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1.
Recent Results Cancer Res ; 215: 181-211, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31605230

RESUMO

ctDNA provided by liquid biopsy offers a promising alternative to tumor biopsy as it gives a non-invasive and «real-time¼ access to the cancer genome and reflects tumor intra and extra heterogeneity. ctDNA has shown growing clinical interest for cancer diagnosis, prognosis, theragnostics, therapeutic monitoring, and clonal evolution tracking. A major technical limit for ctDNA analysis from body fluids is the extremely low proportion of ctDNA compared to non-malignant cell-free DNA, underscoring the need for highly sensitive and specific detection techniques. The control of pre-analytical procedures appears essential for optimal ctDNA analysis and need to be standardized for clinical research applications. This chapter provides insights into major current technologies for ctDNA detection. Overall, PCR-based techniques are able to detect limited molecular alterations and have a high sensitivity suitable for monitoring purposes while NGS-based approaches are broad range molecular screening assays more specifically indicated for treatment selection. We briefly reviewed new technical innovations that are now available for ctDNA detection.


Assuntos
DNA Tumoral Circulante/análise , DNA Tumoral Circulante/isolamento & purificação , Biópsia Líquida/métodos , Neoplasias/diagnóstico , Neoplasias/genética , DNA Tumoral Circulante/genética , Humanos , Neoplasias/sangue , Neoplasias/terapia , Reação em Cadeia da Polimerase , Prognóstico
2.
Recent Results Cancer Res ; 215: 253-261, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31605233

RESUMO

Colorectal cancer is one of the leading cause of death by cancer worldwide in both men and women. Liquid biopsy belongs nowadays to the landscape of cancer management biological tools. In this chapter, we will describe and discuss the actual, potential and future applications of cfDNA analysis in plasma of patients with colorectal cancer in early or metastatic stage. During the last decade, the development of molecular biology assays like digital PCR or next-generation sequencing made the analysis of cfDNA in plasma possible with an excellent sensitivity and applications like early detection, diagnosis, prognosis, response to treatment, monitoring of an emerging resistance, mapping of the disease molecular landscape or evaluation of the residual disease are now feasible. cfDNA detection has several promising applications in the management of patients with colorectal cancer, but prospective randomised clinical trials are still lacking to make liquid biopsy ready for prime-time.


Assuntos
DNA Tumoral Circulante/análise , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Biópsia Líquida , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Neoplasias Colorretais/diagnóstico , Humanos , Prognóstico
3.
Recent Results Cancer Res ; 215: 263-273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31605234

RESUMO

An accurate profiling of the genomic landscape is mandatory to establish the best clinical and therapeutic approach for patients with solid malignancies. Moreover, tumor cells constantly adapt to external pressures-i.e., systemic treatment-with the selection and expansion of resistant subclones and the emergence of heterogeneous overlapping genomic alterations of resistance. The current standard for molecular characterization in cancer is the performance of a tissue tumor biopsy at the time of diagnosis and, when possible, a re-biopsy at the time of progression. However, tissue biopsy is not always feasible or practical and may underestimate tumor heterogeneity and clonal dynamics. Circulating DNA fragments carrying tumor-specific sequence alterations (circulating tumor DNA, ctDNA) are released from cancer cells into the bloodstream, representing a variable and generally small fraction of the total circulating cell-free DNA. Tumor genotyping in ctDNA (liquid biopsy) offers potential advantages versus the standard tumor tissue biopsy, including non-invasiveness and representation of molecular heterogeneity. Technical advances in sequencing platforms have led to dramatic improvements in variant detection sensitivity and specificity that allow for the detection and quantification of low levels of ctDNA. This provides valuable information on both actionable mutations and captures real-time variations in tumor dynamics. Liquid biopsy clinical applications include molecular diagnosis, determination of tumor load as a surrogate marker of early response, monitoring of mutations of resistance to targeted therapy and detection of minimal residual disease after cancer surgery. The aim of this chapter is to provide an overview of the biological rational and technical background of ctDNA analysis, as well as on the main clinical applications of liquid biopsy in dynamic treatment stratification in solid tumors. Special emphasis will be made on the current and potential benefits of the implementation of ctDNA in clinical practice, mainly in melanoma, lung, and colorectal cancer.


