular vesicles (EVs) are characterized by the membrane expression of CD63, CD81 and CD9 tetraspanins. Their size is inferior to 200 nm. They share the same characteristics as the native cells and are found in human fluids. Specific membrane protein biomarkers expressed on small EV are useful for the diagnosis of tumoural pathologies. Clear cell renal cell carcinoma (CCRCC) is diagnosed by imaging examinations and/or tissue biopsy. Carbonic anhydrase IX (CAIX) is a powerful biomarker of CCRCC. The detection of CAIX on small EV from the urine of patients could constitute a liquid biopsy for CCRCC. We have set up a specific protocol for the preparation, the immunostaining characterization and the transmission electron microscopy observation of small EVs isolated from the urine of CCRCC patients. The background labelling was significantly reduced. We successfully detected biomarkers on urinary small EVs from CCRCC patients. This technique could be extended with antibodies directed against other EV biomarkers for the detection and the monitoring of cancer diseases.

Resveratrol Alleviates Inflammation and ER Stress Through SIRT1/NRF2 to Delay Ovarian Aging in a Short-Lived Fish.

Aging is a complex process in which the structure and function of various tissues and organs gradually decline with age, and ovarian aging affects the reproductive capacity of females and induces age-related diseases. Resveratrol, a natural polyphenol compound, extends the life span and has a protective effect on the ovaries of vertebrates. However, the effects and underlying mechanisms of resveratrol delaying ovarian aging are unclear. In this study, using an annual fish Nothobranchius guentheri, we demonstrated that senescence-associated-beta-galactosidase (SA-ß-gal) activity and lipofuscin accumulation increased with age in the ovaries, and resveratrol reversed this phenomenon. Resveratrol increased proliferating cell nuclear antigen (PCNA) expression and the oocyte proportions of the primary growth stage, cortical alveolus stage and vitellogenesis stage, and decreased the number of atretic follicles in the ovaries of 6-, 9-, and 12-month-old fish. Moreover, the expression of SIRT1 and NRF2 decreased and the levels of NF-κB, pro-inflammatory cytokines IL-1ß, TNF-α, and IL-8 and endoplasmic reticulum (ER) stress markers GRP78 and CHOP increased with aging, while resveratrol up-regulated SIRT1 and NRF2 expression and down-regulated NF-κB, IL-1ß, TNF-α, IL-8, GRP78, and CHOP levels in the ovaries of 6- and 9-month-old fish. In HEK293T cells, knockdown SIRT1 decreased NRF2 and increased NF-κB p65, pro-inflammatory cytokines (IL-1ß and TNF-α), and ER stress marker GRP78 expression markedly. Silencing SIRT1 and then treating the cells with resveratrol significantly reversed the phenomenon. Collectively, resveratrol might activate SIRT1/NRF2 to reduce inflammation and ER stress, and finally delay ovarian aging in a short-lived fish. This study highlights the protective effect and mechanism of resveratrol on ovarian aging.

Resveratrol induces DNA damage-mediated cancer cell senescence through the DLC1-DYRK1A-EGFR axis.

Inducing cell senescence is widely regarded as a potent tumor suppression mechanism. Resveratrol has attracted increasing attention for its capacity to prevent and suppress cancer. However, the mechanism of resveratrol on the induction of cancer cell senescence has not been well clarified. Our results showed that resveratrol inhibited cell viability and colony formation and promoted cell senescence along with augmentation of SA-ß-gal activity and modulation of senescence-associated molecular markers p53, p21 and LaminB protein in breast and liver cancer cells. The underlying mechanism was that resveratrol increased ROS generation to enhance tumor suppressor gene DLC1 expression, and DLC1 further inhibited the DYRK1A-EGFR axis to trigger DNA damage accompanied by up-regulation of the DNA double strand break marker protein γH2AX and down-regulation of the DNA repair related proteins p-BRCA1 and RAD51, eventually leading to cancer cell senescence. Resveratrol also effectively inhibited the volume of transplanted tumor with increased SA-ß-gal activity and DLC1 level in a chicken embryo allantoic membrane xenograft tumor model. This is the first report to investigate whether resveratrol induces DNA damage-mediated cancer cell senescence through the DLC1-DYRK1A-EGFR axis, which could provide a solid base for resveratrol's application in cancer prevention and clinical treatment as a food additive or adjuvant therapies.

