RESUMO
High-dose methotrexate treatment requires pharmacological monitoring in order to tailor administration of folinic acid to reduce side effects. The aim of the study was to validate the adaptation of the EMIT reagent on the l'Unicel DxC 600® Beckman Coulter. The establishment of two assays was necessary to obtain a quantification limit as low as possible (0.05 µmol/L). The linearity of the adapted methods extends from 0.05 to 0.25 µmol/L on the one hand, and from 0.25 to 1 µmol/L on the other hand. For each method, fidelity and accuracy were studied and the limits of detection and quantification were quantified. The correlation with the FPIA method was performed on the Abbott TDX(®). The results of all tests are satisfactory with coefficients of variation (CV) of repeatability and reproducibility of less than 6%. However the daily assays are heavy as 66% of blood samples require at least two dosages and 30% a manual dilution.
Assuntos
Análise Química do Sangue/instrumentação , Análise Química do Sangue/métodos , Metotrexato/sangue , Soro/química , Algoritmos , Antimetabólitos Antineoplásicos/análise , Antimetabólitos Antineoplásicos/sangue , Antimetabólitos Antineoplásicos/farmacocinética , Análise Química do Sangue/normas , Calibragem , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Limite de Detecção , Metotrexato/análise , Metotrexato/farmacocinética , Concentração Osmolar , Controle de Qualidade , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Titulometria/instrumentação , Titulometria/métodos , Titulometria/normasRESUMO
Technological advances, in particular the development of high-throughput sequencing, have led to the emergence of a new generation of molecular biomarkers for tumors. These new tools have profoundly changed therapeutic management in oncology, with increasingly precise molecular characterization of tumors leading to increasingly personalized therapeutic targeting. Detection of circulating tumor cells and/or circulating tumor DNA in blood samples -so-called 'liquid biopsies'- can now provide a genetic snapshot of the patient's tumor through an alternative and less invasive procedure than biopsy of the tumor tissue itself. This procedure for characterizing and monitoring the disease in real time facilitates the search for possible relapses, the emergence of resistance, or emergence of a new therapeutic target. In the long term, it might also provide a means of early detection of cancer. These new approaches require the treatment of ever-increasing amounts of clinical data, notably, with the goal of calculating composite clinical-biological predictive scores. The use of artificial intelligence will be unavoidable in this domain, but it raises ethical questions and implications for the health-care system that will have to be addressed.
Assuntos
Inteligência Artificial/tendências , Biomarcadores Tumorais/sangue , Biópsia Líquida , Oncologia/tendências , Neoplasias/sangue , Medicina de Precisão/tendências , Inteligência Artificial/ética , DNA Tumoral Circulante/sangue , Gerenciamento de Dados , Detecção Precoce de Câncer/métodos , Sequenciamento de Nucleotídeos em Larga Escala/tendências , Humanos , Imunoterapia , Biópsia Líquida/métodos , Oncologia/métodos , MicroRNAs/sangue , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Células Neoplásicas CirculantesRESUMO
In oncology, the identification of targets that correlate with a type of cancer has led to a profound change in the notion of "tumor markers". Technological advances, in particular the development of high-throughput sequencing, have led to the emergence of a new generation of molecular biomarkers for tumors. Despite their limited utility for screening and diagnosis, conventional tumor markers remain interesting for evaluation of prognoses, the choice and optimization of treatments, as well as for monitoring the effectiveness of those treatments. In this article, we revisit the conventional serum markers that are enjoying a 'come back' thanks to the development of high-performance scores based on biological, cytological, clinical, or radiological criteria.
