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BACKGROUND: The detection of circulating tumour cells (CTC) is prognostic for disease recurrence in early breast cancer (BC). This study aims to investigate whether this prognostic effect persists or varies over time. METHODS: The study population consisted of prospectively included stage I-III BC patients. The presence of CK19 mRNA-positive CTC in the peripheral blood was evaluated before and after adjuvant chemotherapy, using a real-time RT-PCR assay. Longitudinal samples were collected for a subset of patients. RESULTS: Baseline CTC data were available from 1220 patients, while 1132 had both pre- and post-therapy data. After a median follow-up of 134.1 months, CTC positivity at baseline was associated with shorter overall survival (OS; HRadj = 1.72, 95% CI 1.34-2.21, p < 0.001). For disease-free survival, an interaction with time (p = 0.045) was observed. CTC positivity predicted early (within 5 years; HRadj = 1.76, 95% CI 1.33-2.32, p < 0.001) but not late recurrence (HRadj = 1.10, 95% CI 0.79-1.53, p = 0.577). Following adjuvant chemotherapy, more patients converted from CTC-positive to CTC-negative than vice versa (p < 0.001). Ten-year OS was 68.6% for + /+ and 86.7% for -/- group (p < 0.001). CTC status at follow-up predicted disease recurrence. CONCLUSION: CTC detection pre- and post-adjuvant chemotherapy is prognostic for early relapse, supporting investigations for novel adjuvant therapeutic approaches.
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Neoplasias da Mama , Células Neoplásicas Circulantes , Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Quimioterapia Adjuvante , Feminino , Humanos , Recidiva Local de Neoplasia/patologia , Células Neoplásicas Circulantes/patologia , PrognósticoRESUMO
BACKGROUND: We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). METHODS: Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearchTM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. RESULTS: Using the CellSearchTM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearchTM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearchTM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearchTM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. CONCLUSION: A significant concordance was observed between the CellSearchTM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients.
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Neoplasias da Mama/diagnóstico , Microscopia de Fluorescência , Células Neoplásicas Circulantes/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Diagnóstico Precoce , Feminino , Humanos , Estimativa de Kaplan-Meier , Queratina-18/imunologia , Queratina-18/metabolismo , Queratina-19/genética , Queratina-19/imunologia , Queratina-19/metabolismo , Queratina-8/imunologia , Queratina-8/metabolismo , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , RNA Mensageiro/metabolismoRESUMO
INTRODUCTION: Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement. METHODS: CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test. RESULTS: For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90). CONCLUSIONS: The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required.
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Neoplasias da Mama/patologia , Contagem de Células/instrumentação , Oncologia/instrumentação , Células Neoplásicas Circulantes/patologia , Neoplasias da Mama/sangue , Neoplasias da Mama/metabolismo , Contagem de Células/normas , Feminino , Humanos , Cooperação Internacional , Laboratórios/normas , Oncologia/normas , Metástase Neoplásica , Células Neoplásicas Circulantes/metabolismo , Receptor ErbB-2/metabolismo , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The detection of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTC) before and/or after adjuvant chemotherapy in patients with operable breast cancer is associated with poor clinical outcome. Reliable prognostic markers for late disease relapse are not available. In this study we investigated the value of CTC detection during the first five years of follow-up in predicting late disease relapse. METHODS: Blood was analyzed from 312 women with operable breast cancer who had not experienced disease relapse during the first two years of follow-up. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) for CK-19 mRNA was used to detect CTC three months after the completion of adjuvant chemotherapy and every six months thereafter for a follow-up period of five years. RESULTS: Eighty patients (25.6% of the study population) remained CTC free throughout the five-year period. A change in CTC status was observed in 133 patients (42.6%); 64 patients (20.5%) with initially CK-19 mRNA-positive CTC during the first 24 months turned CTC-negative afterwards while 69 (22.1%) who were initially CTC-negative became CTC-positive. Ninety-nine patients (31.7%) remained persistently CK-19 mRNA-positive. After a median follow-up period of 107 months (range: 38 to 161 months), the persistently CTC-positive patients with either hormonal receptor positive or negative tumors, had a higher risk of late-disease relapse compared to the persistently CTC-negative patients (36.4% versus 11.2%, P <0.001). Multivariate analysis revealed that persistently CTC-positive patients also had a shorter disease-free (P = 0.001) and overall survival (P = 0.001). CONCLUSIONS: Persistent detection of CK-19 mRNA-positive CTC during the first five years of follow-up is associated with an increased risk of late relapse and death in patients with operable breast cancer and indicates the presence of chemo-and hormonotherapy-resistant residual disease. This prognostic evaluation may be useful when deciding on subsequent adjuvant systemic therapy.