Aspirin blocks AMPK/SIRT3-mediated glycolysis to inhibit NSCLC cell proliferation.

Non-small cell lung cancer (NSCLC) has the highest incidence and mortality in the world. Aspirin has been reported to promote apoptosis, inhibit proliferation, stemness, angiogenesis, cancer-associated inflammation and migration in NSCLC. But the effect of aspirin on aerobic glycolysis in NSCLC is less reported. In the present study, we investigated whether aspirin blocked aerobic glycolysis of NSCLC cells to inhibit proliferation. Our results showed that aspirin inhibited viability, PCNA expression, ability of colony formation, dimished extracellular acidification rate (ECAR), oxygen consumption rate (OCR) and production of pyruvic acid and lactic acid, accompanied with reduced mitochondrial membrane potential (MMP), PGC-1α expression and ROS production, indicating mitochondrial dysfunction in NSCLC cells. AMPK and mitochondrial-localized deacetylase sirtuin 3 (SIRT3) were identified as the relevant molecular targets in glycolysis, but mechanism and relationship between AMPK and SIRT3 for aspirin induced glycolysis inhibition remain unknown in cancer cells. The investigation of underlying mechanism indicated that aspirin activated AMPK pathway to inhibit aerobic glycolysis and proliferation by upregulating SIRT3 after application of compound C (CC), an inhibitor of AMPK activity or SIRT3 siRNA. Upon activation of SIRT3, aspirin promoted the release of hexokinase-II (HK-II) from mitochondrial outer membrane to cytosol by deacetylating cyclophilin D (CypD). Consistently, aspirin significantly inhibited the growth of NSCLC xenografts and exhibited antitumor activity probably through AMPK/SIRT3/HK-II pathway in vivo. Collectively, AMPK/SIRT3/HK-II pathway plays a critical role in anticancer effects of aspirin, and our findings might serve as potential target for clinical practice and chemoprevention of aspirin in NSCLC.

Tumor-Derived Exosomal RNA From Fine-Needle Aspiration Supernatant as a Novel Liquid Biopsy for Molecular Diagnosis of Cancer.

Background: We hypothesized that the fine needle aspiration (FNA) supernatant from tumor might contain tumor-derived exosomes. The objective of this pilot study was to test if tumor-derived exosomal RNA could be found in FNA supernatants for molecular diagnosis of cancer. Methods: 10 FNA samples from pancreatic tumor were included. After the routine recuperation of cellular material by centrifugation, the cell-free Cytolyt liquid was collected instead of being discarded. 10 ml Cytolyt was used to isolate the exosomes. Transmission electronic microscopy (TEM) was used to examine the presence of exosomes. The exosomal marker CD63 was analyzed by flow cytometry. The exosomal RNA was extracted. RT-qPCR was performed to detect the GAPDH and the tumor marker of glypican 1 gene expression. Results: TEM confirmed the presence of exosomes from FNA supernatants. Flow cytometry showed a strong positive expression of exosome marker CD63. The concentration of exosomal RNA ranged from 18.81 to 354.75 ng/µl with an average of 81.76 ng/µl. The average exosomal RNA quantity was 1390.01 ng (range from 319.77 to 6030.75 ng) with an average 260/280 ratio of 2.12. GAPDH was detectable in all samples. Exosomal glypican 1 was detected in all samples of pancreatic ductal adenorcarcinomas (3/3) and absent from benign cystic samples (3/3). Furthermore, exosomal glypican 1 was positive in one sample with a non-contributive cytology and in one sample in which no malignant cell was found. Conclusion: This is the first report that the supernatants from FNA biopsy may contain tumor-derived exosomal RNA. These tumor-derived exosomes from FNA may provide a new liquid biopsy for the molecular diagnosis of cancer.

Translationally controlled tumor protein: the mediator promoting cancer invasion and migration and its potential clinical prospects.

