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1.
Cancers (Basel) ; 16(4)2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38398121

RESUMO

Immune checkpoint blockade (ICB) benefits only a subset of advanced cancer patients, and predictive biomarkers for immunotherapy response are needed. Recently, copy number alteration (CNA) burden has been proposed to predict ICB resistance. We assessed this finding using the publicly accessible data for 1661 ICB-treated patients whose tumors were profiled by MSK-IMPACT, an approved targeted assay in clinical care. We tested the hypothesis that the continuous increase in CNA burden is associated with poor overall survival following ICB. In addition, we hypothesized that the combinatorial biomarkers of tumor mutational burden (TMB) and CNA burden would better stratify patients for immune status and ICB response. Of the 1661 cases, 79% (n = 1307) were treated with anti PD-1/PD-L1 and the remaining 21% (n = 354) with anti CTLA-4 or the combination of both. In a multivariate analysis, increase in CNA burden was associated with poor overall survival [HR = 1.52, 95% CI (1.01-2.30), p = 0.04]. The combination of biomarkers TMB and CNA burden stratified patients into four clinically distinct subsets among which "LowTMB/HighCNA" showed the worst survival (p < 0.0001). The four patient subsets had unique CNA profiles and enriched pathways, which could predict transcriptional and phenotypic effects related to immune signaling and CD8+ T-cell abundance in the tumor microenvironment. CNA burden was associated with poor overall survival in patients receiving ICB and could improve patient stratification when incorporated with TMB. These findings may guide patient selection for immunotherapy or alternative strategies.

2.
Biomark Res ; 11(1): 99, 2023 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-37978566

RESUMO

While the field of precision oncology is rapidly expanding and more targeted options are revolutionizing cancer treatment paradigms, therapeutic resistance particularly to immunotherapy remains a pressing challenge. This can be largely attributed to the dynamic tumor-stroma interactions that continuously alter the microenvironment. While to date most advancements have been made through examining the clinical utility of tissue-based biomarkers, their invasive nature and lack of a holistic representation of the evolving disease in a real-time manner could result in suboptimal treatment decisions. Thus, using minimally-invasive approaches to identify biomarkers that predict and monitor treatment response as well as alert to the emergence of recurrences is of a critical need. Currently, research efforts are shifting towards developing liquid biopsy-based biomarkers obtained from patients over the course of disease. Liquid biopsy represents a unique opportunity to monitor intercellular communication within the tumor microenvironment which could occur through the exchange of extracellular vesicles (EVs). EVs are lipid bilayer membrane nanoscale vesicles which transfer a plethora of biomolecules that mediate intercellular crosstalk, shape the tumor microenvironment, and modify drug response. The capture of EVs using innovative approaches, such as microfluidics, magnetic beads, and aptamers, allow their analysis via high throughput multi-omics techniques and facilitate their use for biomarker discovery. Artificial intelligence, using machine and deep learning algorithms, is advancing multi-omics analyses to uncover candidate biomarkers and predictive signatures that are key for translation into clinical trials. With the increasing recognition of the role of EVs in mediating immune evasion and as a valuable biomarker source, these real-time snapshots of cellular communication are promising to become an important tool in the field of precision oncology and spur the recognition of strategies to block resistance to immunotherapy. In this review, we discuss the emerging role of EVs in biomarker research describing current advances in their isolation and analysis techniques as well as their function as mediators in the tumor microenvironment. We also highlight recent lung cancer and melanoma studies that point towards their application as predictive biomarkers for immunotherapy and their potential clinical use in precision immuno-oncology.

