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PURPOSE: We assessed whether preoperativemutational analyses of circulating tumor cells (CTCs) and plasma-cfDNA could be used as minimally invasive biomarkers and as complimentary tools for early prediction of relapse in early-stage non-small -cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Using ddPCR assays, hotspot mutations of BRAF, KRAS, EGFR and PIK3CA were identified in plasma-cfDNA samples and size-based enriched CTCs isolated from the same blood samples of 49 early-stage NSCLC patients before surgery and in a control group of healthy blood donors (n= 22). Direct concordance of the mutational spectrum was further evaluated in 27 patient-matched plasma-cfDNA and CTC-derived DNA in comparison to tissue-derived DNA. RESULTS: The prevalence of detectable mutations of the four tested genes was higher in CTC-derived DNA than in the corresponding plasma-cfDNA (38.8% and 24.5%, respectively).The most commonly mutated gene was PIK3CA, in both CTCs and plasma-cfDNA at baseline and at the time of relapse. Direct comparison of the mutation status of selected drug-responsive genes in CTC-derived DNA, corresponding plasma-cfDNA and paired primary FFPE tissues clearly showed the impact of heterogeneity both within a sample type, as well as between different sample components. The incidence of relapse was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA compared with patients in whom no mutation was detected (p =0.023). Univariate analysis showed a significantly higher risk of progression (HR: 2.716; 95% CI, 1.030-7.165; p =0.043) in patients with detectable mutations in plasma-cfDNA compared with patients with undetectable mutations, whereas the hazard ratio was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA (HR: 3.375; 95% CI, 1.098-10.375; p =0.034). CONCLUSIONS: Simultaneous mutational analyses of plasma-cfDNA and CTC-derived DNA provided complementary molecular information from the same blood sample and greater diversity in genomic information for cancer treatment and prognosis. The detection of specific mutations in ctDNA and CTCs in patients with early-stage NSCLC before surgery was independently associated with disease recurrence, which represents an important stratification factor for future trials.
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PURPOSE: Metabolic reprogramming is now characterized as one of the core hallmarks of cancer, and it has already been shown that the altered genomic profile of metabolically rewired cancer cells can give valuable information. In this study, we quantified three Metabolism-Related Gene (MRG) transcripts in the circulating tumor cells (CTCs) of early stage NSCLC patients and evaluated their associations with epithelial and EMT markers. EXPERIMENTAL DESIGN: We first developed and analytically validated highly sensitive RT-qPCR assays for the quantification of HK2, MCT1 and PHGDH transcripts, and further studied the expression of MRGs in CTCs that were isolated using a size-dependent microfluidic device (Parsortix, Angle) from the peripheral blood of: (a) 46 NSCLC patients at baseline, (b) 39/46 of these patients one month after surgery, (c) 10/46 patients at relapse and (d) 10 pairs of cancerous and adjacent non-cancerous FFPE tissues from the same NSCLC patients. Epithelial and EMT markers were also evaluated. RESULTS: MCT1 and HK2 were differentially expressed between HD and NSCLC patients. An overexpression of MCT1 was detected in 15/46 (32.6%) and 3/10 (30%) patients at baseline and at progression disease (PD), respectively, whereas an overexpression of HK2 was detected in 30.4% and 0% of CTCs in the same group of samples. The expression levels of all tested MRGs decreased in CTCs one month after surgery, but a significant increase was noticed at the time of relapse for PHGDH and MCT1 only. The expression levels of HK2 and MCT1 were associated with the overexpression of mesenchymal markers (TWIST-1 and VIM). CONCLUSION: An overexpression of MRGs was observed at a high frequency in the CTCs isolated from early NSCLC patients, thereby supporting the role of MRGs in metastatic processes. The glycolytic and mesenchymal subpopulation of CTCs was significantly predominant compared to CTCs that were glycolytic but not mesenchymal-like. Our data indicate that MRGs merit further evaluation through large and well-defined cohort studies.