Translationally controlled tumor protein (TCTP) is a highly conserved multifunctional protein localized in the cytoplasm and nucleus of eukaryotic cells. It is secreted through exosomes and its degradation is associated with the ubiquitin-proteasome system (UPS), heat shock protein 27 (Hsp27), and chaperone-mediated autophagy (CMA). Its structure contains three α|-helices and eleven ß|-strands, and features a helical hairpin as its hallmark. TCTP shows a remarkable similarity to the methionine-R-sulfoxide reductase B (MsrB) and mammalian suppressor of Sec4 (Mss4/Dss4) protein families, which exerts guanine nucleotide exchange factor (GEF) activity on small guanosine triphosphatase (GTPase) proteins, suggesting that some functions of TCTP may at least depend on its GEF action. Indeed, TCTP exerts GEF activity on Ras homolog enriched in brain (Rheb) to boost the growth and proliferation of Drosophila cells. TCTP also enhances the expression of cell division control protein 42 homolog (Cdc42) to promote cancer cell invasion and migration. Moreover, TCTP regulates cytoskeleton organization by interacting with actin microfilament (MF) and microtubule (MT) proteins and inducing the epithelial-mesenchymal transition (EMT) process. In essence, TCTP promotes cancer cell movement. It is usually highly expressed in cancerous tissues and thus reduces patient survival; meanwhile, drugs can target TCTP to reduce this effect. In this review, we summarize the mechanisms of TCTP in promoting cancer invasion and migration, and describe the current inhibitory strategy to target TCTP in cancerous diseases.

Comprehensive Evaluation of the m6A Regulator Prognostic Risk Score in the Prediction of Immunotherapy Response in Clear Cell Renal Cell Carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) is known for its high drug resistance. The tumor-immune crosstalk mediated by the epigenetic regulation of N6-methyladenosine (m6A) modification has been demonstrated in recent studies. Therefore, m6A modification-mediated immune cell infiltration characteristics may be helpful to guide immunotherapy for ccRCC. Methods: This study comprehensively analyzed m6A modifications using the clinical parameters, single-cell RNA sequencing data, and bulk RNA sequencing data from the TCGA-ccRC cohort and 13 external validation cohorts. A series of bioinformatic approaches were applied to construct an m6A regulator prognostic risk score (MRPRS) to predict survival and immunotherapy response in ccRCC patients. Immunological characteristics, enriched pathways, and mutation were evaluated in high- and low-MRPRS groups. Results: The expressional alteration landscape of m6A regulators was profiled in ccRCC cell clusters and tissue. The 8 regulator genes with minimal lambda were integrated to build an MRPRS, and it was positively correlated with immunotherapeutic response in extent validation cohorts. The clinicopathological features and immune infiltration characteristics could be distinguished by the high- and low-MRPRS. Moreover, the MRPRS-mediated mutation pattern has an enhanced response to immune checkpoint blockade in the ccRCC and pan-cancer cohorts. Conclusions: The proposed MRPRS is a promising biomarker to predict clinical outcomes and therapeutic responses in ccRCC patients.

Resveratrol drives cancer cell senescence via enhancing p38MAPK and DLC1 expressions.

Pro-senescence therapy is a recently proposed anti-cancer strategy and has been shown to effectively inhibit cancer. Resveratrol is gaining attention for its cancer preventive and suppressive properties. The mechanisms of resveratrol in cancer suppression by inducing cancer cell senescence are unclear. Our results showed that resveratrol induced cell senescence along with an increase of SA-ß-Gal activity and inhibition of colony formation in breast and lung cancer cells. The underlying mechanisms were that resveratrol induced ER-stress by increasing SIRT1 to promote p38MAPK expression and by reducing NO level to up-regulate DLC1 expression, and ER-stress further resulted in DNA damage and mitochondrial dysfunction, eventually leading to cancer cell senescence. Our findings on resveratrol's induction of cancer cell senescence via activating ER-stress through the SIRT1/p38MAPK and NO/DLC1 pathways provide a solid base for its clinical application and its preventive application as a food additive.

Nanoscale Covalent Organic Frameworks with Donor-Acceptor Structures as Highly Efficient Light-Responsive Oxidase-like Mimics for Colorimetric Detection of Glutathione.