Assuntos
DNA Tumoral Circulante/genética , Biópsia Líquida , Neoplasias/genética , Neoplasias/terapia , DNA Tumoral Circulante/análise , DNA Tumoral Circulante/sangue , Humanos , Mutação , Neoplasia Residual/sangue , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Neoplasias/sangue , Neoplasias/diagnóstico
4.
Recent Results Cancer Res ; 215: 319-344, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31605237

RESUMO

Extracellular micro- and nanoscale membrane vesicles produced by different cells progressively attract the attention of the scientific community. They function as mediators of intercellular communication and transport genetic material and signaling molecules between the cells. In the context of keeping homeostasis, the extracellular vesicles contribute to the regulation of various systemic and local processes. Vesicles released by the tumor and activated stromal cells exhibit multiple functions including support of tumor growth, preparation of the pre-metastatic niches, and immune suppression. Considerable progress has been made regarding the criteria of classification of the vesicles according to their origin, content, and function: Exosomes, microvesicles, also referred to as microparticles or ectosomes, and large oncosomes were defined as actively released vesicles. Additionally, apoptotic bodies represented by a highly heterogeneous population of particles produced during apoptosis, the programmed cell death, should be considered. Because the majority of isolation techniques do not allow the separation of different types of vesicles, a joined term "extracellular vesicles" (EVs) was recommended by the ISEV community for the definition of vesicles isolated from either the cell culture supernatants or the body fluids. Because EV content reflects the content of the cell of origin, multiple studies on EVs from body fluids in the context of cancer diagnosis, prediction, and prognosis were performed, actively supporting their high potential as a biomarker source. Here, we review the leading achievements in EV analysis from body fluids, defined as EV-based liquid biopsy, and provide an overview of the main EV constituents: EV surface proteins, intravesicular soluble proteins, EV RNA including mRNA and miRNA, and EV DNA as potential biomarkers. Furthermore, we discuss recent developments in technology for quantitative EV analysis in the clinical setting and future perspectives toward miniaturized high-precision liquid biopsy approaches.


Assuntos
Vesículas Extracelulares , Biópsia Líquida/métodos , Biópsia Líquida/tendências , Neoplasias/diagnóstico , Neoplasias/patologia , Apoptose , Micropartículas Derivadas de Células , Exossomos , Humanos
5.
Recent Results Cancer Res ; 215: 347-368, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31605238

RESUMO

Next-generation sequencing of DNA and RNA obtained from liquid biopsies of cancer patients may reveal important insights into disease progression and metastasis formation, and it holds the promise to enable new methods for noninvasive screening and clinical decision support. However, implementing liquid biopsy sequencing protocols is challenged by capturing circulating tumor cells or cell-free tumor DNA from blood samples, by amplifying genomic DNA and RNA in a reliable and unbiased manner, and by extracting biologically meaningful signals from the noisy sequencing data. In this chapter, we discuss computational methods for the analysis of DNA and RNA sequencing data obtained from liquid biopsies, addressing these challenges.


Assuntos
DNA Tumoral Circulante/análise , DNA Tumoral Circulante/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biópsia Líquida , Neoplasias/diagnóstico , Neoplasias/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , DNA Tumoral Circulante/sangue , Humanos
6.
Cancer Treat Rev ; 79: 101893, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31499407

