Comparative Analysis of Cell-Free miR-205-5p, let-7f-5p, and miR-483-5p Expression in Ovarian Cell Cultures and Plasma Samples of Patients with Ovarian Cancer

The term liquid biopsy reveals a non-invasive diagnostic method that might be based on the quantification of cell-free microRNAs in body fluids. However, the identification of candidates for liquid biopsy is challenging. Our aim was to compare the cell-free expression of miR-483-5p, miR-205-5p, and let-7f-5p in ovarian cell cultures and plasma samples of patients with ovarian cancer. Both the intracellular and cell-free expression of miR-205-5p and let-7f-5p proved to be higher in the Estrogen Receptor α (ERα) expressing PEO1 cell-line than in the estrogen non-sensitive A2780. Moreover, the expression of let-7f-5p was up-regulated in response to estradiol exposure that was diminished after the addition of an ERα selective antagonist. MiR-483-5p had lower intracellular and cell-free expression in PEO1. All these miRNAs had detectable expression level in plasma samples, among which miR-205-5p proved to be overexpressed in the plasma samples of patients with ovarian tumors compared to healthy controls and possessed an acceptable diagnostic potential with ROC-AUC 0.683 (95% CI 0.57–0.795). Functional annotation clustering of the target genes of miR-205-5p revealed several clusters involved in cancer development. We suggest that miR-205-5p might be a promising biomarker candidate in ovarian cancer that should be further analyzed in larger sample size.

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Potential clinical applications of circulating cell-free DNA in ovarian cancer patients

Expert Reviews in Molecular Medicine ◽

10.1017/erm.2018.5 ◽

2018 ◽

Vol 20 ◽

Cited By ~ 4

Author(s):

Ana Barbosa ◽

Ana Peixoto ◽

Pedro Pinto ◽

Manuela Pinheiro ◽

Manuel R. Teixeira

Keyword(s):

Ovarian Cancer ◽

Cancer Patients ◽

Liquid Biopsy ◽

Clinical Applications ◽

Epigenetic Alterations ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna ◽

Potential Use ◽

Circulating Cell Free Dna

AbstractCirculating cell-free DNA (cfDNA) consists of small fragments of DNA that circulate freely in the bloodstream. In cancer patients, a fraction of cfDNA is derived from tumour cells, therefore containing the same genetic and epigenetic alterations, and is termed circulating cell-free tumour DNA. The potential use of cfDNA, the so-called ‘liquid biopsy’, as a non-invasive cancer biomarker has recently received a lot of attention. The present review will focus on studies concerning the potential clinical applications of cfDNA in ovarian cancer patients.

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Circulating miRNA Profiling in Plasma Samples of Ovarian Cancer Patients

International Journal of Molecular Sciences ◽

10.3390/ijms20184533 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4533 ◽

Cited By ~ 8

Author(s):

András Penyige ◽

Éva Márton ◽

Beáta Soltész ◽

Melinda Szilágyi-Bónizs ◽

Róbert Póka ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Patients ◽

Target Genes ◽

Enrichment Analysis ◽

Differentially Expressed ◽

Cancer Development ◽

Plasma Samples ◽

Circulating Mirna ◽

Differentially Expressed Mirnas ◽

Potential Biomarkers

Ovarian cancer is one of the most common cancer types in women characterized by a high mortality rate due to lack of early diagnosis. Circulating miRNAs besides being important regulators of cancer development could be potential biomarkers to aid diagnosis. We performed the circulating miRNA expression analysis in plasma samples obtained from ovarian cancer patients stratified into FIGO I, FIGO III, and FIGO IV stages and from healthy females using the NanoString quantitative assay. Forty-five miRNAs were differentially expressed, out of these 17 miRNAs showed significantly different expression between controls and patients, 28 were expressed only in patients, among them 19 were expressed only in FIGO I patients. Differentially expressed miRNAs were ranked by the network-based analysis to assess their importance. Target genes of the differentially expressed miRNAs were identified then functional annotation of the target genes by the GO and KEGG-based enrichment analysis was carried out. A general and an ovary-specific protein–protein interaction network was constructed from target genes. Results of our network and the functional enrichment analysis suggest that besides HSP90AA1, MYC, SP1, BRCA1, RB1, CFTR, STAT3, E2F1, ERBB2, EZH2, and MET genes, additional genes which are enriched in cell cycle regulation, FOXO, TP53, PI-3AKT, AMPK, TGFβ, ERBB signaling pathways and in the regulation of gene expression, proliferation, cellular response to hypoxia, and negative regulation of the apoptotic process, the GO terms have central importance in ovarian cancer development. The aberrantly expressed miRNAs might be considered as potential biomarkers for the diagnosis of ovarian cancer after validation of these results in a larger cohort of ovarian cancer patients.