Although organic artificial enzymes have been reported as biomimetic oxidation catalysts and are widely used for colorimetric biosensors, developing organic artificial enzymes with high enzymatic activity is still a challenge. Two-dimensional (2D) covalent organic frameworks (COFs) have shown superior potential in biocatalysts because of their periodic π-π arrays, tunable pore size and structure, large surface area, and thermal stability. The interconnection of electron acceptor and donor building blocks in the 2D conjugated COF skeleton can lead to narrower band gaps and efficient charge separation and transportation and thus is helpful to improve catalytic activity. Herein, a donor-acceptor 2D COF was synthesized using tetrakis(4-aminophenyl)pyrene (Py) as an electron donor and thieno[3,2-b]thiophene-2,5-dicarbaldehyde (TT) as an electron acceptor. Under visible light irradiation, the donor-acceptor 2D COF exhibited superior enzymatic catalytic activity, which could catalyze the oxidation of chromogenic substrates such as 3,3',5,5'-tetramethylbenzidine (TMB) by the formation of superoxide radicals and holes. Based on the above property, the photoactivated donor-acceptor 2D COF with enzyme-like catalytic properties was designed as a robust colorimetric probe for cheap, highly sensitive, and rapid colorimetric detection of glutathione (GSH); the corresponding linear range of GSH was 0.4-60 µM, and the limit of detection was 0.225 µM. This study not only presents the construction of COF-based light-activated nanozymes for environmentally friendly colorimetric detection of GSH but also provides a smart strategy for improving nanozyme activity.

Urinary Exosomes Diagnosis of Urological Tumors: A Systematic Review and Meta-Analysis.

PURPOSE: Exosomes could be released directly into the urine by the urological tumoral cells, so testing urinary exosomes has great potential for non-invasive diagnosis and monitor of urological tumors. The objective of this study is to systematically review and meta-analysis of urinary exosome for urological tumors diagnosis. MATERIALS AND METHODS: A systematic review of the recent English-language literature was conducted according to the PRISMA statement recommendations (CRD42021250613) using PubMed, Embase, Cochrane Library, Web of Science, and Scopus databases up to April 30, 2021. Risk-of-bias assessment was performed according to the QUADAS 2 tool. The true diagnostic value of urinary exosomes by calculating the number of true positive, false positive, true negative, and false negative, diagnoses by extracting specificity and sensitivity data from the selected literature. RESULTS: Sixteen eligible studies enrolling 3224 patients were identified. The pooled sensitivity and specificity of urinary exosomes as a diagnostic tool in urological tumors were 83% and 88%, respectively. The area under the summary receiver operating characteristic curve was 0.92 (95% CI: 0.89-0.94). Further subgroup analyses showed that our results were stable irrespective of the urinary exosome content type and tumor type. CONCLUSION: Urinary exosomes may serve as novel non-invasive biomarkers for urological cancer detection. Future clinical trial designs must validate and explore their utility in treatment decision-making. SYSTEMATIC REVIEW REGISTRATION: [ https://www.crd.york.ac.uk/prospero/], identifier [CRD42021250613].

Meta-Analysis of the Diagnostic Value of Cell-free DNA for Renal Cancer.

Cell-free DNA (cf-DNA) has been reported to represent a suitable material for liquid biopsy in the diagnosis and prognosis of various cancers. We performed a meta-analysis of published data to investigate the diagnostic value of cf-DNA for renal cancer (RCa). Systematic searches were conducted using Pubmed, Embase databases, Web of Science, Medline and Cochrane Library to identify relevant publications until the 31st March 2021. For all patients, we evaluated the true diagnostic value of cf-DNA by calculating the number of true positive, false positive, true negative, and false negative, diagnoses by extracting specificity and sensitivity data from the selected literature. In total, 8 studies, featuring 754 RCa patients, and 355 healthy controls, met our inclusion criteria. The overall diagnostic sensitivity and specificity for cf-DNA was 0.71 (95% confidence interval (CI), 0.55-0.83) and 0.79 (95% CI, 0.66-0.88), respectively. The pooled positive likelihood ratio and pooled negative likelihood ratio were 3.42 (95% CI, 2.04-5.72) and 0.36 (95% CI, 0.23-0.58), respectively. The area under the summary receiver operating characteristic curve was 0.82 (95% CI, 0.79-0.85), and the diagnostic odds ratio was 7.80 (95% CI, 4.40-13.85). Collectively, our data demonstrate that cf-DNA has high specificity and sensitivity for diagnosing RCa. Therefore, cf-DNA is a useful biomarker for the diagnosis of RCa.