3.
Sci Rep ; 13(1): 9251, 2023 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-37286718

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) has a high fatality rate, mainly due to its asymptomatic nature until late-stage disease and therefore delayed diagnosis that leads to a lack of timely treatment intervention. Consequently, there is a significant need for better methods to screen populations that are at high risk of developing PDAC. Such advances would result in earlier diagnosis, more treatment options, and ultimately better outcomes for patients. Several recent studies have applied the concept of liquid biopsy, which is the sampling of a biofluid (such as blood plasma) for the presence of disease biomarkers, to develop screening approaches for PDAC; several of these studies have focused on analysis of extracellular vesicles (EVs) and their cargoes. While these studies have identified many potential biomarkers for PDAC that are present within EVs, their application to clinical practice is hindered by the lack of a robust, reproducible method for EV isolation and analysis that is amenable to a clinical setting. Our previous research has shown that the Vn96 synthetic peptide is indeed a robust and reproducible method for EV isolation that has the potential to be used in a clinical setting. We have therefore chosen to investigate the utility of the Vn96 synthetic peptide for this isolation of EVs from human plasma and the subsequent detection of small RNA biomarkers of PDAC by Next-generation sequencing (NGS) analysis. We find that analysis of small RNA from Vn96-isolated EVs permits the discrimination of PDAC patients from non-affected individuals. Moreover, analyses of all small RNA species, miRNAs, and lncRNA fragments are most effective at segregating PDAC patients from non-affected individuals. Several of the identified small RNA biomarkers have been previously associated with and/or characterized in PDAC, indicating the validity of our findings, whereas other identified small RNA biomarkers may have novel roles in PDAC or cancer in general. Overall, our results provide a basis for a clinically-amendable detection and/or screening strategy for PDAC using a liquid biopsy approach that relies on Vn96-mediated isolation of EVs from plasma.


Assuntos
Carcinoma Ductal Pancreático , Vesículas Extracelulares , MicroRNAs , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Vesículas Extracelulares/genética , Vesículas Extracelulares/patologia , MicroRNAs/genética , Peptídeos/genética , Análise de Sequência de RNA , Biomarcadores Tumorais/genética , Neoplasias Pancreáticas
4.
Front Bioinform ; 3: 1127661, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37252342

RESUMO

RNA sequencing analysis is an important field in the study of extracellular vesicles (EVs), as these particles contain a variety of RNA species that may have diagnostic, prognostic and predictive value. Many of the bioinformatics tools currently used to analyze EV cargo rely on third-party annotations. Recently, analysis of unannotated expressed RNAs has become of interest, since these may provide complementary information to traditional annotated biomarkers or may help refine biological signatures used in machine learning by including unknown regions. Here we perform a comparative analysis of annotation-free and classical read-summarization tools for the analysis of RNA sequencing data generated for EVs isolated from persons with amyotrophic lateral sclerosis (ALS) and healthy donors. Differential expression analysis and digital-droplet PCR validation of unannotated RNAs also confirmed their existence and demonstrates the usefulness of including such potential biomarkers in transcriptome analysis. We show that find-then-annotate methods perform similarly to standard tools for the analysis of known features, and can also identify unannotated expressed RNAs, two of which were validated as overexpressed in ALS samples. We demonstrate that these tools can therefore be used for a stand-alone analysis or easily integrated into current workflows and may be useful for re-analysis as annotations can be integrated post hoc.

5.
Methods Mol Biol ; 2508: 341-352, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35737249

RESUMO

Extracellular vesicles (EVs) are membrane-bound nanoparticles that carry DNA, RNA, and protein cargoes and are found in a variety of biofluids. EVs, along with cell-free DNA (cfDNA), have attracted interest as a source of biomarker material for liquid biopsy, a process in which a sample of body fluid is used for the detection or monitoring of disease. The Vn96 synthetic peptide facilitates the isolation of both EVs and cfDNA from multiple body fluids, including human plasma, placing it as a versatile tool for the capture of multiple biomarker materials for disease detection and/or treatment monitoring. In this chapter, we describe an optimized protocol for Vn96-mediated isolation of EVs and cfDNA from human plasma samples, as well as downstream methods for EV enumeration and DNA, RNA, and protein extraction from the material captured by Vn96 for use in biomarker discovery or detection.


Assuntos
Ácidos Nucleicos Livres , Vesículas Extracelulares , Biomarcadores/metabolismo , Ácidos Nucleicos Livres/metabolismo , DNA/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Peptídeos/metabolismo , Proteínas/metabolismo , RNA/metabolismo
7.
J Extracell Vesicles ; 10(11): e12138, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34478244

RESUMO

Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy-based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently-available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non-toxic, and/or clinically-amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non-immunogenicity, biodegradability, and low toxicity. Chitosan-precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic-based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture-conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method.