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Neoplasias da Mama/patologia , Queratina-19/genética , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Feminino , Seguimentos , Humanos , Queratina-19/sangue , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Prognóstico , RNA Mensageiro/sangue , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: Detection of CTCs represents a poor prognostic factor in patients with early and metastatic breast cancer (mBC) and treatment with everolimus-exemestane (E/E) is an established effective treatment in hormone receptor-positive/HER2-negative mBC patients. The effect of E/E on CTCs in mBC patients was prospectively investigated. METHODS: CTCs from 50 pre-treated patients with mBC receiving E/E were analyzed using the CellSearch (CS) platform and triple immunofluorescence (IF) staining for cytokeratin, M30 and Ki67 expression to assess their proliferative and apoptotic status. RESULTS: CTCs (by CS) were detected in 64% of patients before treatment and E/E administration resulted in their decreased prevalence [(n = 18; 36%, p = 0.004) and (n = 7; 19.4%, p = 0.019) post-1st and post-3rd treatment cycle, respectively] whereas it was significantly increased at disease progression (PD: 61%) compared to post-1st and post-3rd cycle (p = 0.049 and p = 0.021, respectively). Ki67-positive CTCs were detected in 60%, 60%, 17% and 50% of patients before treatment, post-1st, post-3rd cycle and at PD, respectively, while the opposite was observed for M30-positive CTCs (0% at baseline, 10% after the 1st cycle, 50% after the 3rd cycle and 0% at PD). The detection of even ≥ 1 CTC/5 ml after one cycle was associated with decreased PFS (3.3 vs 9.0 months, p = 0.025) whereas the detection of even ≥ 2 CTCs at PD was associated with decreased OS (32.4 vs 19.5 months; p = 0.009). CONCLUSIONS: The combination of E/E resulted in early elimination of proliferating CTCs in mBC patients and this effect was associated with a favorable clinical outcome.
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Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Células Neoplásicas Circulantes/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Androstadienos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Everolimo/administração & dosagem , Feminino , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Estudos Prospectivos , Receptor ErbB-2/metabolismo , Terapia de Salvação , Resultado do TratamentoRESUMO
To evaluate whether HER2 mRNA could be used as a marker of circulating tumor cells (CTCs) in women with operable breast cancer. A nested RT-PCR assay was developed and used for the detection of HER2 mRNA-positive CTCs. Blood from 216 women with early breast cancer obtained before adjuvant treatment was tested for HER2 mRNA-positive cells to assess their prognostic value. Nested RT-PCR for HER2 mRNA showed high sensitivity whereas no HER2 mRNA-positive cells could be identified in the blood of healthy donors. HER2 mRNA-positive CTCs were detected in 53 (24.5%) of 216 patients and HER2 mRNA detection was associated with reduced disease-free survival (DFS; P < 0.0001) and overall survival (OS; P = 0.004). In multivariate analysis, detection of HER2 mRNA-positive CTCs emerged as independent prognostic factor for DFS (P = 0.0001) and OS (P = 0.003). HER2 mRNA could be a valuable prognostic marker for the detection of CTCs in early breast cancer patients.