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Infecções por Vírus Epstein-Barr , Neoplasias Nasofaríngeas , DNA , DNA Viral/genética , Infecções por Vírus Epstein-Barr/diagnóstico , Herpesvirus Humano 4/genética , Humanos , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/terapia , PrognósticoRESUMO
Over the last decade, liquid biopsy has gained much attention as a powerful tool in personalized medicine, since it enables monitoring cancer evolution and follow-up of cancer patients in real time. Through minimally invasive procedures, liquid biopsy provides important information through the analysis of Circulating Tumor Cells (CTCs), and circulating tumor-derived material like circulating tumor DNA (ctDNA), circulating miRNAs (cfmiRNAs) and extracellular vehicles (EVs). CTCs and ctDNA analysis has already an important impact on the prognosis, detection of minimal residual disease (MRD), treatment selection and monitoring of cancer patients, while recent data show also its potential for early cancer diagnosis (Figure 1). Numerous clinical trials include now a liquid biopsy arm, and functional studies mainly based on CTC derived cell-lines and CTC derived explants (CDx) provide important insight on the metastatic process. The recent findings in the field of liquid biopsy and the benefits and main clinical applications of CTC and ctDNA analysis in solid tumors are summarized in this review.
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DNA Tumoral Circulante , Células Neoplásicas Circulantes , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Humanos , Biópsia Líquida/métodos , Células Neoplásicas Circulantes/patologia , PrognósticoRESUMO
The analysis of circulating cell free DNA is an important tool for the analysis of tumor resistance, tumor heterogeneity, detection of minimal residual disease and detection of allograft rejection in kidney or heart transplant patients. The proper use of this technique is important, and starts with considering pre-analytic aspects. The current paper addresses some important technical considerations to ensure the proper and harmonized use of cfDNA techniques.
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Ácidos Nucleicos Livres , DNA Tumoral Circulante , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Testes Diagnósticos de Rotina , Humanos , Neoplasia ResidualRESUMO
Purpose: Monocarboxylate transporter 4 (MCT4) can influence the amount of lactate in the tumor microenvironment and further control cancer cell proliferation, migration, and angiogenesis. We investigated for the first time the expression of MCT4 in circulating tumor cells (CTCs) derived from early stage Non-Small Cell Lung Cancer patients (NSCLC) and whether this is associated with clinical outcome. Experimental Design: A highly sensitive RT-qPCR assay for quantification of MCT4 transcripts was developed and validated and applied to study MCT4 expression in CTC isolated through the Parsortix size-dependent microfluidic device from 53 and 9 peripheral blood (PB) samples of NSCLC patients at baseline (pre-surgery) and at relapse, respectively, as well as the "background noise" was evaluated using peripheral blood samples from 10 healthy donors (HD) in exactly the same way as patients. Results: MCT4 was differentially expressed between HD and NSCLC patients. Overexpression of MCT4 was detected in 14/53 (26.4%) and 3/9 (33.3%) patients at baseline and at progression disease (PD), respectively. The expression levels of MCT4 was found to increase in CTCs at the time of relapse. Kaplan-Meier analysis showed that the overexpression of MCT4 was significantly (P = 0.045) associated with progression-free survival (median: 12.5 months, range 5-31 months). Conclusion: MCT4 overexpression was observed at a high frequency in CTCs from early NSCLC patients supporting its role in metastatic process. MCT4 investigated as clinically relevant tumor biomarker characterizing tumor aggressiveness and its potential value as target for cancer therapy. We are totally convinced that MCT4 overexpression in CTCs merits further evaluation as a non-invasive circulating tumor biomarker in a large and well-defined cohort of patients with NSCLC.
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Molecular diversity and continuing evolution of metastatic tumors are not easily captured by tissue biopsies. Development of non-invasive diagnostic tools, such asanalysis of circulating tumor DNA (ctDNA), Circulating Tumor Cells (CTCs) and exosomes provides the opportunity to assess a blood sample in order to monitor tumor change and extract molecular information from cancers at a given time. "Liquid biopsy", which refers to molecular analysis of tumor's genetic features based on circulating genetic material in the peripheral blood, is already used to monitor disease response and track mechanisms of drug resistance in solid tumors. Head and Neck Squamous Cell Carcinoma (HNSCC) is a malignancy associated with advanced disease at presentation and dismal outcomes; furthermore, there is lack of biomarkers to monitor disease burden. Incorporation of liquid biopsy in the management of HNSCC might help identify patients with occult metastatic disease earlier and in a non-invasive manner. Herein, we aim to review current knowledge regarding CTCs and ctDNA in HNSCC and address open questions in this fast-evolving field of research.