Assuntos
Quitosana/metabolismo , Vesículas Extracelulares/metabolismo , Biópsia Líquida/métodos , Proteômica/métodos , Linhagem Celular Tumoral , Humanos
8.
Biosens Bioelectron ; 194: 113585, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34517262

RESUMO

Extracellular vesicles (EVs) are the cell-derived vesicles which play a critical role in cell-to-cell communication, and disease progression. These vesicles contain a myriad of substances like RNA, DNA, proteins, and lipids from their origin cells, offering a good source of biomarkers. The existing methods for the isolation of EVs are time-consuming, lack yield and purity, and expensive. In this work, we present a magnetic particle based liquid biopsy chip for the isolation of EVs by using a synthetic peptide, Vn96. To ensure capture efficiency, a 3D mixer is integrated in the chip, along with a sedimentation unit, which allows EV-captured magnetic particles to settle in it based on gravity assisted sedimentation. The captured EVs are then isolated for their elution and validation. The EVs are characterized by the scanning electron microscopy (SEM) measurements and the ability of capture and isolation of EVs is validated by the nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM). The DNA content of the EVs is further characterized by the absolute quantification of a housekeeping gene (RNase P) copies using droplet digital PCR (ddPCR). The results show that the chip can capture and isolate the EVs, without affecting their morphology. Thus, the liquid biopsy chip can be considered as a potential point of care device for diagnostics in a clinical setting.


Assuntos
Técnicas Biossensoriais , Vesículas Extracelulares , Amplificação de Genes , Biópsia Líquida , Fenômenos Magnéticos
9.
Sci Rep ; 11(1): 8085, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850235

RESUMO

Extracellular vesicles (EVs) have been recognized as a rich material for the analysis of DNA, RNA, and protein biomarkers. A remaining challenge for the deployment of EV-based diagnostic and prognostic assays in liquid biopsy testing is the development of an EV isolation method that is amenable to a clinical diagnostic lab setting and is compatible with multiple types of biomarker analyses. We have previously designed a synthetic peptide, known as Vn96 (ME kit), which efficiently isolates EVs from multiple biofluids in a short timeframe without the use of specialized lab equipment. Moreover, it has recently been shown that Vn96 also facilitates the co-isolation of cell-free DNA (cfDNA) along with EVs. Herein we describe an optimized method for Vn96 affinity-based EV and cfDNA isolation from plasma samples and have developed a multiparametric extraction protocol for the sequential isolation of DNA, RNA, and protein from the same plasma EV and cfDNA sample. We are able to isolate sufficient material by the multiparametric extraction protocol for use in downstream analyses, including ddPCR (DNA) and 'omic profiling by both small RNA sequencing (RNA) and mass spectrometry (protein), from a minimum volume (4 mL) of plasma. This multiparametric extraction protocol should improve the ability to analyse multiple biomarker materials (DNA, RNA and protein) from the same limited starting material, which may improve the sensitivity and specificity of liquid biopsy tests that exploit EV-based and cfDNA biomarkers for disease detection and monitoring.


Assuntos
Ácidos Nucleicos Livres , Vesículas Extracelulares , Biomarcadores Tumorais , Humanos , Biópsia Líquida , RNA
10.
Neurosci Lett ; 750: 135813, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33705931

RESUMO

DNA methylation has garnered much attention in recent years for its diagnostic potential in multiple conditions including cancer and neurodegenerative diseases. Conversely, advances regarding the potential diagnostic relevance of DNA methylation status have been sparse in the field of amyotrophic lateral sclerosis (ALS) even though patients diagnosed with this condition would significantly benefit from improved molecular assays aimed at furthering the current diagnostic and therapeutic options available. This review will provide an overview of the current diagnostic approaches available for ALS diagnosis and discuss the potential clinical usefulness of DNA methylation. We will also present examples of DNA methylation as a diagnostic tool in various types of cancer and neurodegenerative conditions and expand on how circulating cfDNA methylation may be leveraged for the early detection of ALS. In general, this article will reinforce the importance of cfDNA methylation as diagnostic tools and will further highlight its clinical relevance for persons diagnosed with ALS.