RESUMO

BACKGROUND: The management of locally advanced rectal cancer (RC) is an evolving clinical field where the multidisciplinary approach can reach its best, and liquid biopsy for obtaining tumor-derived component such as circulating tumor DNA (ctDNA) might provide complementary informations. METHODS: A systematic review of studies available in literature of liquid biopsy in non-metastatic RC has been performed according to PRISMA criteria to assess the role of ctDNA as a diagnostic, predictive and prognostic biomarker in this setting. RESULTS: Twenty-five publications have been retrieved, of which 8 full-text articles, 7 abstracts and 10 clinical trials. Results have been categorized into three groups: diagnostic, predictive and prognostic. Few but promising data are available about the use of liquid biopsy for early diagnosis of RC, with the main limitation of sensitivity due to low concentrations of ctDNA in this setting. In terms of prediction of response to chemoradiation, still inconclusive data are available about the utility of a pre-treatment liquid biopsy, whereas some studies report a positive correlation with a dynamic (pre/post-treatment) monitoring. The presence of minimal residual disease by ctDNA was consistently associated with worse prognosis across studies. CONCLUSIONS: The use of liquid biopsy for monitoring response to chemoradiation and assess the risk of disease recurrence are the most advanced potential applications for liquid biopsy in RC, with implications also in the context of non-operative management strategies.


Assuntos
Biomarcadores Tumorais , Biópsia Líquida , Neoplasias Retais/diagnóstico , DNA Tumoral Circulante , DNA de Neoplasias , Humanos , Biópsia Líquida/métodos , Metástase Neoplásica , Estadiamento de Neoplasias , Células Neoplásicas Circulantes , Prognóstico , Neoplasias Retais/etiologia , Neoplasias Retais/mortalidade , Neoplasias Retais/terapia , Recidiva , Resultado do Tratamento
7.
Gan To Kagaku Ryoho ; 46(9): 1361-1366, 2019 Sep.
Artigo em Japonês | MEDLINE | ID: mdl-31530771

RESUMO

This year, the genetic testing for cancer genome medicine approved in Japan, and the clinical basis for this medical area is developed. Focus will likely fall not only on genetic analysis in tissues, but also on genomic analysis by liquid biopsy using patient body fluids such as blood, saliva and urine. The advantages of liquid biopsy are the fact that it is minimally invasive and the possibility of broadening the diagnosis and selection of therapeutic agents, even in cases in which it is difficult to collect tumor tissues. Liquid biopsies of cancer patients will enable, circulating tumor cell(CTC), cell free DNA(cfDNA), circulating tumor cell DNA(ctDNA), exosomes and microRNA(miRNA). The potential usefulness of liquid biopsy for cancer diagnosis, recurrence prediction and monitoring of treatment effect has been mentioned in various manuscripts. In immunotherapy, liquid genetic biomarkers for predicting the therapeutic effect of immune checkpoint inhibitors are under development worldwide. Although issues such as standardization of the measurement methods and the samples used remain, early diagnosis of cancer by liquid biopsy may become a reality in the near future.


Assuntos
Genômica , Biomarcadores Tumorais , Humanos , Imunoterapia , Japão , Biópsia Líquida , Recidiva Local de Neoplasia
8.
Cancer Sci ; 110(10): 3235-3243, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368627

RESUMO

Cytology is widely conducted for diagnosis of urothelial bladder cancer; however, its sensitivity is still low. Recent studies show that liquid biopsies can reflect tumor genomic profiles. We aim to investigate whether plasma or urine is more suitable for detecting tumor-derived DNA in patients with early-stage urothelial bladder cancer. Targeted sequencing of 71 genes was carried out using a total of 150 samples including primary tumor, urine supernatant, urine precipitation, plasma and buffy coat from 25 patients with bladder cancer and five patients with cystitis and benign tumor. We compared mutation profiles between each sample, identified tumor-identical mutations and compared tumor diagnostic sensitivities between urine and conventional cytology. We identified a total of 168 somatic mutations in primary tumor. In liquid biopsies, tumor-identical mutations were found at 53% (89/168) in urine supernatant, 48% (81/168) in urine precipitation and 2% (3/168) in plasma. The high variant allele fraction of urine was significantly related to worse clinical indicators such as tumor invasion and cytological examination. Although conventional cytology detected tumor cells in only 22% of non-invasive tumor, tumor diagnostic sensitivity increased to 67% and 78% using urine supernatant and precipitation, respectively. Urine is an ideal liquid biopsy for detecting tumor-derived DNA and more precisely reflects tumor mutational profiles than plasma. Genomic analysis of urine is clinically useful for diagnosis of superficial bladder cancer at early stage.