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Liquid biopsy for patients with IBD-associated neoplasia

BMC Cancer ◽

10.1186/s12885-020-07699-z ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Hideaki Kinugasa ◽

Sakiko Hiraoka ◽

Kazuhiro Nouso ◽

Shumpei Yamamoto ◽

Mami Hirai ◽

...

Keyword(s):

Liquid Biopsy ◽

Target Genes ◽

Tumor Tissue ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Tissue Samples ◽

Non Invasive ◽

Tumor Tissues ◽

Inflammatory Bowel

Abstract Background It is often difficult to diagnose inflammatory bowel disease (IBD)-associated neoplasia endoscopically due to background inflammation. In addition, due to the absence of sensitive tumor biomarkers, countermeasures against IBD-associated neoplasia are crucial. The purpose of this study is to develop a new diagnostic method through the application of liquid biopsy. Methods Ten patients with IBD-associated cancers and high-grade dysplasia (HGD) with preserved tumor tissue and blood were included. Tumor and non-tumor tissues were analyzed for 48 cancer-related genes using next-generation sequencing. Simultaneously, circulating tumor DNA (ctDNA) was analyzed for mutations in the target genes using digital PCR. Results Out of 10 patients, seven had IBD-related cancer and three had IBD-related HGD. Two patients had carcinoma in situ; moreover, three had stageII and two had stage III. To avoid false positives, the mutation rate cutoff was set at 5% based on the control results; seven of 10 (70%) tumor tissue samples were mutation-positive. Mutation frequencies for each gene were as follows: TP53 (20.9%; R136H), TP53 (25.0%; C110W), TP53 (8.5%; H140Q), TP53 (31.1%; R150W), TP53 (12.8%; R141H), KRAS (40.0%; G12V), and PIK3CA (34.1%; R 88Q). The same mutations were detected in the blood of these seven patients. However, no mutations were detected in the blood of the remaining three patients with no tumor tissue mutations. The concordance rate between tumor tissue DNA and blood ctDNA was 100%. Conclusion Blood liquid biopsy has the potential to be a new method for non-invasive diagnosis of IBD-associated neoplasia.

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Diagnostic potential for a serum miRNA neural network for detection of ovarian cancer

eLife ◽

10.7554/elife.28932 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 38

Author(s):

Kevin M Elias ◽

Wojciech Fendler ◽

Konrad Stawiski ◽

Stephen J Fiascone ◽

Allison F Vitonis ◽

...

Keyword(s):

Neural Network ◽

Ovarian Cancer ◽

Predictive Value ◽

Clinical Samples ◽

Small Rna Sequencing ◽

Serum Samples ◽

Non Invasive ◽

Metastatic Lesions ◽

Diagnostic Potential ◽

The Neural Network

Recent studies posit a role for non-coding RNAs in epithelial ovarian cancer (EOC). Combining small RNA sequencing from 179 human serum samples with a neural network analysis produced a miRNA algorithm for diagnosis of EOC (AUC 0.90; 95% CI: 0.81–0.99). The model significantly outperformed CA125 and functioned well regardless of patient age, histology, or stage. Among 454 patients with various diagnoses, the miRNA neural network had 100% specificity for ovarian cancer. After using 325 samples to adapt the neural network to qPCR measurements, the model was validated using 51 independent clinical samples, with a positive predictive value of 91.3% (95% CI: 73.3–97.6%) and negative predictive value of 78.6% (95% CI: 64.2–88.2%). Finally, biologic relevance was tested using in situ hybridization on 30 pre-metastatic lesions, showing intratumoral concentration of relevant miRNAs. These data suggest circulating miRNAs have potential to develop a non-invasive diagnostic test for ovarian cancer.

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Disease Monitoring in Multiple Myeloma Patients Using Liquid Biopsy and Next Generation Sequencing of IGH Gene Rearrangements

Blood ◽

10.1182/blood.v128.22.4430.4430 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4430-4430

Author(s):

Silvia Gimondi ◽

Giulia Biancon ◽

Antonio Vendramin ◽

Silvia Zaninelli ◽

Sara Rizzitano ◽

...