Assuntos
Biomarcadores Tumorais/sangue , Oncologia/métodos , Proteínas de Neoplasias/sangue , Neoplasias/sangue , Medicina de Precisão/métodos , França , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/diagnóstico , Neoplasias/diagnóstico , Neoplasias/imunologia , Neoplasias/terapia , Especificidade de Órgãos , Valor Preditivo dos Testes , Prognóstico , Reprodutibilidade dos Testes , Resultado do TratamentoRESUMO
AIM: Despite unprecedented results of anti-programmed death protein (ligand) 1 (PD-(L)1) immune checkpoint inhibitor in the oncology's armamentarium, immune-related adverse events (irAEs) represent a therapeutic hurdle. Currently, there is no consensual recommendation on a routinely monitored biomarker to early detect irAE. Biological markers such as serum creatine phosphokinase (CPK) are commonly used to measure muscular tissue injury. The potential of routine serum CPK monitoring to predict cardiac or neuromuscular irAE in patients treated with immunotherapy remains unknown. METHODS: In this retrospective study between January 2016 and December 2018 at Gustave Roussy Cancer Campus, 1151 cancer patients treated with anti-PD-(L)1 immunotherapy were systematically monitored with serum CPK measurements before each immunotherapy cycle. We considered significant CPK increases according to Common Terminology Criteria for Adverse Events v5.0 (CTCAEV5) of grade ≥2 severity. Comparisons were performed in patients with immune-related CPK (ir-CPK) elevations symptomatic versus asymptomatic. RESULTS: Overall, 53 of 1151 (4.6%) patients showed a CPK increase. Elevations of CPK were deemed to be immunotherapy-related in 31 of 1151 (2.7%) patients. Among them, 12 of 31 (38.7%) patients experienced symptomatic cardiac or neuromuscular irAE, whereas the other 19 of 31 (61.3%) patients remained asymptomatic. In patients with symptomatic irAE, the mean ir-CPK level was higher compared with asymptomatic patients (1271 versus 771 UI/L, P value = 0.02). In the asymptomatic group, all patients experienced a spontaneous resolution of the ir-CPK increase, and none required medical intervention. CONCLUSION: Most patients with immune-related CPK increase remained asymptomatic. The CPK serum increase did not alter the clinical management of asymptomatic patients. The results of this study did not support a significant clinical interest for a systematic routine CPK monitoring in patients amenable to anti-PD-(L)1 immunotherapy.
Assuntos
Cardiotoxicidade/diagnóstico , Creatina Quinase/sangue , Inibidores de Checkpoint Imunológico/efeitos adversos , Neoplasias/tratamento farmacológico , Doenças Neuromusculares/diagnóstico , Antígeno B7-H1/antagonistas & inibidores , Biomarcadores/sangue , Cardiotoxicidade/sangue , Cardiotoxicidade/imunologia , Estudos de Viabilidade , Feminino , Humanos , Masculino , Memória Episódica , Pessoa de Meia-Idade , Neoplasias/sangue , Doenças Neuromusculares/sangue , Doenças Neuromusculares/induzido quimicamente , Doenças Neuromusculares/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Estudos RetrospectivosAssuntos
Adenoma/diagnóstico , Adenoma/patologia , Colina/análogos & derivados , Neoplasias das Paratireoides/diagnóstico , Neoplasias das Paratireoides/patologia , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , Adenoma/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Imagem Multimodal , Estadiamento de Neoplasias , Neoplasias das Paratireoides/diagnóstico por imagemRESUMO
BACKGROUND: No sensitive, specific marker able to discriminate favourable or unfavourable outcome of fever of unknown origin (FUO) at diagnosis has been identified. Procalcitonin, a recently assessed infection marker, may be useful in predicting the outcome of FUO. METHODS: We conducted a prospective study examining the following variables: age 0.5-22 years; solid tumour diagnosis; chemotherapy-related grade-4 febrile neutropenia (FN). A complete clinical, bacteriological and biological evaluation was performed at hospital admission (H0). Other investigations depended on clinical status. FUO was considered to be of unfavourable outcome if the fever was persistent or re-appeared at day 3 (or later), or if secondary clinical or microbiological infection occurred. To validate the results of the analysis the data set was randomly split into a training set and a validation set. RESULTS: Out of 172 episodes of FN, 136 episodes were classified as FUO (80%). Seventy-two (53%) were included in this study. PCT values were significantly higher in episodes of unfavourable outcome (P < 0.001). None of the other prediction candidates appeared to be significantly linked to the risk of unfavourable outcome. In the validation set, the best PCT cut-off was 0.12 micro/L, which was associated with a sensitivity of 80% and specificity of 64%. CONCLUSIONS: PCT-H0 level can predict FUO outcome. A protocol based on PCT-H0 measurement, integrating clinical and bacteriological evaluation, facilitates shorter hospital stays and less antibiotic treatment. Patients with a PCT-H0 value <0.12 micro/L could benefit from an outpatient treatment starting at H48 thus reducing hospitalisation costs and improving quality of life.