Assuntos
Esclerose Lateral Amiotrófica/sangue , Ácidos Nucleicos Livres/sangue , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/genética , Animais , Biomarcadores/sangue , Ácidos Nucleicos Livres/genética , Metilação de DNA , Humanos
11.
Anal Biochem ; 619: 114137, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33582115

RESUMO

Several common reagents for the alkylation of cysteine residues of model intact proteins were evaluated for reaction speed, yield of alkylated product and degree of over-alkylation using an online LC-MS platform. The efficiency of the alkylation reaction is found to be dependent on the (1) reagent, (2) peptide/protein, (3) reagent concentration and (4) reaction time. At high reagent concentrations, iodoacetic acid was found to produce significant levels of over-alkylation products wherein methionine residues become modified. For optimal performance of the alkylation reaction, we found the use of a cocktail of chloroacetamide, bromoacetamide and iodoacetamide worked best. The alkylating efficiency of each haloacetamide is a balance between the characteristics of the halogen leaving group and the steric hindrance of the alkylation site on the peptide or protein. A key aspect of using a cocktail of haloacetamides is that they all produce the same modification (+57.0209 Da) to the cysteine residues of the protein while the alkylation efficiency of each site may differ for each of the three reagents. Over-alkylation effects appear to be lower with the cocktail due to a lower concentration of each reagent. The haloacetamide cocktail could be useful when considering complex mixtures of proteins.


Assuntos
Acetamidas/química , Cisteína/química , Iodoacetamida/química , Proteínas/química , Alquilação , Cromatografia Líquida , Espectrometria de Massas em Tandem
12.
Sci Rep ; 11(1): 1537, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452395

RESUMO

PAX5 and EBF1 work synergistically to regulate genes that are involved in B lymphocyte differentiation. We used the KIS-1 diffuse large B cell lymphoma cell line, which is reported to have elevated levels of PAX5 expression, to investigate the mechanism of EBF1- and PAX5-regulated gene expression. We demonstrate the lack of expression of hallmark B cell genes, including CD19, CD79b, and EBF1, in the KIS-1 cell line. Upon restoration of EBF1 expression we observed activation of CD19, CD79b and other genes with critical roles in B cell differentiation. Mass spectrometry analyses of proteins co-immunoprecipitated with PAX5 in KIS-1 identified components of the MLL H3K4 methylation complex, which drives histone modifications associated with transcription activation. Immunoblotting showed a stronger association of this complex with PAX5 in the presence of EBF1. Silencing of KMT2A, the catalytic component of MLL, repressed the ability of exogenous EBF1 to activate transcription of both CD19 and CD79b in KIS-1 cells. We also find association of PAX5 with the MLL complex and decreased CD19 expression following silencing of KMT2A in other human B cell lines. These data support an important role for the MLL complex in PAX5-mediated transcription regulation.


Assuntos
Linfoma de Células B/genética , Fator de Transcrição PAX5/metabolismo , Transativadores/metabolismo , Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Humanos , Ativação Linfocitária , Linfoma de Células B/metabolismo , Metiltransferases/metabolismo , Fator de Transcrição PAX5/genética , Transativadores/genética
13.
Am J Med Genet A ; 185(4): 1211-1215, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33421311

RESUMO

NRG1 is a gene that encodes for a protein that binds to a receptor of the tyrosine kinase family which is essential for the survival of the central nervous system development during embryogenesis. Mutation of the NRG1 gene causes aganglionosis, which leads to Hirschsprung disease. Two brothers of Acadian descent presented with a history of Hirschsprung disease, in association with other anomalies including congenital heart disease, learning difficulties, developmental issues, and hypopigmented hair patch. Molecular analysis in both siblings revealed a heterozygous pathogenic mutation in the NGR1 gene (c.235C>T [p.Arg79*]), that was inherited from an unaffected father. This family expands our knowledge about the phenotypic spectrum associated with pathogenic mutation in the NRG1 gene with intrafamilial variability and the likely reduced penetrance for the phenotypic expression.