Keyword(s):

Liquid Biopsy ◽

Tumor Burden ◽

Disease Monitoring ◽

Gene Rearrangements ◽

Plasma Samples ◽

Time Points ◽

Non Invasive ◽

Generation Sequencing ◽

Over Time

Abstract Background: Current criteria used to assess response lag behind the extraordinary evolution in the treatment of multiple myeloma (MM) patients (pts), and more sensitive techniques are being explored to detect true minimal residual disease (MRD) for new complete remission (CR) definitions. In recent years, next generation sequencing (NGS) technologies have emerged. NGS of immunoglobulin (IgH) gene rearrangements are very sensitive and also allow the identification of small subclonal population that can be monitored over time during treatment, something not possible with flow cytometry or PCR. However, the patchy pattern of bone marrow infiltration observed in MM leads to some degree of uncertainty regarding MRD-negative results, irrespectively of the technique adopted. This highlights the value of applying "liquid biopsy" as a non-invasive strategy for monitoring MRD through the analysis of circulating cell-free tumor DNA (ctDNA). The objective of the current study was to measure residual tumor burden in sequential plasma samples of a cohort of MM pts by NGS of the IgH gene rearrangements. Methods: We retrospectively analyzed 14 MM pts homogeneously treated between 2011 and 2015 with all clinical data available. We obtained serial tumor and plasma samples at diagnosis and at specified time points during treatment cycles and up to 24 months of follow-up. Genomic DNA (gDNA) was extracted from immunomagnetically selected CD138+ plasma cells at diagnosis (n=14). ctDNA was extracted from 500uL of plasma (Qiagen) at diagnosis (n=14) and at follow-up time points (n=58). IgH gene rearrangements were amplified, quality assessed (Agilent hsDNA kit) and sequenced on Ion Torrent PGM as previously described (Gimondi et al., ASH 2015). Raw reads were filtered for quality, length (>250bp) and presence of both forward and reverse primers. Reads were subsequently aligned using IgBlast against IMGT germline database and aggregated into clonotypes based on identity of CDR3, V and J segments (MigMap). Post-processing analyses were performed using VDJtools and customized R scripts. Results: PCR products quality assessment from ctDNA amplification of the entire IgH-VDJ region revealed the presence of both short (150-250bp) and long amplicons (310-360bp). Raw reads were subjected to filtering using our custom bioinformatic workflow to retain only complete IgH-VDJ gene rearrangements and discard low-quality reads. Three pts could not be evaluated due to low quality sequencing reads in all samples. At least 3 follow-up time points were available for all the remaining 11 pts whereas 6 pts had 4 time points. At diagnosis, both plasma and tumor samples revealed a high level of heterogeneity (range 1980-7753 clonotypes) with only a small fraction of shared clonotypes (346±262, mean±SD). Among the shared ones, the clonotype with the highest frequency in plasma corresponded to the tumor-associated one identified in CD138+ cells. Interestingly, in the plasma of 3 pts, additional clonotypes were detected at relatively high frequencies (range 1-16%) suggesting the presence of subclones. IgH-NGS at follow-up time points revealed that the clonotype identified at diagnosis (range 4-31% of total reads) could be easily tracked over time in plasma samples, at frequencies as low as 0.00001%. Frequencies of the tumor-associated IgH gene rearrangement in plasma showed a patient-specific modulation and reflected the tumor burden assessed according to the International Myeloma Working Group-Uniform Response Criteria. At the time of CR, the tumor-associated clonotype was undetectable in the plasma of pts who would not subsequently relapse. In patients that would lately experience progressive disease, the tumor specific clonotype was still detectable at low frequencies (range 0.00001-0.03%) in all plasma samples suggesting that liquid biopsy can be used for MRD monitoring. Conclusions: Despite the limited number of pts and follow-up samples analyzed, we demonstrate that NGS of IgH gene rearrangements from ctDNA can be used for MM disease monitoring, thus representing a non-invasive alternative strategy for clinical management. The analysis of retrospectively collected plasma samples revealed that ctDNA quality is essential for a NGS characterization of IgH gene rearrangements. Plasma samples collection and processing represent critical steps that need to be considered designing prospective liquid biopsy studies. Disclosures No relevant conflicts of interest to declare.

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Estrogen receptor-α mediates gene expression changes and growth response in ovarian cancer cells exposed to estrogen

Endocrine Related Cancer ◽

10.1677/erc.1.01039 ◽

2005 ◽

Vol 12 (4) ◽

pp. 851-866 ◽

Cited By ~ 94

Author(s):

Amanda J M O’Donnell ◽

Kenneth G Macleod ◽

David J Burns ◽

John F Smyth ◽

Simon P Langdon

Keyword(s):

Gene Expression ◽

Ovarian Cancer ◽

Estrogen Receptor ◽

Ovarian Carcinoma ◽

Significant Role ◽

Target Genes ◽

De Novo ◽

Estrogen Receptor Α ◽

Ovarian Cancer Cells ◽

Real Time Quantitative Pcr

Estrogens play a significant role in the development, growth, invasion and metastasis of ovarian tumors. The transcriptional program regulated by 17β-estradiol (E2) in human ovarian cancer cell lines was analyzed using cDNA microarrays containing 1200 cancer-related genes. Twenty-eight transcripts had at least a threefold change in expression in E2-treated PEO1 ovarian carcinoma cells compared with controls. These differences were confirmed by real-time quantitative PCR and shown to be dependent upon the expression of functional estrogen receptor-α (ERα). Consistent with this, these gene expression changes were blocked by the anti-estrogen tamoxifen. The use of ERα- and ERβ-specific ligands allowed molecular dissection of the E2 response and showed that ERα activation was responsible for the observed changes in gene expression, whereas ERβ played no significant role. Inhibition of de novo protein synthesis by cycloheximide was used to distinguish between primary and secondary target genes regulated by E2. Actinomycin D was used to show that changes in gene expression levels induced by E2 were a result of changes in transcription and not due to changes in mRNA stability. The results presented here demonstrate that estrogen-driven growth of epithelial ovarian carcinoma is mediated by activation of ERα-mediated, and not ERβ-mediated, transcription.