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Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Células Neoplásicas Circulantes/metabolismo , RNA Mensageiro/análise , Receptor ErbB-2/genética , Adulto , Idoso , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Feminino , Imunofluorescência , Humanos , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Microscopia Confocal , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Células Neoplásicas Circulantes/patologia , Prognóstico , RNA Mensageiro/sangue , Receptor ErbB-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
PURPOSE: To investigate the prognostic value of the molecular detection of circulating tumor cells (CTCs) using three markers [cytokeratin 19 (CK19), mammaglobin A (MGB1), and HER2] in early breast cancer. EXPERIMENTAL DESIGN: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected using real-time (CK19) and nested (MGB1 and HER2) reverse transcription-PCR in the peripheral blood of 175 women with stage I to III breast cancer before the initiation of adjuvant chemotherapy. The detection of CTCs was correlated with clinical outcome. In 10 patients, immunofluorescence staining experiments were done to investigate the coexpression of cytokeratin, MGB1, and HER2 in CTCs. RESULTS: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected in 41.1%, 8%, and 28.6% of the 175 patients, respectively. Patients had one of the following molecular profiles: CK19mRNA+/MGB1mRNA+/HER2mRNA+ (n = 8), CK19mRNA+/MGB1mRNA+/HER2mRNA- (n = 1), CK19mRNA+/MGB1mRNA-/HER2mRNA+ (n = 42), CK19mRNA+/MGB1mRNA-/HER2mRNA- (n = 21), CK19mRNA-/MGB1mRNA+/HER2mRNA- (n = 5), and CK19mRNA-/MGB1mRNA-/HER2mRNA- (n = 98). Double-immunofluorescence experiments confirmed the following CTC phenotypes: CK+/MGB1+, CK+/MGB1-, CK-/MGB1+, CK+/HER2+, CK+/HER2-, MGB1+/HER2-, and MGB1+/HER2+. In univariate analysis, the detection of CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells was associated with shorter disease-free survival (DFS; P < 0.001, P = 0.001, and P < 0.001, respectively), whereas the detection of CK19mRNA+ and MGB1mRNA+ cells was associated with worse overall survival (P = 0.044 and 0.034, respectively). In multivariate analysis, estrogen receptor-negative tumors and the detection of CK19mRNA+ and MGB1mRNA+ cells were independently associated with worse DFS. CONCLUSION: The detection of peripheral blood CK19mRNA+ and MGB1mRNA+ cells before adjuvant chemotherapy predicts poor DFS in women with early breast cancer.
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Neoplasias da Mama/diagnóstico , Queratina-19/análise , Proteínas de Neoplasias/análise , Células Neoplásicas Circulantes , Receptor ErbB-2/análise , Uteroglobina/análise , Adulto , Idoso , Biomarcadores Tumorais/análise , Neoplasias da Mama/sangue , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Progressão da Doença , Feminino , Humanos , Queratina-19/sangue , Mamoglobina A , Pessoa de Meia-Idade , Proteínas de Neoplasias/sangue , Receptor ErbB-2/sangue , Uteroglobina/sangueRESUMO
Sustainable management of water resources calls for integration of ideas and approaches and revolves around assessment of causal-effect relationships as tools towards defining informed mitigation options and planning. The current paper presents a new holistic approach developed within the Globaqua Coordination Project that combines indicator-based well-established and tested concepts towards developing informed Programmes of Measures and River basin management plans: a. The DPSIR framework that has been engaged as central instrument to address the Water Framework Directive requirements and the concepts embedded in the Integrated Water Resource Management; b. The Ecosystem Services Approach emphasizing on the links between ecosystem services, changes in ecosystems and human well-being, c. Scenario assessment for valuation of future conditions to ensure the sustainability in the use of water resources. The implementation of the new combined framework in two river basins, Ebro in Spain and Evrotas in Greece, stressed the need for revised options targeting elimination of water pollution, measures to ensure water supply that covers the demand even under conditions of climate change and increased water stress and the need for improved valuation of environmental and resource use costs.