Assuntos
Biomarcadores Tumorais/urina , Mutação , Análise de Sequência de DNA/métodos , Neoplasias da Bexiga Urinária/diagnóstico , Urina/química , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Detecção Precoce de Câncer , Feminino , Humanos , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/urina
9.
Gynecol Oncol ; 155(1): 135-143, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31434614

RESUMO

OBJECTIVE: The altered miRNAs expression in cervical cancer tissue can be a critical player during tumorigenesis, may contribute to tumor cell heterogeneity and may determine distinct phenotypes within the tumor. Recent studies have highlighted the role of circulating miRNAs as a minimally-invasive biomarker and its potential as biosignature to complement routine tissue-based procedures. METHODS: In order to determine whether miRNAs in serum can indicate changes in cervical tissue specimens, we performed small RNA sequencing and selected miRNAs were validated using qRT-PCR in serum and tissue specimens (n = 115). Further, luciferase assay were performed to investigate the interactions between hsa-miR-409-3p and hsa-miR-454-3p binding sites on 3'UTR region of MTF2 and ST18 respectively. RESULTS: We have identified a total of 14 differentially expressed miRNAs common in serum and tissue specimens. Among them, hsa-miR-17-5p, hsa-miR-32-5p and hsa-miR-454-3p were upregulated while, hsa-miR-409-3p was downregulated in serum and tissue of cervical cancer subjects. Our in-silico small RNA sequencing data analysis identified isomiRs and classified miRNA into clusters and subtypes (exonic, intronic and intergenic) with respect to the expression status in serum and tissue specimens. Expression level of hsa-miR-409-3p and hsa-miR-454-3p were inversely correlated with their target genes MTF2 and ST18 levels respectively in human cervical cancer specimens. Luciferase assay demonstrated that hsa-miR-409-3p and hsa-miR-454-3p functionally interacts with 3'-UTR of MTF2 and ST18 respectively to decrease their activity. CONCLUSION: Our results support the significant role of circulating miRNAs in disease dissemination and their potential utility as biosignatures of clinical relevance.


Assuntos
MicroRNAs/biossíntese , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasia Intraepitelial Cervical/genética , Neoplasia Intraepitelial Cervical/metabolismo , Neoplasia Intraepitelial Cervical/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida , MicroRNAs/sangue , MicroRNAs/genética , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima , Neoplasias do Colo do Útero/sangue , Neoplasias do Colo do Útero/metabolismo
10.
Cancer Sci ; 110(10): 3375-3381, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31436356

RESUMO

Cell-free DNA (cfDNA) analysis to detect circulating tumor DNA has been focused on monitoring malignant lymphomas. However, clonal hematopoiesis of indeterminate potential (CHIP)-associated mutations can also be detected by cfDNA analysis. Our aim is to investigate the origin of mutations detected in cfDNA among B-cell lymphoma patients. MYD88/CD79B, DNMT3A, and TP53 were chosen as genes of interest, representing each of the following categories: lymphoma driver genes, CHIP-related genes, and genes shared between lymphoma and CHIP. Seventy-five B-cell lymphoma patients were included in this retrospective study. Serum cfDNAs at time of complete metabolic response (CMR) were sequenced for TP53 (N = 75) and DNMT3A (N = 49). MYD88 p.L265P and CD79B p.Y196C/H mutations were analyzed in diffuse large B-cell lymphoma (DLBCL) patients whose tumor samples were available (N = 29). Two and seven mutations in TP53 and DNMT3A, respectively, were detected in cfDNA at CMR. These mutations were detected in either bone marrow mononuclear cells (BMMC) or PBMC. Although four DNMT3A mutations were also detected in tumors, median variant allele frequencies in the tumors (<1.0%) were significantly lower than those in both BMMC (6.1%) and serum (5.2%) obtained before the therapy. Conversely, five MYD88 and three CD79B mutations detected in tumors were confirmed in cfDNA before therapy, but not in BMMC nor in cfDNA at CMR. Thus, all TP53 and DNMT3A mutations detected in cfDNA at remission seemed to originate from CHIP rather than from residual disease. Results of liquid biopsy should be carefully interpreted, especially in genes shared between lymphomas and CHIP.