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Carcinoma de Células Escamosas/diagnóstico , DNA de Neoplasias/sangue , Neoplasias de Cabeça e Pescoço/diagnóstico , Biópsia Líquida/estatística & dados numéricos , Células Neoplásicas Circulantes , Biomarcadores Tumorais/sangue , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/patologia , Transição Epitelial-Mesenquimal , Neoplasias de Cabeça e Pescoço/sangue , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Prognóstico , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
BACKGROUND: Successful application of programmed death 1 (PD1) checkpoint inhibitors in the clinic may ultimately benefit from appropriate patient selection based upon predictive biomarkers. Molecular characterization of circulating tumor cells (CTC) is crucial for the investigation of molecular-targeted therapies while predictive biomarkers for response to PD1 checkpoint inhibitors are lacking. We sought to assess whether overexpression of PD-L1 in CTCs could be detected at baseline and at different timepoints during treatment in a prospective cohort of head and neck squamous cell carcinoma (HNSCC) patients and used to predict clinical outcome after treatment with curative intent. PATIENTS AND METHODS: We developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA expression in EpCAM(+) CTCs. In a prospective cohort of 113 locally advanced HNSCC patients treated with curative intent we evaluated PD-L1 expression in the EpCAM(+) CTC fraction at baseline, after 2 cycles of induction chemotherapy (week 6) and at the end of concurrent chemoradiotherapy (week 15). RESULTS: PD-L1 overexpression was found in 24/94 (25.5%) patients at baseline, 8/34 (23.5%) after induction chemotherapy and 12/54 (22.2%) patients at the end of treatment. Patients with CTCs overexpressing PD-L1 at end of treatment had shorter progression-free survival (P = 0.001) and overall survival (P < 0.001). Multivariate analysis revealed that PD-L1 overexpression at end of treatment was independent prognostic factor for progression-free survival and overall survival. The absence of PD-L1 overexpression at the end of treatment was strongly associated with complete response with an odds ratio = 16.00 (95% CI = 2.76-92.72, P = 0.002). CONCLUSIONS: We demonstrate that detection of CTCs overexpressing PD-L1 is feasible and may provide important prognostic information in HNSCC. Our results suggest that adjuvant PD1 inhibitors deserve evaluation in HNSCC patients in whom PD-L1(+) CTCs are detected at the end of curative treatment.
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Antígeno B7-H1/metabolismo , Carcinoma de Células Escamosas/sangue , Neoplasias de Cabeça e Pescoço/sangue , Células Neoplásicas Circulantes/metabolismo , Idoso , Antígeno B7-H1/genética , Carcinoma de Células Escamosas/patologia , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Reprodutibilidade dos Testes , Carcinoma de Células Escamosas de Cabeça e Pescoço , Análise de SobrevidaRESUMO
BACKGROUND: Liquid biopsy is based on minimally invasive blood tests and has the potential to characterize the evolution of a solid tumor in real time, by extracting molecular information from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Epigenetic silencing of tumor and metastasis suppressor genes plays a key role in survival and metastatic potential of cancer cells. Our group was the first to show the presence of epigenetic alterations in CTCs. METHODS: We present the development and analytical validation of a highly specific and sensitive Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA) for the simultaneous detection of the methylation status of three tumor and metastasis suppressor genes (CST6, SOX17 and BRMS1) in liquid biopsy material (CTCs, corresponding ctDNA) and paired primary breast tumors. RESULTS: In the EpCAM-positive CTCs fraction we observed methylation of: a) CST6, in 11/30(37%) and 11/30(37%), b) BRMS1 in 8/30(27%) and 11/30(37%) c) SOX17 in 8/30(27%) and 13/30(43%) early breast cancer patients and patients with verified metastasis respectively. In ctDNA we observed methylation of: a) CST6, in 5/30(17%) and 10/31(32%), b) BRMS1 in 8/30 (27%) and 8/31 (26%) c) SOX17 in 5/30(17%) and 13/31(42%) early breast cancer patients and patients with verified metastasis respectively. CONCLUSIONS: Our results indicate a high cancerous load at the epigenetic level in EpCAM-positive CTCs fractions and corresponding ctDNA in breast cancer. The main principle of the developed methodology has the potential to be extended in a large number of gene-targets and be applied in many types of cancer.