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The aim of the study was to investigate the effect of 2nd-line pazopanib on the different CTCs subpopulations in SCLC patients and evaluate the clinical relevance of their changes. Different CTCs subpopulations were evaluated before pazopanib initiation (n = 56 patients), after one-cycle (n = 35) and on disease progression (n = 45) by CellSearch and double immunofluorescence using anti-CKs and anti-Ki67, anti-M30 or anti-Vimentin antibodies. Before treatment, CTCs were detected in 50% of patients by CellSearch whereas 53.4%, 15.5% and 74.1% patients had CK+/Ki67+, CK+/M30+ and CK+/Vim+ CTCs, respectively. One pazopanib cycle significantly decreased the number of CTCs as detected by CellSearch (p = 0.043) as well as the number of CK+/Ki67+ (p < 0.001), CK+/M30+ (p = 0.015) and CK+/Vim+ (p < 0.001) cells. On disease progression, both the incidence and CTC numbers were significantly increased (CellSearch, p = 0.027; CK+/Ki67+, p < 0.001; CK+/M30+, p = 0.001 and CK+/Vim+, p < 0.001). In multivariate analysis, the detection of CK+/Vim+ CTCs after one treatment cycle (HR: 7.9, 95% CI: 2.9-21.8; p < 0.001) and CTCs number on disease progression, as assessed by CellSearch, (HR: 2.0, 95% CI: 1.0-6.0; p = 0.005) were emerged as independent factors associated with decreased OS. In conclusion, pazopanib can eliminate different CTC subpopulations in patients with relapsed SCLC. The analysis of CTCs could be used as a dynamic biomarker of treatment efficacy.
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Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/patologia , Pirimidinas/uso terapêutico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/patologia , Sulfonamidas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Contagem de Células , Linhagem Celular Tumoral , Feminino , Imunofluorescência , Células HeLa , Humanos , Indazóis , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
PURPOSE: The aim of this study was to evaluate the clinical relevance of the simultaneous detection of cytokeratin (CK)-19 messenger RNA (mRNA)- and HER2 mRNA-positive cells in peripheral blood of women with early-stage breast cancer. PATIENTS AND METHODS: CK-19 mRNA- and HER2 mRNA-positive cells were detected using a real-time and a nested reverse-transcriptase polymerase chain reaction assay, respectively, in a cohort of 185 women with early-stage breast cancer before the initiation of any adjuvant systemic treatment. Detection of CK-19 mRNA- and HER2 mRNA-positive cells in the peripheral blood was correlated with clinical outcome. RESULTS: Overall, 63 of the 185 patients (34%) had detectable CK-19 mRNA-positive cells, and 33 (52.3%) also had detectable HER2 mRNA-positive cells. Patients with CK-19/HER2 mRNA-negative cells showed a trend toward longer disease-free survival (DFS) compared with patients with CK-19 mRNA-positive/HER2 mRNA-negative cells (P = .054) and had longer DFS than patients with CK-19/HER2 mRNA-positive cells (P < .001). Similarly, overall survival (OS) was higher in patients with CK-19/HER2 mRNA-negative cells compared with patients with CK-19 mRNA-positive/HER2 mRNA-negative cells (P = .039) or CK-19/HER2 mRNA-positive cells (P < .001). Patients with CK-19/HER2 mRNA-positive cells had shorter DFS but not OS compared with patients with CK-19 mRNA-positive/HER2 mRNA-negative cells. In multivariate analysis, the simultaneous detection of CK-19 mRNA- and HER2 mRNA-positive cells was independently associated with early relapse. CONCLUSION: The simultaneous detection of CK-19 mRNA- and HER2 mRNA-positive cells in peripheral blood predicts poor clinical outcome for women with early-stage breast cancer.
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Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Queratina-19/sangue , RNA Mensageiro/sangue , Receptor ErbB-2/sangue , Adulto , Idoso , Neoplasias da Mama/patologia , Estudos de Coortes , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Células Neoplásicas Circulantes/metabolismo , Prognóstico , Receptores de Estrogênio/sangue , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The aim of the study was to evaluate the phenotypic CTCs heterogeneity (TTF-1+ and/or CD56+) in SCLC patients and correlate it with the CellSearch. Peripheral blood was obtained from 108 consecutive patients. CTCs were detected by CellSearch and double-immunofluorescence using anti-CD45, anti-TTF-1 and anti-CD56 antibodies. Before chemotherapy TTF-1+/CD45-, CD56+/CD45- and TTF-1+/CD56+ CTCs were detected in 66(61.1%), 55(50.9%) and 46(42.6%) patients, respectively; 60.2% of patients were CellSearch+. Among the 22 patients with 0 CTCs/7.5 ml on CellSearch, TTF-1+/CD45-, CD56+/CD45- and TTF-1+/CD56+ CTCs were detected in 8(36.4%), 6(27.3) and 6(27.3%) patients, respectively; no CK+/EpCAM+ or TTF1+/EpCAM+ CTCs were detected in these patients. One-chemotherapy cycle decreased both the number of positive patients (p < 0.001) and their CTC number (p < 0.001), irrespectively of their phenotype and the detection method. The incidence and number of the different CTC subpopulations on PD, was significantly increased at their baseline levels. Multivariate analysis revealed that the increased number of CTCs at baseline and on PD were significantly associated with decreased PFS (p = 0.048) and OS (p = 0.041), respectively. There is an important CTC heterogeneity in such patients according to the expression of TTF-1 and CD56 which could detect EpCAM- CTC subpopulations and, thus, undetectable by CellSearch. These CTC subpopulations are dynamically correlated with treatment efficacy and disease-progression.