Overexpression of Human Aspartyl (Asparaginyl) ß-hydroxylase in NSCLC: Its Diagnostic Value by Means of Exosomes of Bronchoalveolar Lavage.

The human aspartyl ß-hydroxylase (ASPH) is overexpressed in tumor tissues. Bronchoalveolar lavage (BAL) is a diagnostic procedure for infections and malignancies. The aim of this study was to investigate whether tumor exosomes carrying ASPH gene marker were present in bronchoalveolar fluid of patients with non-small cell lung cancer (NSCLC). A tissue microarray analysis was applied to explore the expression of ASPH in different histologic NSCLC. The human NSCLC cell lines and normal bronchial cell lines were used to study exosomal ASPH exprerssion. A total of 27 NSCLC, 21 benign tumor, and 15 healthy controls underwent BAL. Immunohistochemistry was performed to study the ASPH expression in malignant and normal lung tissues. The expression characteristics of ASPH in different NSCLC and normal bronchial cells and pneumocytes were confirmed by cell blocks. A reverse transcription-quantitative polymerase chain reaction was carried out to study the levels of exosomal ASPH expression. Immunohistochemical staining of tissue microarray demonstrated that overexpression of ASPH was found in NSCLC tissues including adenocarcinoma, large cell carcinoma, and squamous cell carcinoma, but absent in adjacent normal tissues. All NSCLC specimens exhibited high levels of ASPH immunoreactivity, while nonmalignant and normal lung tissues exhibited a very low level of expression. Overexpression of ASPH was found in exosomes from NSCLC cell lines but absent from the normal bronchial cell line NL-20. ASPH level from BAL exosomes was significantly increased in NSCLC patients compared with that from nonmalignant or health group. Our method of isolation of BAL exosomes was easily performed in the clinical laboratory. BAL exosomal ASPH can be a potential biomarker for NSCLC diagnosis.

Presence of Urinary Exosomes for Liquid Biopsy of Clear Cell Renal Cell Carcinoma: Protocol for a Pilot Feasibility Study.

BACKGROUND: Approximately 70%-80% of kidney cancers are clear cell renal cell carcinomas (CCRCCs). Patient management is based on imaging (abdominal ultrasound and computerized tomography), surgical excision of the tumor, and pathological analysis. A tissue biopsy is therefore necessary to confirm the diagnosis and avoid unnecessary nephrectomy. For metastatic cancers, a tissue biopsy is essential for establishing the targeted therapy. This biopsy of tumor material is invasive and painful. Other techniques such as liquid biopsy would help reduce the need for tissue biopsy. The development of a simple biological test for diagnosis is essential. CA9 is a powerful marker for the diagnosis of CCRCC. Exosomes have become a major source of liquid biopsy because they carry tumor proteins, RNA, and lipids. Urine is the most convenient biological liquid for exosome sampling. OBJECTIVE: The aim of this study (PEP-C study) is mainly to determine whether it is possible to detect urinary exosomal CA9 for the molecular diagnosis of CCRCC. METHODS: This study will include 60 patients with CCRCC and 40 noncancer patients. Exosomes will be isolated from urine samples and exosomal CA9 will be detected by transmission electron microscopy, flow cytometry, and reverse transcription-quantitative polymerase chain reaction. RESULTS: This study is currently underway with funding support from the CHU Saint-Etienne of France. CONCLUSIONS: We expect to demonstrate that urinary tumor exosomes could be a novel liquid biopsy to diagnose CCRCC and to guide clinicians in treatment decision-making. TRIAL REGISTRATION: ClinicalTrials.gov NCT04053855; https://clinicaltrials.gov/ct2/show/NCT04053855. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/24423.