Assuntos
Predisposição Genética para Doença , Doença de Hirschsprung/genética , Neuregulina-1/genética , Adolescente , Povo Asiático , Heterozigoto , Doença de Hirschsprung/patologia , Humanos , Masculino , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética
14.
Anal Sci Adv ; 2(5-6): 299-307, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38716156

RESUMO

Peptide and protein quantitation by liquid chromatography-mass spectrometry relies on the assumption of linear signal response with concentration. At low concentrations, analyte adsorption to pipette tips, sample vials and equipment can have significant deleterious effects on signal response. Meanwhile at high concentrations, linearity breaks down due to competitive ionization, signal suppression, and the formation of peptide or protein multimers. These effects result in calibration curves that are more sigmoidal than linear. Linearity at low protein levels for identification and quantitation is of paramount importance in the discovery and validation of biomarker molecules. Herein, we demonstrate the benefits of using commercial low-bind microcentrifuge tubes and LC vials on the response of a 27-mer peptide, Vn96, and the intact proteins apomyoglobin and carbonic anhydrase. Linear curves were acquired for Vn96 while apomyoglobin required the addition of intact carbonic anhydrase as an adsorption competitor to achieve linearity. A linear calibration curve for carbonic anhydrase was also acquired by using the polypeptide ubiquitin as an adsorption competitor and internal standard. Linear response was recorded for approximately two orders of magnitude for apomyoglobin and carbonic anhydrase and three orders of magnitude for Vn96 with detection limits ranging from 0.33 to 19 fmol/µL. Finally, we used low-bind vials for the online enzymatic digestion of apomyoglobin where a high concentration of apomyoglobin acted as an adsorption blocker for the low level trypsin enzyme. Fortunately, the liberated tryptic peptides showed no affinity for the walls of the low-bind vials. In this study, we take a comprehensive approach to combat analyte adsorption by showing the significance of utilizing low-bind vials and adsorption competitors to greatly improve upon signal sensitivity at low concentrations of target molecules. The use of these methodologies should improve the low-level detection of molecules by mass spectrometry.

15.
Int J Mol Sci ; 21(23)2020 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-33260345

RESUMO

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS.


Assuntos
Ácidos Nucleicos Livres/genética , Vesículas Extracelulares/química , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Mutação/genética , Peptídeos/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/genética , Feminino , Humanos , Biópsia Líquida , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética
16.
Int J Mol Sci ; 21(21)2020 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-33172003

RESUMO

Serum prostate-specific antigen (sPSA) testing has helped to increase early detection of and decrease mortality from prostate cancer. However, since sPSA lacks specificity, an invasive prostate tissue biopsy is required to confirm cancer diagnosis. Using urinary extracellular vesicles (EVs) as a minimally invasive biomarker source, our goal was to develop a biomarker panel able to distinguish prostate cancer from benign conditions with high accuracy. We enrolled 56 patients in our study, 28 negative and 28 positive for cancer based on tissue biopsy results. Using our Vn96 peptide affinity method, we isolated EVs from post-digital rectal exam urines and used quantitative polymerase chain reaction to measure several mRNA and miRNA targets. We identified a panel of seven mRNA biomarkers whose expression ratio discriminated non-cancer from cancer with an area under the curve (AUC) of 0.825, sensitivity of 75% and specificity of 84%. We also identified two miRNAs whose combined score yielded an AUC of 0.744. A model pairing the seven mRNA and two miRNA panels yielded an AUC of 0.843, sensitivity of 79% and specificity of 89%. Addition of EV-derived PCA3 levels and clinical characteristics to the biomarker model further improved test accuracy. An AUC of 0.955, sensitivity of 86% and specificity of 93% were obtained. Hence, Vn96-isolated urinary EVs are a clinically applicable and minimally invasive source of mRNA and miRNA biomarkers with potential to improve on the accuracy of prostate cancer screening and diagnosis.