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Antígeno CD56/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Pulmonares/diagnóstico , Células Neoplásicas Circulantes/metabolismo , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Fatores de Transcrição/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/mortalidadeRESUMO
INTRODUCTION: To evaluate the incidence of central nervous system (CNS) involvement in patients with breast cancer treated with a taxane-based chemotherapy regimen and to determine predictive factors for CNS relapse. METHODS: The medical files of patients with early breast cancer (n = 253) or advanced stage breast cancer (n = 239) as well of those with other solid tumors (n = 336) treated with or without a taxane-based chemotherapy regimen during a 42-month period were reviewed. HER2/neu overexpression was identified by immunohistochemistry, whereas cytokeratin 19 (CK-19) mRNA-positive circulating tumor cells (CTCs) in the peripheral blood were identified by real-time PCR. RESULTS: The incidence of CNS relapse was similar in patients suffering from breast cancer or other solid tumors (10.4% and 11.4%, respectively; P = 0.517). The incidence of CNS relapse was significantly higher in breast cancer patients with advanced disease (P = 0.041), visceral disease and bone disease (P = 0.036), in those who were treated with a taxane-containing regimen (P = 0.024), in those with HER2/neu-overexpressing tumors (P = 0.022) and, finally, in those with detectable CK-19 mRNA-positive CTCs (P = 0.008). Multivariate analysis revealed that the stage of disease (odds ratio, 0.23; 95% confidence interval, 0.007-0.23; P = 0.0001), the HER2/neu status (odds ratio, 29.4; 95% confidence interval, 7.51-101.21; P = 0.0001) and the presence of CK-19 mRNA-positive CTCs (odds ratio, 8.31; 95% confidence interval, 3.97-12.84; P = 0.001) were independent predictive factors for CNS relapse. CONCLUSION: CNS relapses are common among breast cancer patients treated with a taxane-based chemotherapy regimen, patients with HER2/neu-positive tumor and patients with CK-19 mRNA-positive CTCs.
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Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Neoplasias do Sistema Nervoso Central/fisiopatologia , Células Neoplásicas Circulantes , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias do Sistema Nervoso Central/secundário , Feminino , Humanos , Queratina-19 , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Valor Preditivo dos Testes , RNA Mensageiro , Receptor ErbB-2/biossíntese , Taxoides/uso terapêuticoRESUMO
PURPOSE: The purpose of this research was to develop a quantitative real-time reverse transcription-PCR (RT-PCR) for CK19-mRNA and evaluate its clinical potential for the molecular detection of occult carcinoma cells in the peripheral blood of breast cancer patients. EXPERIMENTAL DESIGN: The method is based on real-time monitoring during PCR of fluorescently labeled specific hybridization probes for CK19-mRNA. The breast cancer cell line MCF-7 was used for the development and analytical evaluation of the assay. We analyzed blood samples from 89 healthy blood donors, 77 patients with early breast cancer (stage I-II) postoperatively, and 47 patients with previously untreated metastatic disease (stage IV) before and after chemotherapy. All of the samples were also analyzed by nested RT-PCR. RESULTS: The method is highly sensitive and specific, because only 2 of 89 (2.2%) of the healthy control subjects had detectable CK19-mRNA+ cells. In 77 patients with early breast cancer, CK19-mRNA+ cells were detected in 24 (31.2%) before and 5 (6.5%) after adjuvant chemotherapy, and their levels differed significantly (P < 0.001, Wilcoxon test). In 47 patients with verified metastases 19 (40.4%) and 20 (42.6%) were found positive before and after chemotherapy, and no significant difference in CK19-mRNA+ cell levels was observed (P = 0.96, Wilcoxon test). Results obtained by the proposed real-time RT-PCR method correlated well with those obtained for the same samples by nested RT-PCR [concordance in 312 of 337 (92.6%); P = 0.69, McNemar test]. CONCLUSIONS: The developed method is highly sensitive and specific, and can be used for high-throughput continuous monitoring and quantification of circulating epithelial cells in the peripheral blood of breast cancer patients.