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Biomarcadores Tumorais/genética , Biópsia , Neoplasias da Mama/genética , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Biomarcadores Tumorais/sangue , Neoplasias da Mama/diagnóstico , Linhagem Celular Tumoral , Metilação de DNA/genética , DNA de Neoplasias/sangue , Feminino , Humanos , Reação em Cadeia da PolimeraseRESUMO
Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p < 0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed (p = 0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA (p = 0.033) as well as CA-19.9 (p = 0.032) levels, was noticed. The Kaplan-Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p = 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments.
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Carcinoma/genética , Metilação de DNA , DNA de Neoplasias/genética , Genes APC , Regiões Promotoras Genéticas , Neoplasias Gástricas/genética , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Antígeno CA-19-9/sangue , Antígeno Carcinoembrionário/análise , Carcinoma/sangue , Carcinoma/mortalidade , Carcinoma/secundário , Carcinoma/cirurgia , DNA de Neoplasias/sangue , Feminino , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Neoplasias Gástricas/sangue , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/cirurgiaRESUMO
BACKGROUND: Breast-cancer metastasis suppressor 1 (BRMS1) gene encodes for a predominantly nuclear protein that differentially regulates the expression of multiple genes, leading to suppression of metastasis without blocking orthotropic tumour growth. The aim of the present study was to evaluate for the first time the prognostic significance of BRMS1 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of non-small cell lung cancer (NSCLC) patients. Towards this goal, we examined the methylation status of BRMS1 promoter in NSCLC tissues, matched adjacent non-cancerous tissues and corresponding cfDNA as well as in an independent cohort of patients with advanced NSCLC and healthy individuals. METHODS: Methylation of BRMS1 promoter was examined in 57 NSCLC tumours and adjacent non-cancerous tissues, in cfDNA isolated from 48 corresponding plasma samples, in cfDNA isolated from plasma of 74 patients with advanced NSCLC and 24 healthy individuals. RESULTS: The BRMS1 promoter was highly methylated both in operable NSCLC primary tissues (59.6%) and in corresponding cfDNA (47.9%) but not in cfDNA from healthy individuals (0%), while it was also highly methylated in cfDNA from advanced NSCLC patients (63.5%). In operable NSCLC, Kaplan-Meier estimates were significantly different in favour of patients with non-methylated BRMS1 promoter in cfDNA, concerning both disease-free interval (DFI) (P=0.048) and overall survival (OS) (P=0.007). In advanced NSCLC, OS was significantly different in favour of patients with non-methylated BRMS1 promoter in their cfDNA (P=0.003). Multivariate analysis confirmed that BRMS1 promoter methylation has a statistical significant influence both on operable NSCLC patients' DFI time and OS and on advanced NSCLC patients' PFS and OS. CONCLUSIONS: Methylation of BRMS1 promoter in cfDNA isolated from plasma of NSCLC patients provides important prognostic information and merits to be further evaluated as a circulating tumour biomarker.