Assuntos
Vesículas Extracelulares/genética , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , Idoso , Área Sob a Curva , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/urina , Detecção Precoce de Câncer , Vesículas Extracelulares/metabolismo , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Projetos Piloto , Próstata/patologia , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/urina , RNA Mensageiro/genética , Sensibilidade e Especificidade
17.
Cells ; 9(8)2020 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-32784507

RESUMO

Accurate risk classification of men with localized high-risk prostate cancer directly affects treatment management decisions and patient outcomes. A wide range of risk assessments and classifications are available. However, each one has significant limitations to distinguish between indolent and aggressive prostate cancers. Circulating tumor cells (CTCs) may provide an alternate additional source, beyond tissue biopsies, to enable individual patient-specific clinical assessment, simply because CTCs can reveal both tumor-derived and germline-specific genetic information more precisely than that gained from a single diagnostic biopsy. In this study, we combined a filtration-based CTC isolation technology with prostate cancer CTC immunophenotyping to identify prostate cancer CTCs. Next, we performed 3-D telomere profiling prior to laser microdissection and single-cell whole-exome sequencing (WES) of 21 CTCs and 4 lymphocytes derived from 10 localized high-risk prostate cancer patient samples. Localized high-risk prostate cancer patient CTCs present a high number of telomere signals with lower signal intensities (short telomeres). To capture the genetic diversity/heterogeneity of high-risk prostate cancer CTCs, we carried out whole-exome sequencing. We identified 202,241 single nucleotide variants (SNVs) and 137,407 insertion-deletions (indels), where less than 10% of these genetic variations were within coding regions. The genetic variation (SNVs + indels) and copy number alteration (CNAs) profiles were highly heterogeneous and intra-patient CTC variation was observed. The pathway enrichment analysis showed the presence of genetic variation in nine telomere maintenance pathways (patients 3, 5, 6, and 7), including an important gene for telomere maintenance called telomeric repeat-binding factor 2 (TRF2). Using the PharmGKB database, we identified nine genetic variations associated with response to docetaxel. A total of 48 SNVs can affect drug response for 24 known cancer drugs. Gene Set Enrichment Analysis (GSEA) (patients 1, 3, 6, and 8) identified the presence of CNAs in 11 different pathways, including the DNA damage repair (DDR) pathway. In conclusion, single-cell approaches (WES and 3-D telomere profiling) showed to be useful in unmasking CTC heterogeneity. DDR pathway mutations have been well-established as a target pathway for cancer therapy. However, the frequent CNA amplifications found in localized high-risk patients may play critical roles in the therapeutic resistance in prostate cancer.


Assuntos
Variações do Número de Cópias de DNA , Células Neoplásicas Circulantes/metabolismo , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias da Próstata/patologia , Análise de Célula Única
18.
BMC Med Genet ; 20(1): 78, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068177

RESUMO

BACKGROUND: Few manuscripts have reported phenotypes of skeletal muscle myopathies caused by mutations in the head region of slow/cardiac beta-myosin heavy chain (MyHCI). Among the patients, some of them showed the phenotype of skeletal muscle weakness with the obvious clinical features of cardiomyopathy while others showed pure skeletal muscle weakness with no symptoms of cardiac involvement. Genotype-phenotype relationship regarding the effect of a mutation on MyHCI is complex. Questions regarding why some mutations cause cardiomyopathy or skeletal muscle disorders alone or a combination of both still need to be answered. More findings in genetic variation are needed to extend knowledge of mutations in the MYH7 gene linked to skeletal muscle disorders. CASE PRESENTATION: Here we present a female adult patient with a phenotype of childhood onset of muscular disorders and predominant involvement of thigh muscles with biopsy showing intrasarcoplasmic inclusion bodies. Whole exome sequencing showed that variant c.1370 T > G (p.Ile457Arg) in the MYH7 gene is a missense mutation possibly linked to the clinical findings. Our patient likely shows an uncharacteristic myosin storage myopathy associated with respiratory and cardiac involvement linked to a missense mutation in the head of MyHCI. CONCLUSIONS: Given this mutation is located within the motor domain of MyHCI, this might affect the regulation of myosin mechano-chemical activity during the contractile cycle. Consequently, this potentially damaging effect can be easily amplified within the network of ~ 300-myosin molecules forming the thick filament and therefore become cumulatively deleterious, affecting, in turn, the overall organization and performance of sarcomere.