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MiR-93/miR-375: Diagnostic Potential, Aggressiveness Correlation and Common Target Genes in Prostate Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms21165667 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5667

Author(s):

Ewa Ciszkowicz ◽

Paweł Porzycki ◽

Małgorzata Semik ◽

Ewa Kaznowska ◽

Mirosław Tyrka

Keyword(s):

Prostate Cancer ◽

Operating Characteristic ◽

Target Genes ◽

Diagnostic Value ◽

Tissue Samples ◽

Non Invasive ◽

Diagnostic Potential ◽

Diagnostic Applications ◽

First Time ◽

Common Target

Dysregulation of miRNAs has a fundamental role in the initiation, development and progression of prostate cancer (PCa). The potential of miRNA in gene therapy and diagnostic applications is well documented. To further improve miRNAs’ ability to distinguish between PCa and benign prostatic hyperplasia (BPH) patients, nine miRNA (-21, -27b, -93, -141, -205, -221, -182, -375 and let-7a) with the highest reported differentiation power were chosen and for the first time used in comparative studies of serum and prostate tissue samples. Spearman correlations and response operating characteristic (ROC) analyses were applied to assess the capability of the miRNAs present in serum to discriminate between PCa and BPH patients. The present study clearly demonstrates that miR-93 and miR-375 could be taken into consideration as single blood-based non-invasive molecules to distinguish PCa from BPH patients. We indicate that these two miRNAs have six common, PCa-related, target genes (CCND2, MAP3K2, MXI1, PAFAH1B1, YOD1, ZFYVE26) that share the molecular function of protein binding (GO:0005515 term). A high diagnostic value of the new serum derived miR-182 (AUC = 0.881, 95% confidence interval, CI = 0.816–0.946, p < 0.0001, sensitivity and specificity were 85% and 79%, respectively) is also described.

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Aberrant Methylation Status of Tumour Suppressor Genes in Ovarian Cancer Tissue and Paired Plasma Samples

International Journal of Molecular Sciences ◽

10.3390/ijms20174119 ◽

2019 ◽

Vol 20 (17) ◽

pp. 4119 ◽

Cited By ~ 2

Author(s):

Dana Dvorská ◽

Dušan Braný ◽

Bálint Nagy ◽

Marián Grendár ◽

Robert Poka ◽

...

Keyword(s):

Ovarian Cancer ◽

Tumour Suppressor ◽

Tumour Suppressor Genes ◽

Aberrant Methylation ◽

Potential Candidate ◽

Plasma Samples ◽

Cdh1 Gene ◽

Suppressor Genes ◽

Non Invasive ◽

Malignant Lesions

Ovarian cancer is a highly heterogeneous disease and its formation is affected by many epidemiological factors. It has typical lack of early signs and symptoms, and almost 70% of ovarian cancers are diagnosed in advanced stages. Robust, early and non-invasive ovarian cancer diagnosis will certainly be beneficial. Herein we analysed the regulatory sequence methylation profiles of the RASSF1, PTEN, CDH1 and PAX1 tumour suppressor genes by pyrosequencing in healthy, benign and malignant ovarian tissues, and corresponding plasma samples. We recorded statistically significant higher methylation levels (p < 0.05) in the CDH1 and PAX1 genes in malignant tissues than in controls (39.06 ± 18.78 versus 24.22 ± 6.93; 13.55 ± 10.65 versus 5.73 ± 2.19). Higher values in the CDH1 gene were also found in plasma samples (22.25 ± 14.13 versus 46.42 ± 20.91). A similar methylation pattern with positive correlation between plasma and benign lesions was noted in the CDH1 gene (r = 0.886, p = 0.019) and malignant lesions in the PAX1 gene (r = 0.771, p < 0.001). The random forest algorithm combining methylation indices of all four genes and age determined 0.932 AUC (area under the receiver operating characteristic (ROC) curve) prediction power in the model classifying malignant lesions and controls. Our study results indicate the effects of methylation changes in ovarian cancer development and suggest that the CDH1 gene is a potential candidate for non-invasive diagnosis of ovarian cancer.

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