Assuntos
Células Clonais/química , DNA (Citosina-5-)-Metiltransferases/genética , Hematopoese , Linfoma de Células B/genética , Mutação , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/química , Ácidos Nucleicos Livres/genética , Feminino , Frequência do Gene , Humanos , Leucócitos Mononucleares/química , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Monócitos/química , Indução de Remissão , Estudos Retrospectivos , Análise de Sequência de DNA/métodos
11.
Cancer Treat Rev ; 78: 31-41, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31326635

RESUMO

Current classification and treatment of lung cancer rely increasingly on molecular and genetic testing. Obtaining tumor tissue is not always feasible and multiple biopsies are undesirable. In response to the demand for non-invasive molecular and genetic testing in cancer care, several liquid biopsy technologies, including circulating DNA (ctDNA), have been developed. ctDNA analysis is now technically feasible to be carried out in large scales and integrated into clinical practice owing to the advances in technology. Despite the challenges in improving test accuracy and cost-effectiveness, there are huge potentials for ctDNA analysis in lung cancer management. This review focuses on the clinical utility of ctDNA analysis in lung cancer, including early detection, monitoring treatment response and detecting residual disease, identification of genetic determinants for targeted therapy, and predicting efficacy of immune checkpoint blockade.


Assuntos
Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/sangue , DNA de Neoplasias/sangue , Neoplasias Pulmonares/diagnóstico , Humanos , Biópsia Líquida , Neoplasias Pulmonares/sangue
12.
Anal Bioanal Chem ; 411(23): 6039-6047, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31304564

RESUMO

Circulating tumor DNA (ctDNA) is a tumor-derived fragmented DNA in the bloodstream that is not associated with cells. It has been greatly focused in the recent decade because of its potential clinical utility for liquid biopsies. Development of ctDNA analytical techniques with high sensitivity and cost-efficiency will undoubtedly promote the clinical spread of ctDNA testing. In this paper, we propose a novel flow cytometry-based ctDNA sensing strategy which combines enzyme-free amplification and magnetic separation. The target DNA is capable of triggering a hybridization chain reaction, producing a fluorescent long linear assembly of DNA, which can be further captured by magnetic beads to present fluorescent signals using flow cytometry. In comparison with some conventional methods, our strategy has the advantages of easy operation and cost-efficiency, and thereby shows a promising application in clinical diagnosis. Graphical abstract.


Assuntos
DNA Tumoral Circulante/sangue , Citometria de Fluxo/métodos , Imãs/química , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA Tumoral Circulante/análise , Humanos , Limite de Detecção , Biópsia Líquida/métodos , Hibridização de Ácido Nucleico/métodos
14.
Vet Clin North Am Small Anim Pract ; 49(5): 781-791, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31280902

RESUMO

Molecular diagnostics have revolutionized human oncology to allow early detection, targeted therapy, monitoring throughout treatment, and evidence of recurrence. By identifying genetic signatures associated with cancers, liquid biopsy techniques have been developed to diagnose and monitor cancer in noninvasive or minimally invasive ways. These techniques offer new opportunities for improving cancer screening, diagnosis, and monitoring the impact of therapy on the patients over time. Liquid biopsy also drives drug development programs. Similar diagnostics hold promise for comparable results in the veterinary field. Several noninvasive/minimally invasive techniques have been described in veterinary medicine that could be referred to as liquid biopsy.