Assuntos
Miosinas Cardíacas/genética , Doenças Musculares/congênito , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/genética , Adulto , Criança , Feminino , Humanos , Pessoa de Meia-Idade , Doenças Musculares/genética , Fenótipo
19.
Carcinogenesis ; 40(8): 1010-1020, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30605519

RESUMO

Recent studies have enabled the identification of important factors regulating cancer progression, such as paired box gene 5 (Pax-5). This transcription factor has consistently been associated to B-cell cancer lesions and more recently solid tumors including breast carcinoma. Although Pax-5 downstream activity is relatively well characterized, aberrant Pax-5 expression in a cancer-specific context is poorly understood. To investigate the regulation of Pax-5 expression, we turned to micro RNAs (miRNAs), small non-coding RNA molecules that regulate key biological processes. Extensive studies show that miRNA deregulation is prevalent in cancer lesions. In this study, we aim to elucidate a causal link between differentially expressed miRNAs in cancer cells and their putative targeting of Pax-5-dependent cancer processes. Bioinformatic prediction tools indicate that miRNAs 484 and 210 are aberrantly expressed in breast cancer and predicted to target Pax-5 messenger RNA (mRNA). Through conditional modulation of these miRNAs in breast cancer cells, we demonstrate that miRNAs 484 and 210 inhibit Pax-5 expression and regulate Pax-5-associated cancer processes. In validation, we show that these effects are probably caused by direct miRNA/mRNA interaction, which are reversible by Pax-5 recombinant expression. Interestingly, miRNAs 484 and 210, which are both overexpressed in clinical tumor samples, are also modulated during epithelial-mesenchymal transitioning and hypoxia that correlate inversely to Pax-5 expression. This is the first study demonstrating the regulation of Pax-5 expression and function by non-coding RNAs. These findings will help us better understand Pax-5 aberrant expression within cancer cells, creating the possibility for more efficient diagnosis and treatments for cancer patients.


Assuntos
Neoplasias da Mama/genética , MicroRNAs/genética , Fator de Transcrição PAX5/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , RNA Mensageiro/genética , Fatores de Transcrição/genética , Hipóxia Tumoral/genética
20.
Brain Res ; 1708: 100-108, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30552897

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with the progressive death of motor neurons. Mean survival for a patient diagnosed with ALS is between 2 and 5 years. Early and efficient diagnosis of the various forms of ALS remains a significant challenge, resulting in a need to identify clinically-relevant biomarkers in readily accessible body fluids. microRNAs (miRNAs) are short, evolutionarily conserved non-coding RNA molecules involved in post-transcriptional regulation of gene expression that have received interest as disease biomarkers. This study was undertaken to identify an ALS-associated miRNA signature in extracellular vesicles (EVs), which can cross the blood-brain barrier and enter the circulatory system, obtained from plasma samples of persons diagnosed and living with ALS (PALS). Next-generation sequencing was used to identify differentially expressed miRNAs recovered from EVs of PALS and healthy controls. High-throughput sequencing data for select miRNA targets was subsequently validated by droplet digital PCR (ddPCR). This approach revealed elevated levels of 5 miRNAs and reduced levels of 22 miRNAs in EVs collected from PALS as compared with healthy controls subjects. miRNAs with relevance to ALS were found to be deregulated, including miR-9-5p, miR-183-5p, miR-338-3p and miR-1246. MiR-15a-5p and miR-193a-5p were identified for their diagnostic potential of ALS and association with disability progression, respectively. Functional assessment of transcripts targeted by select ALS-associated miRNAs revealed processes such as transcriptional regulation and protein ubiquitination. These data identify an ALS-associated miRNAs signature in EVs of PALS and further strengthen the potential diagnostic relevance of these small molecules for this condition.


Assuntos
Esclerose Lateral Amiotrófica/genética , Vesículas Extracelulares/genética , MicroRNAs/genética , Adulto , Idoso , Esclerose Lateral Amiotrófica/sangue , Esclerose Lateral Amiotrófica/diagnóstico , Biomarcadores/sangue , Progressão da Doença , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Transcriptoma/genética
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