Assuntos
Calcitonina/sangue , Febre de Causa Desconhecida/diagnóstico , Neoplasias , Neutropenia/diagnóstico , Precursores de Proteínas/sangue , Adolescente , Biomarcadores/sangue , Peptídeo Relacionado com Gene de Calcitonina , Criança , Pré-Escolar , Feminino , Febre de Causa Desconhecida/sangue , Humanos , Lactente , Modelos Logísticos , Masculino , Neutropenia/sangue , Estudos Prospectivos , Fatores de Risco , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Adulto JovemRESUMO
The quantification of very low concentrations of circulating tumor DNA (ctDNA) biomarkers from liquid biopsies has become an important requirement for clinical diagnostics and personalized medicine. In particular, the simultaneous detection of wild-type (WT) dsDNA and their cancer-related counterparts presenting single-point mutations with simple, sensitive, specific, and reproducible technologies is paramount for ctDNA assays in clinical practice. Here, we present the development and evaluation of an amplified dsDNA assay based on a combination of isothermal rolling circle amplification (RCA) and time-gated Förster resonance energy transfer (TG-FRET) between a Tb donor and two dye (Cy3.5 and Cy5.5) acceptors. The RCA-FRET assay is free of washing and separation steps and can quantify both WT and mutated (MT) (V600E) dsDNA in the BRAF gene from a single sample in the 75 fM to 4.5 pM (4.5 × 105 to 2.7 × 107 copies) concentration range. This assay includes all steps from denaturation of the dsDNA targets to the final duplexed quantification of WT and MT targets. High assay performance at different dsDNA sequence lengths and high target specificity even in the presence of a large excess of nonspecific cell-free DNA from human plasma samples demonstrated the applicability to clinical samples. The RCA-FRET single-point mutation sensor has the potential to become an important complementary technique for analyzing liquid biopsies in advanced cancer diagnostics.
Assuntos
Técnicas Biossensoriais/métodos , DNA Tumoral Circulante/sangue , Proteínas Proto-Oncogênicas B-raf/genética , Sequência de Bases , Carbocianinas/química , DNA Tumoral Circulante/genética , Sondas de DNA/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico , Mutação PuntualRESUMO
The eukaryotic topoisomerase II is an ubiquitous nuclear enzyme involved in vital cellular functions. It is also the target for some of the most active anticancer drugs. In the various crystal structures of yeast topoisomerase II, the 701-748 segment homologous to the human topoisomerase II alpha 724-771 segment folds into a compact alpha(2)beta(1)alpha(3)talpha(4) conformation, hereafter termed alpha(2)HTH module (helix turn helix (HTH), alpha(3)talpha(4)). The crystal structure of gyrase A has suggested a model wherein HTH is involved in both the enzyme dimerization and the binding to DNA. These two properties were investigated in solution, using the recombinant alpha(2)HTH module of human topoisomerase II alpha and its synthetic components HTH, alpha(4), alpha(3) and turn. The homology-based structure model of human alpha(2)HTH superposed that of yeast in the crystal structure with a rmsd of 1.03 A. Circular dichroism spectra showed that the helical content of human alpha(2)HTH in solution is similar to that of its counterpart within yeast topoisomerase II in the solid state. The chemical cross-linking data indicated that alpha(2)HTH self-associated into dimers while gel mobility shift assays and anisotropy fluorescence titrations demonstrated that alpha(2)HTH, HTH and alpha(4), but not alpha(3), bind efficiently to DNA (dissociation constants of 3.10(-7) M for alpha(2)HTH and alpha(4), of 3.10(-6) M for HTH and of only 1.10(-5) M for alpha(3)). Correlatively, alpha(2)HTH, alpha(4) and HTH, but not alpha(3), were able to inhibit topoisomerase II in DNA relaxation assays, stipulating that alpha(4) is the DNA recognition helix. All suggests that the alpha(2)HTH module once separated from the whole protein conserves a compact conformation, integral to specific dimerization and DNA recognition. The module may thus be used for the search of drugs efficient in hindering topoisomerase II dimerization or binding to DNA.
Assuntos
Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Volta-Hélice , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , DNA/química , DNA/metabolismo , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Dimerização , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Alinhamento de Sequência , Inibidores da Topoisomerase IIRESUMO
The peptide denoted K159 (30 residues) derives from the catalytic core (CC) sequence of HIV-1 integrase (IN, residues 147-175). In the crystal structure of CC, the corresponding segment belongs to the alpha4 helix (residues 148-168, including residues Glu 152, Lys 156 and Lys 159, crucial for enzyme activity and DNA recognition), a loop (residues 169-171) and a part of the alpha5 helix (171-175), involved in enzyme dimerization. We used the fluorescence and the circular dichroism (CD) properties in the near-UV of the aromatic side chain of a tyrosine residue added at the C-terminal end of K159 in order to analyze the behavior of the concentrated and diluted peptide in aqueous trifluoroethanol (TFE), in an attempt to connect the information obtainable at high (NMR), medium (CD) and low (fluorescence) concentrations of the peptide. Altogether, the C-terminal tyrosine residue provided indirect information on the global conformation of K159 and on the local orientation and environment of the residue. The propensity of TFE to stabilize alpha-helical conformations in peptides was confirmed in CD and fluorescence experiments at relatively high (20-160 microM) and low (2-16 microM) concentrations, respectively. At relatively high concentration, stabilization of the peptide into alpha-helical conformation favored its auto-association likely in parallel coiled-coil dimers, as pointed out in our previous work [Eur. J. Biochem. 253 (1998) 236]. This was further confirmed by ANS (1-anilinonaphtalene-8-sulfonic acid) analysis and fluorescence temperature coefficient measurement. With diluted K159, a Stern-Volmer analysis with positively and negatively charged quenchers indicated that, when the intermolecular interactions were absent, the tyrosine was in a positively charged environment, as if the peptide folded into a U-shaped conformation similar to that present in the crystal structure of the enzyme.