Assuntos
Doenças do Cão/diagnóstico , Biópsia Líquida/veterinária , Neoplasias/veterinária , Animais , Carcinoma de Células de Transição/diagnóstico , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/veterinária , Doenças do Cão/tratamento farmacológico , Doenças do Cão/genética , Cães , Detecção Precoce de Câncer/métodos , Detecção Precoce de Câncer/veterinária , Feminino , Humanos , Leucemia/diagnóstico , Leucemia/veterinária , Biópsia Líquida/métodos , Linfoma/diagnóstico , Linfoma/tratamento farmacológico , Linfoma/veterinária , Masculino , Terapia de Alvo Molecular/veterinária , Mutação , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias Uretrais/diagnóstico , Neoplasias Uretrais/genética , Neoplasias Uretrais/veterinária , Neoplasias da Bexiga Urinária/veterinária
15.
Biochim Biophys Acta Rev Cancer ; 1872(1): 49-59, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31152821

RESUMO

Cancer, a local disease at an early stage, systemically evolves as it progresses by triggering alterations in surrounding microenvironment, disturbing immune surveillance and further disseminating its molecular contents into circulation. This pathogenic characteristic of cancer makes the use of biofluids such as blood/serum/plasma, urine, tear and cerebrospinal fluids credible surrogates harboring tumor tissue-derived molecular alterations for the detection of cancer. Most importantly, a number of recent reports have credentialed the clinical validity of saliva for the detection of systemic diseases including cancers. In this review, we discussed the validity of saliva as credible biofluid and clinical sample type for the detection of cancers. We have presented the molecular constituents of saliva that could mirror the systemic status of our body and recent findings of salivaomics associated with cancers. Recently, liquid biopsy to detect cancer-derived circulating tumor DNA has emerged as a credible cancer-detection tool with potential benefits in screening, diagnosis and also risk management of cancers. We have further presented the clinical validity of saliva for liquid biopsy of cancers and a new technology platform based on electrochemical detection of cancer-derived ctDNA in saliva with superior sensitivity and point-of-care potential. The clinical utilities of saliva for the detection of cancers have been evidenced, but biological underpinning on the existence of molecular signatures of cancer-origin in saliva, such as via exosomal distribution, should be addressed in detail.


Assuntos
Biomarcadores Tumorais/genética , Detecção Precoce de Câncer , Neoplasias/diagnóstico , Saliva/química , Biomarcadores Tumorais/química , DNA Tumoral Circulante/química , DNA Tumoral Circulante/genética , Humanos , Biópsia Líquida , Neoplasias/química , Neoplasias/genética , Neoplasias/metabolismo , Saliva/metabolismo , Microambiente Tumoral/genética
16.
Cancer Sci ; 110(8): 2590-2599, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31169336

RESUMO

Liquid biopsy of circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) is gaining attention as a method for real-time monitoring in cancer patients. Conventional methods based upon epithelial cell adhesion molecule (EpCAM) expression have a risk of missing the most aggressive CTC subpopulations due to epithelial-mesenchymal transition and may, thus, underestimate the total number of actual CTC present in the bloodstream. Techniques utilizing a label-free inertial microfluidics approach (LFIMA) enable efficient capture of CTC without the need for EpCAM expression. In this study, we optimized a method for analyzing genetic alterations using next-generation sequencing (NGS) of extracted ctDNA and CTC enriched using an LFIMA as a first-phase examination of 30 patients with head and neck cancer, esophageal cancer, gastric cancer and colorectal cancer (CRC). Seven patients with advanced CRC were enrolled in the second-phase examination to monitor the emergence of alterations occurring during treatment with epidermal growth factor receptor (EGFR)-specific antibodies. Using LFIMA, we effectively captured CTC (median number of CTC, 14.5 cells/mL) from several types of cancer and detected missense mutations via NGS of CTC and ctDNA. We also detected time-dependent genetic alterations that appeared during anti-EGFR therapy in CTC and ctDNA from CRC patients. The results of NGS analyses indicated that alterations in the genomic profile revealed by the liquid biopsy could be expanded by using a combination of assays with CTC and ctDNA. The study was registered with the University Hospital Medical Information Network Clinical Trials Registry (ID: UMIN000014095).