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Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA/genética , Proteínas de Neoplasias/sangue , Adulto , Idoso , Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Ilhas de CpG , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , Estadiamento de Neoplasias , Células Neoplásicas Circulantes , Regiões Promotoras Genéticas , Proteínas RepressorasRESUMO
Deregulation of miRNAs expression levels has been detected in many human tumor types, and recent studies have demonstrated the critical roles of miRNAs in cancer pathogenesis. Numerous recent studies have shown that miRNAs are rapidly released from tissues into the circulation in many pathological conditions. The high relative stability of miRNAs in biofluids such as plasma and serum, and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNAs as promising non-invasive new tumor biomarkers. In this study, we used liquid bead array technology (Luminex) to profile the expression of 320 mature miRNAs in a pilot testing group of 19 matched fresh frozen cancerous and non-cancerous tissues from NSCLC patients. We further validated our results by RT-qPCR for differentially expressed miRNAs in an independent group of 40 matched fresh frozen tissues, 37 plasma samples from NSCLC patients and 28 healthy donors. We found that eight miRNAs (miR-21, miR-30d, miR-451, miR-10a, miR-30e-5p and miR-126*, miR-126, miR-145) were differentially expressed by three different statistical analysis approaches. Two of them (miR-10a and miR-30e-5p) are reported here for the first time. Bead-array results were further verified in an independent group of 40 matched fresh frozen tissues by RT-qPCR. According to RT-qPCR miR-21 was significantly up-regulated (P = 0.010), miR-126* (P = 0.002), miR-30d (P = 0.012), miR-30e-5p (P < 0.001) and miR-451 (P < 0.001) were down-regulated, while miR-10a was not differentiated (P = 0.732) in NSCLC tissues. However, in NSCLC plasma samples, only three of these miRNAs (miR-21, miR-10a, and miR-30e-5p) displayed differential expression when compared to plasma of healthy donors. High expression of miR-21 was associated with DFI and OS both in NSCLC tissues (P = 0.022 and P = 0.037) and plasma (P = 0.045 and P = 0.065), respectively. Moreover, we report for the first time that low expression of miR-10a in NSCLC plasma samples was associated with worse DFI (P = 0.050) and high expression of miR-30e-5p was found to be associated with shorter OS (P = 0.048). In conclusion, circulating miR-21, miR-10a and miR-30e-5p in plasma should be further evaluated as potential non-invasive biomarkers in NSCLC.
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Carcinoma Pulmonar de Células não Pequenas/genética , Perfilação da Expressão Gênica , Neoplasias Pulmonares/genética , MicroRNAs/genética , Adulto , Idoso , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Análise por Conglomerados , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/mortalidade , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Reprodutibilidade dos Testes , Fatores de RiscoRESUMO
Blood testing for circulating tumour cells (CTC) has emerged as one of the hottest fields in cancer research. CTC detection and enumeration can serve as a 'liquid biopsy' and an early marker of response to systemic therapy, whereas their molecular characterisation has a strong potential to be translated to individualised targeted treatments and spare breast cancer (BC) patients unnecessary and ineffective therapies. Different analytical systems for CTC detection and isolation have been developed and new areas of research are directed towards developing novel assays for CTC molecular characterisation. Molecular characterisation of single CTC holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. The application of extremely powerful next-generation sequencing technologies in the area of CTC molecular characterisation in combination with reliable single CTC isolation opens new frontiers for the management of patients in the near future. This review is mainly focused on the clinical potential of the molecular characterisation of CTC in BC.
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Biomarcadores Tumorais/isolamento & purificação , Neoplasias da Mama/genética , Carcinoma/genética , Células Neoplásicas Circulantes/patologia , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biópsia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/diagnóstico , Carcinoma/metabolismo , Carcinoma/patologia , Carcinoma Intraductal não Infiltrante/diagnóstico , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Ensaios Clínicos como Assunto , Feminino , Humanos , Metástase Neoplásica , Células Neoplásicas Circulantes/química , Células Neoplásicas Circulantes/metabolismo , PrognósticoRESUMO
OBJECTIVES: To develop a multiplex PCR-coupled liquid bead array assay for the expression of VEGF splice variants. DESIGN AND METHODS: The assay was based on the combination of multiplex PCR with liquid bead array technology, and optimized and evaluated in terms of analytical sensitivity, specificity, and reproducibility using the MCF-7 cell line. Clinical performance was evaluated in 16 pairs of fresh frozen cancerous and corresponding noncancerous adjacent tissues from NSCLC patients. RESULTS: The assay is highly sensitive, reproducible and can detect specifically VEGF splice variants in clinical samples. When applied in 32 clinical samples it gave comparable results to RT-qPCR (concordance of 81%, 75%, 88% and 81% for PBGD, VEGF(121), VEGF(165), and VEGF(189) respectively). CONCLUSIONS: The developed assay can simultaneously detect three VEGF splice variants with high specificity and sensitivity and can be further used to evaluate the role of each individual VEGF splice variant in molecular therapies targeted against VEGF.