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Neoplasias da Mama/sangue , Queratinas/sangue , Células Neoplásicas Circulantes , RNA Mensageiro/análise , Adolescente , Adulto , Idoso , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Primers do DNA , Feminino , Humanos , Queratinas/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Neoplásico , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
PURPOSE: The detection of disseminated occult breast cancer cells in peripheral blood and bone marrow is associated with poor prognosis. Since a high proportion of these cells express the HER-2 receptor, we evaluated the effectiveness of the anti-HER-2 antibody trastuzumab (Herceptin) administration to eliminate them. EXPERIMENTAL DESIGN: Thirty patients with prior chemotherapy exposure were recruited to the study on the basis of having detectable cytokeratin-19 (CK-19) mRNA transcripts by nested reverse transcription (RT)-PCR in the peripheral blood and/or bone marrow. There were 13 patients with stage I, II, or III breast cancer and 17 with stage IV disease. They were treated in two cohorts with either 4 to 8 weekly infusions of trastuzumab at 2 mg/kg (4 mg/kg loading dose; 20 patients) or 2 to 3 infusions every 3 weeks at 6 mg/kg (8 mg/kg loading dose; 10 patients). All of the patients' samples were also analyzed for HER-2 by nested RT-PCR, but detectable HER-2 messenger RNA (mRNA) was not required for inclusion in the study. After trastuzumab infusions, patients were closely monitored by nested RT-PCR and real-time RT-PCR for the detection of CK-19 mRNA-positive cells. RESULTS: Before trastuzumab infusions, CK-19 mRNA-positive cells were detected in the peripheral blood (n = 10), bone marrow (n = 14), or both (n = 6). In 25 of 30 patients (83%), HER-2 mRNA expression was detected by nested RT-PCR in the pretrastuzumab CK-19-positive sample. After trastuzumab infusions, overall, 28 of 30 (93%) patients became CK-19 mRNA negative by nested RT-PCR and 20 of 30 (67%) by real-time RT-PCR. After a median follow-up of 6 months (range 2 to 22+), the median duration of CK-19 mRNA negativity by nested RT-PCR was 9, 12, and 6 months for stage I/II, III, and IV disease, respectively. CONCLUSIONS: Therapy-resistant CK-19 mRNA-positive cells in the peripheral blood and bone marrow can be effectively targeted by trastuzumab administration. Further studies are needed to evaluate the prognostic significance of the disappearance of these cells.