Assuntos
DNA Tumoral Circulante/genética , DNA de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Molécula de Adesão da Célula Epitelial/genética , Transição Epitelial-Mesenquimal/genética , Receptores ErbB/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Biópsia Líquida/métodos , Masculino , Pessoa de Meia-Idade , Mutação/genética
17.
J Cancer Res Clin Oncol ; 145(8): 2071-2082, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154543

RESUMO

PURPOSE: Fluorescence in situ hybridization (FISH) using tumor tissue is the gold standard for detection of anaplastic lymphoma kinase (ALK) rearrangement in non-small cell lung cancer (NSCLC). However, this method often is not repeatable due to difficulties in the acquisition of tumor tissues. Blood-based liquid biopsy using reverse transcription polymerase chain reaction (RT-PCR) is expected to be useful to overcome this limitation. Here, we investigated the feasibility of liquid biopsy using plasma and platelets for detection of ALK rearrangement and prediction of ALK inhibitor treatment outcomes. METHODS: ALK-FISH assays were performed in 1128 tumor specimens of NSCLC between January 2015 and June 2018. We retrospectively analyzed formalin-fixed paraffin-embedded (FFPE) tissues from previously confirmed FISH-positive (n = 199) and -negative (n = 920) cases. We recruited patients who had available tissue specimens and agreed to venous sampling. RNA was extracted from FFPE blocks, plasma, and platelets. Fusion RNA of echinoderm microtubule-associated protein-like 4 (EML4)-ALK was detected by quantitative PCR. RESULTS: Thirty-three FISH-positive and 28 FISH-negative patients were enrolled. In validation, data compared with FISH, RT-PCR using FFPE tissues showed 54.5% sensitivity, 78.6% specificity, and 75.5% accuracy. Liquid biopsy had higher sensitivity (78.8%), specificity (89.3%) and accuracy (83.6%). Higher positivity for liquid biopsy was shown in subgroups with delayed (≥ 6 months from diagnosis) blood sampling (plasma, 85.7%; platelets, 87.0%). In 26 patients treated with crizotinib, the platelet-positive subgroup showed longer median duration of treatment (7.2 versus 1.5 months), longer median progression-free survival (5.7 months versus 1.7 months), a higher overall response rate (70.6% versus 11.1%), and a higher disease control rate (88.2% versus 44.4%) than the platelet-negative subgroup. CONCLUSION: Liquid biopsy could have applications in the diagnosis of ALK-positive NSCLC, even when using RT-PCR, and platelets can be useful for predicting treatment outcomes of ALK inhibitors.


Assuntos
Quinase do Linfoma Anaplásico/genética , Plaquetas/química , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Análise Mutacional de DNA/métodos , Neoplasias Pulmonares/diagnóstico , Plasma/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Química do Sangue/métodos , Plaquetas/patologia , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , DNA de Neoplasias/análise , Resistencia a Medicamentos Antineoplásicos/genética , Estudos de Viabilidade , Feminino , Humanos , Hibridização in Situ Fluorescente , Biópsia Líquida , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Mutação , Células Neoplásicas Circulantes/patologia , Plasma/citologia , Inibidores de Proteínas Quinases/uso terapêutico , Estudos Retrospectivos , Sensibilidade e Especificidade
18.
Crit Rev Oncol Hematol ; 141: 36-42, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31212145

RESUMO

Liquid biopsy can quantify and qualify cell-free (cfDNA) and tumour-derived (ctDNA) DNA fragments in the bloodstream. CfDNA quantification and mutation analysis can be applied to diagnosis, follow-up and therapeutic management as novel oncologic biomarkers. However, some tumor-types release a low amount of DNA into the bloodstream, hampering diagnosis through standard liquid biopsy procedures. Several tumors, as such as brain, kidney, prostate, and thyroid cancer, are in direct contact with other body fluids and may be alternative sources for cfDNA and ctDNA. Non-blood sources of cfDNA/ctDNA useful as novel oncologic biomarkers include cerebrospinal fluids, urine, sputum, saliva, pleural effusion, stool and seminal fluid. Seminal plasma cfDNA, which can be analyzed with cost-effective procedures, may provide powerful information capable to revolutionize prostate cancer (PCa) patient diagnosis and management. In the near future, cfDNA analysis from non-blood biological liquids will become routine clinical practice for cancer patient diagnosis and management.