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Reação em Cadeia da Polimerase Multiplex/métodos , Fator A de Crescimento do Endotélio Vascular/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Análise por Pareamento , Microesferas , Reação em Cadeia da Polimerase Multiplex/normas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Reprodutibilidade dos Testes , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
MicroRNAs (miRNAs) are small RNAs that do not code for proteins, but function by controlling protein expression of other genes. miRNAs have been shown to control cell growth, differentiation and apoptosis. Shortly after their discovery, miRNAs have been found to be associated with cancer. Earlier reports have shown that human cancers frequently show a distorted expression profile of miRNAs. In this review, the biogenesis of miRNAs and potential mechanisms of their dysregulation and involvement in cancer pathogenesis are discussed. The current literature on potential applications of miRNAs in the field of clinical oncology from diagnostic to prognostic and predictive applications at the tissue, and more recently, serum levels, is reviewed. The potential therapeutic applications of miRNAs and RNAi in the field of cancer are summarised. Finally, some of the potential challenges that face the transition of miRNAs from a research setting into a clinical application are highlighted, with a future prospective of the incorporation of miRNAs in cancer patient management.
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MicroRNAs/genética , Neoplasias/genética , RNA Neoplásico/genética , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Humanos , Masculino , Neoplasias/diagnóstico , Neoplasias/terapiaRESUMO
BACKGROUND: We evaluated the prognostic significance of KLK10 exon 3 methylation in patients with early-stage breast cancer since it has been shown to have a significant impact on biological characteristics of breast tumors. MATERIALS AND METHODS: Using methylation-specific PCR, we evaluated the specificity of KLK10 methylation in 10 breast tumors and matching normal tissues, 10 breast fibroadenomas, 11 normal breast tissues and in a testing group of 35 patients. The prognostic significance of KLK10 methylation was validated in an independent cohort of 93 patients. RESULTS: KLK10 was not methylated in normal breast tissues and fibroadenomas while it was in 5 of 10 breast tumors and in 1 of 10 matching normal tissues. In the testing group of 35 patients, KLK10 methylation was detected in 70.0% of patients who relapsed (P = 0.001) and in 77.8% of patients who died (P = 0.025). In the independent cohort, 53 of 93 (57.0%) patients were found positive for KLK10 methylation. During the follow-up period, 24 of 93 (25.8%) patients relapsed and 19 of 93 (20.4%) died. Disease-free interval (DFI) and overall survival (OS) were significantly associated with KLK10 methylation (P = 0.0025 and P = 0.003). Multivariate analysis revealed that KLK10 methylation was an independent prognostic factor for DFI and OS. CONCLUSION: KLK10 exon 3 methylation provides important prognostic information in early breast cancer patients.
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Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Metilação de DNA , Calicreínas/genética , Biomarcadores Tumorais/metabolismo , Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Calicreínas/metabolismo , Prognóstico , Análise de SobrevidaRESUMO
PURPOSE: To investigate and compare the diagnostic value of the detection of cytokeratin 19 (CK-19), carcinoembryonic antigen (CEA) and maspin mRNA by nested RT-PCR in the peripheral blood of women with breast cancer. MATERIALS AND METHODS: The tumor cell lines MCF-7 and LOVO were used in an experimental tumor cell dilution model to determine the sensitivity of the nested RT-PCR for the 3 detection markers. RT-PCR analysis was performed in the peripheral blood of 54 healthy female blood donors, 28 patients with hematological malignancies, 31 with metastatic colorectal cancer, 75 with operable and 50 with metastatic breast cancer before receiving any cytotoxic chemotherapy, as well as in the bone marrow aspirates of 61 breast cancer patients. RESULTS: Nested RT-PCR for CK-19 mRNA presented the highest sensitivity by detecting 1 tumor cell amongst 10(6) PBMC in 4 out of 5 experiments. CK-19 mRNA was detected in the peripheral blood of 3.7% of female blood donors, 14.3% of hematological malignancies, 32% of operable and 42% of metastatic breast cancer patients. CEA mRNA was undetectable in the blood of female blood donors but was detected in blood samples of 3.5% of hematological malignancies, 19.3% of colorectal cancer and 10% of breast cancer patients. Maspin mRNA was undetectable in the blood of female blood donors, patients with hematological malignancies and colorectal cancer but was detected