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Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/administração & dosagem , Medula Óssea/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Queratinas/genética , Células Neoplásicas Circulantes/efeitos dos fármacos , RNA Mensageiro/metabolismo , Adulto , Idoso , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/metabolismo , Quimioterapia Adjuvante , Estudos de Coortes , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , RNA Neoplásico/sangue , Receptor ErbB-2/genética , TrastuzumabRESUMO
BACKGROUND: Previous studies showed that molecular detection of CK-19 mRNA in peripheral blood and the mitotic index of primary tumors have prognostic value in early breast cancer. The aim of this study was to assess the association between these variables. PATIENTS AND METHODS: The primary tumors of 223 operable breast cancer patients (92 premenopausal and 131 postmenopausal) were evaluated for the MAI classified as either ≤ 5 per 10, 6 to 10 per 10 and > 10 per 10 or < 10 per 10 and ≥ 10 per 10 mitoses per high power field using a standardized protocol previously reported. Peripheral blood was also collected before and after the end of adjuvant chemotherapy for detection of CK-19 mRNA-positive cells using reverse transcription polymerase chain reaction previously described. RESULTS: After a median follow-up of 118 months, 75 patients (33.6%) experienced disease relapse and 56 (25.1%) died of breast cancer. MAI was strongly associated with disease-free survival (DFS) and overall survival (OS) (P < .001 for DFS and OS together). Detecting CK-19 mRNA-positive cells in the peripheral blood before but not after adjuvant chemotherapy was associated with marginally worse DFS (P = .055) and OS (P = .059). Cox regression analysis revealed that MAI and CK-19 mRNA-positive cell detection before adjuvant chemotherapy were independent variables associated with decreased DFS (P < .001 and P = .038, respectively) and OS (P < .001 and P = .029, respectively). There was no significant interaction between MAI and detection of CK-19 mRNA-positive cells. CONCLUSION: MAI of the primary tumor and detection of CK-19 mRNA-positive cells in the blood before adjuvant chemotherapy in early breast cancer patients are 2 independent prognostic factors associated with clinical outcome.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Carcinoma Lobular/patologia , Queratina-19/genética , Índice Mitótico , Células Neoplásicas Circulantes/patologia , RNA Mensageiro/genética , Adulto , Idoso , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/genética , Carcinoma Lobular/mortalidade , Quimioterapia Adjuvante , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de SobrevidaRESUMO
INTRODUCTION: Circulating tumor cells (CTCs) could represent a non-invasive source of cancer cells used for longitudinal monitoring of the tumoral mutation status throughout the course of the disease. The aims of the present study were to investigate the detection of KRAS mutations in CTCs from patients with metastatic colorectal cancer (mCRC) and to compare their mutation status during treatment or disease progression with that of the corresponding primary tumors. MATERIALS AND METHODS: Identification of the seven most common KRAS mutations on codons 12 and 13 was performed by Peptide Nucleic Acid (PNA)-based qPCR method. The sensitivity of the assay was determined after isolation of KRAS mutant cancer cells spiked into healthy donors' blood, using the CellSearch Epithelial Cell kit. Consistent detection of KRAS mutations was achieved in samples containing at least 10 tumor cells/7.5 ml of blood. RESULTS: The clinical utility of the assay was assessed in 48 blood samples drawn from 31 patients with mCRC. All patients had PIK3CA and BRAF wild type primary tumors and 14 KRAS mutant tumors. CTCs were detected in 65% of specimens obtained from 74% of patients. KRAS mutation analysis in CTC-enriched specimens showed that 45% and 16.7% of patients with mutant and wild type primary tumors, respectively, had detectable mutations in their CTCs. Assessing KRAS mutations in serial blood samples revealed that individual patient's CTCs exhibited different mutational status of KRAS during treatment. CONCLUSIONS: The current findings support the rationale for using the CTCs as a dynamic source of tumor cells which, by re-evaluating their KRAS mutation status, could predict, perhaps more accurately, the response of mCRC patients to targeted therapy.
Assuntos
Adenocarcinoma/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais/genética , Células Neoplásicas Circulantes , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bevacizumab , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Feminino , Fluoruracila/administração & dosagem , Genótipo , Células HCT116 , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Panitumumabe , Proteínas Proto-Oncogênicas p21(ras)RESUMO
PURPOSE: To evaluate the clinical relevance of circulating CEACAM5mRNA-positive cells in patients with operable colorectal cancer (CRC). METHODS: Peripheral blood was obtained from 265 patients with operable CRC before the initiation of adjuvant systemic therapy from 96 normal donors and RNA prepared from the Lovo and ARH-77 CRC and leukemic cell lines, respectively, was used as positive and negative controls. The detection of CEACAM5mRNA-positive cells was done using a real-time PCR assay. The association with known prognostic factors and the effect of CEACAM5mRNA-positive cells on patients' prognosis was investigated. RESULTS: The analytical detection limit of the method was found to correspond to 0.7 Lovo cell equivalence/5 µg RNA, with a sensitivity of 1 tumor cell/10(5) normal cells and a specificity of 97%. Ninety-eight (37%) patients had detectable circulating CEACAM5mRNA-positive cells. Detection of CEACAM5mRNA-positive cells was significantly associated with higher relapse rate (P < 0.001), decreased disease-free survival (DFS; P < 0.001), higher death rate (P = 0.017), and decreased median overall survival (P = 0.025). Multivariate analysis revealed that the detection of circulating CEACAM5mRNA-positive cells was an independent prognostic factor for decreased DFS [HR = 3.4; 95% CI: 2.0-5.9; P < 0.001]. CONCLUSIONS: Detection of peripheral blood CEACAM5mRNA-positive cells is an adverse prognostic factor correlated with poor clinical outcome in patients with operable CRC.