Assuntos
Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/isolamento & purificação , Perfilação da Expressão Gênica/métodos , Oncologia/métodos , Neoplasias/diagnóstico , Patologia Clínica/métodos , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/isolamento & purificação , Ácidos Nucleicos Livres/análise , DNA Tumoral Circulante/análise , DNA Tumoral Circulante/isolamento & purificação , DNA Tumoral Circulante/urina , Análise Mutacional de DNA/métodos , Fezes/química , Feminino , Humanos , Biópsia Líquida , Masculino , Oncologia/tendências , Neoplasias/genética , Neoplasias/patologia , Neoplasias/urina , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Urinálise/métodos
19.
Anal Chim Acta ; 1076: 154-161, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203960

RESUMO

Cancer cell detection in liquid biopsies has been a widely studied application in many microfluidic devices. The use of a common antibody, such as the epithelial cell adhesion molecule (Anti-EpCAM) or other specific antibodies, has facilitated the detection and study of many cancers. However, the use of such antibodies requires a priori knowledge of the cancer source, and many cancer subtypes are missed in screening applications. There remains a need to study a wider range of cancers that maintain the streamlined antibody approach in cell affinity separations. The Human transferrin receptor (CD71) has recently been demonstrated as a cancer cell affinity target in blood samples. CD71 expression in blood cells is low, whereas proliferating cancer cells have a higher expression of the surface protein. CD71 expression is variable with cell cycle, which can impact cell separations. In this work, we investigated the effects of cell cycle and CD71 expression on cell capture metrics. Six cancer cell lines were isolated from blood via CD71 affinity capture, with a capture efficiency and purity that varied with CD71 expression. Despite variation in CD71 expression, the affinity was sufficient to isolate cancer cells spiked into blood; under optimal conditions, CD71-based capture resulted in capture purity >80%. We conclude that CD71 affinity separations show potential as a biomarker for cancer studies without sacrificing sensitivity and selectivity, and that cancer cells can be isolated from liquid biopsies over a range of expression of the target protein.


Assuntos
Antígenos CD/imunologia , Biomarcadores Tumorais/imunologia , Células Neoplásicas Circulantes/imunologia , Receptores da Transferrina/imunologia , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Dispositivos Lab-On-A-Chip , Ligantes , Biópsia Líquida , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
20.
Anal Chim Acta ; 1076: 82-90, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203967

RESUMO

We present a method to preserve and process urine proteins for proteomic analysis in a filter aided sample preparation (FASP) format. The method combines concentration of urine proteins in ultrafiltration devices, their thermal stabilization, allowing long term storage of the samples, and filter aided sample preparation. Proteomes of four different urines were preserved during 48 h and 6 months using (i) the classic freeze preservation at -20 °C, (ii) snap-heated freeze-free preservation at laboratory temperature (20 °C) and (iii) snap-heated preservation at -60 °C. The three storage methods can effetely preserve the urine proteome for at least 6 months without significant alterations. Abundances of more than 500 proteins and specially 24 selected -cleared or -approved protein assayed in serum or plasma were found similar within the three preservation methods assessed. The new method here proposed dramatically simplifies the conditions for preserving the urine proteome for biobanks in terms of space and storage, including lowering the risks of sample degradation caused by misfunction of the freezer. Furthermore, the shipping of large number of samples can be made without the need of freezing. The application of the FASP format to isolate and preserve the proteins facilitates long-term storage and processing of proteome of urine samples.


Assuntos
Proteoma/química , Manejo de Espécimes/métodos , Urina/química , Lesão Renal Aguda/urina , Adulto , Biomarcadores/análise , Análise por Conglomerados , Calefação , Humanos , Biópsia Líquida , Masculino , Estudo de Prova de Conceito , Proteoma/análise , Proteoma/isolamento & purificação , Adulto Jovem
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