in 9.3% of operable and 14% of metastatic breast cancer patients. Maspin mRNA positivity correlated with tumor size in patients with early stage breast cancer (p = 0.057). The detection rates of CK-19 and maspin mRNA in bone marrow aspirates were 33% and 11% for operable and 62% and 9% for metastatic breast cancer, respectively. During follow-up, 27.4% of blood samples were positive for CK-19 mRNA versus 10.7% for maspin mRNA in patients with operable breast cancer with a concordance rate of only 12.7% for positives and 86% for negatives. CONCLUSION: RT-PCR positivity for CK-19 mRNA is the most sensitive detection marker for occult tumor cells in operable and metastatic breast cancer, although nested RT-PCR for maspin mRNA appears to be more specific.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Antígeno Carcinoembrionário/sangue , Queratinas/sangue , Células Neoplásicas Circulantes/metabolismo , Serpinas/sangue , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Medula Óssea/patologia , Neoplasias da Mama/patologia , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/genética , Feminino , Genes Supressores de Tumor , Humanos , Queratinas/biossíntese , Queratinas/genética , Metástase Neoplásica , Estadiamento de Neoplasias , Células Neoplásicas Circulantes/patologia , Biossíntese de Proteínas , Proteínas/genética , RNA Mensageiro/sangue , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Serpinas/biossíntese , Serpinas/genética , Células Tumorais CultivadasRESUMO
BACKGROUND: The purpose of this study was to evaluate the prognostic significance of the molecular detection of cytokeratin 19 (CK-19) mRNA-positive cells in the peripheral blood of women with operable breast cancer after the completion of adjuvant chemotherapy. PATIENTS AND METHODS: Blood from 161 patients with stage I and II breast cancer, obtained after the completion of adjuvant chemotherapy, was tested by nested RT-PCR for CK-19 mRNA detection. Using univariate and multivariate analyses possible interactions with other prognostic factors and association of CK-19 mRNA detection with risk of relapse, disease-free interval (DFI) and overall survival were investigated. RESULTS: After completion of adjuvant chemotherapy, 27.3% of patients had peripheral blood CK-19 mRNA-positive cells; there was no association of this finding with any other prognostic factors or the type of chemotherapy regimen used. For patients with less than four involved axillary lymph nodes the risk of relapse was 3.81 [95% confidence interval (CI) 1.06-13.71] times higher, and the DFI was significantly reduced (P = 0.028) if CK-19 mRNA-positive cells were detectable in the blood after the completion of adjuvant chemotherapy. In contrast, for patients with four or more involved lymph nodes, the presence of CK-19 mRNA-positive cells after adjuvant chemotherapy did not significantly affect the risk of relapse or DFI. Furthermore, the risk of relapse was higher (hazards ratio 3.70; 95% CI 1.09-13.89) and the DFI was reduced (P = 0.022) for patients with detectable CK-19 mRNA-positive cells following adjuvant cyclophosphamide, methotrexate and 5-fluorouracil (CMF) as compared with epirubicin, cyclophosphamide and 5-fluorouracil (FEC) or sequential taxotere-epirubicin and cyclophosphamide (T/EC) chemotherapy. CONCLUSIONS: The detection of CK-19 mRNA-positive cells in the peripheral blood after adjuvant chemotherapy may be of clinical relevance for patients with early breast cancer and less than four involved axillary lymph nodes.
Assuntos
Neoplasias da Mama/sangue , Queratinas/genética , Células Neoplásicas Circulantes , RNA Mensageiro/sangue , RNA Neoplásico/sangue , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Quimioterapia Adjuvante , Primers do DNA/química , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Taxa de SobrevidaRESUMO
BRCA1 and BRCA2 genes were screened for loss-of-function mutations in a series of 85 patients having at least one first- or second-degree relative affected by breast and/or ovarian cancer. All BRCA1 exons and BRCA2 exons 10 and 11 were screened with a combination of methods including SSCP, PTT and direct sequencing. We have found disease-associated mutations in 14 families (16.5%), eleven in BRCA1 and three in BRCA2. The known founder mutation 5382insC of BRCA1 was identified in seven unrelated families. The other mutations identified include the non-sense R1751X, the splice junction variant 5586G>A of BRCA1 and three frameshifts, 2024del5, 3034del4, and 6631del5, of BRCA2. Nine out of these 14 families had a family history of three or more breast/ovarian cancer cases. A large number of polymorphic or unclassified variants is also reported. Combined with our previously published data 5382insC was found in nine out of 20 families (45%), suggesting that this mutation may represent a common founder mutation in the Greek population.