Assuntos
Antígeno Carcinoembrionário/genética , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Idoso , Antígeno Carcinoembrionário/sangue , Estudos de Casos e Controles , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/cirurgia , Feminino , Seguimentos , Proteínas Ligadas por GPI/sangue , Proteínas Ligadas por GPI/genética , Humanos , Masculino , Análise Multivariada , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/sangue , Recidiva , Análise de Sobrevida , Resultado do TratamentoRESUMO
The effect of hydroxyethyl starches (HES) on blood coagulation is affected by their molecular weight, their molar substitution and the C2/C6 ratio. The solutions of 6% HES 130/0.4 and 6% HES 130/0.42 have similar molecular weight and molar substitution but different C2/C6 ratio and plant origin. In the present study, the comparative effect of 6% HES 130/0.4 versus 6% HES 130/0.42 on blood coagulation was investigated in vitro. Thirty milliliter of blood was obtained from 10 healthy volunteers and was diluted by 10, 30 and 50% using either 6% HES 130/0.4 or HES 130/0.42, respectively. Blood coagulation was assessed using thrombelastography measurements (clotting time, clot formation time, maximal clot firmness and alpha-angle). The assessment of platelet function was performed with whole blood aggregometry after adding thrombin-receptor-activating protein. No differences were noted between respective dilutions of the two HES. Both colloids produced significant reductions below the reference values range in clotting time at 10, 30 and 50% dilutions. The 50% dilution of both colloids resulted in significant reduction of maximal clot firmness, alpha-angle and platelet aggregation. The present study showed that the corn-derived 6% HES 130/0.4 and the potato-derived 6% HES 130/0.42 have the same effect on blood coagulation in vitro.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Derivados de Hidroxietil Amido/química , Derivados de Hidroxietil Amido/farmacologia , Substitutos do Plasma/química , Substitutos do Plasma/farmacologia , Adulto , Feminino , Humanos , Derivados de Hidroxietil Amido/administração & dosagem , Masculino , Substitutos do Plasma/administração & dosagem , Valores de Referência , Tempo de Coagulação do Sangue TotalRESUMO
PURPOSE: To evaluate the prognostic significance of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTCs) in peripheral blood of women with early-stage breast cancer after the completion of adjuvant chemotherapy. PATIENTS AND METHODS: Blood was obtained from 437 patients with early breast cancer before the start and after the completion of adjuvant chemotherapy, and the presence of CK-19 mRNA-positive CTCs was assessed by real-time reverse transcriptase polymerase chain reaction. Interaction with known prognostic factors and association of CTCs with clinical outcome were investigated. RESULTS: CK-19 mRNA-positive CTCs were detected before chemotherapy in 179 patients (41.0%). After adjuvant chemotherapy, a significant change in CK-19 status was observed, as status for 51% of patients with initially CK-19 mRNA-positive disease turned negative, and status for 22% of patients with initially CK-19 mRNA-negative disease became positive (McNemar test P = .004). The detection of CK-19 mRNA-positive CTCs postchemotherapy was associated with involvement of more than three axillary lymph nodes (P = .026). Clinical relapses and disease-related deaths were significantly increased in patients with detectable postchemotherapy CK-19 mRNA-positive CTCs (both P < .001, respectively). Disease-free and overall survival were significantly reduced in patients with detectable CK-19 mRNA-positive CTCs postchemotherapy (P < .001 and P = .001, respectively). In multivariate analysis, the detection of CK-19 mRNA-positive CTCs before and after adjuvant chemotherapy was an independent factor associated with reduced disease-free survival (P < .001) and overall survival (P = .003). CONCLUSION: The detection of CK-19 mRNA-positive CTCs in the blood after adjuvant chemotherapy is an independent risk factor indicating the presence of chemotherapy-resistant residual disease.