Assuntos
Domínio Catalítico , Fragmentos de Peptídeos/química , Conformação Proteica , Tirosina/química , Dicroísmo Circular , Integrase de HIV/química , Humanos , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos/efeitos dos fármacos , Espectrometria de Fluorescência , Trifluoretanol/farmacologiaRESUMO
The aim of the present study was to evaluate the clinical performance of the CA 15-3 assay on Elecsys systems in an international multicenter study (11 centers). A total of 1326 single samples (272 apparently healthy individuals, 34 pregnant women, 308 benign diseases, 273 cancers other than breast, 439 breast cancer) and 538 serial samples of 98 breast cancer patients during follow-up were analyzed. 95% of values in healthy individuals were below 25 kU/L, and 88% in benign breast diseases, respectively. In malignant breast disease at primary diagnosis the value distribution of Elecsys CA 15-3, sensitivity at 95% specificity, as well as the areas under the curve in ROC analysis were clearly correlated to tumor stages: UICC I to IV 88 to 25% of values < 25 kU/L, sensitivity 7 to 78%, areas under the curve 0.53 to 0.94. During follow-up, sensitivity/specificity for detection of recurrences were 90%/71%. In metastatic disease clinical progression/response to therapy were indicated in 91%/78% of patients at a specificity of 92%/78%. The findings indicate that the Elecsys CA 15-3 assay is very suitable in routine work for detection of recurrences as well as for therapy control in metastatic breast cancer.
Assuntos
Antígenos de Neoplasias/sangue , Biomarcadores Tumorais/sangue , Neoplasias da Mama/diagnóstico , Mucina-1/sangue , Adolescente , Adulto , Neoplasias da Mama/cirurgia , Europa (Continente) , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/imunologia , Valor Preditivo dos Testes , Gravidez/sangue , Valores de Referência , Estudos Retrospectivos , Estados UnidosAssuntos
Seminoma/diagnóstico por imagem , Neoplasias Testiculares/diagnóstico por imagem , alfa-Fetoproteínas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Radiografia , Seminoma/sangue , Seminoma/patologia , Neoplasias Testiculares/sangue , Neoplasias Testiculares/patologiaRESUMO
Ovarian yolk sac tumour (OYST) is a very rare malignancy arising most often in young women. Preoperative clinical, biological (alpha-foetoprotein) and radiological findings should help to establish the diagnosis of OYST, in order to propose adequate surgical treatment. The aim of surgery is to remove the primary tumour, to obtain an accurate histological diagnosis and to assess the disease extent. In young women, fertility-sparing surgery should be performed, in order to preserve the possibility of pregnancy later on. Chemotherapy has substantially modified the prognosis of these tumours, and practically all patients will be cured. The overall 5-year survival rate is 94% when patients are treated with BEP chemotherapy. Depending on the clinical situation, two to four cycles of the BEP regimen should be administered after surgery. Identification of prognostic factors may help to propose risk-adapted treatment in order to increase the cure rate in patients with a poor prognosis and to decrease toxicity in patients with a low risk of relapse. Fertility preservation represents a major objective in women treated for OYSTs.
Assuntos
Tumor do Seio Endodérmico/terapia , Doenças Raras/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Terapia Combinada/métodos , Tumor do Seio Endodérmico/diagnóstico , Tumor do Seio Endodérmico/mortalidade , Feminino , Fertilidade , Humanos , Doenças Raras/diagnóstico , Doenças Raras/mortalidade , Taxa de Sobrevida , Fatores de Tempo , alfa-Fetoproteínas/análiseRESUMO
BACKGROUND: Ovarian yolk sac tumour (OYST) is a very rare malignancy arising in young women. Our study aimed to evaluate long-term outcomes and to identify prognostic parameters likely to help make appropriate risk-based decisions about therapy in this disease. METHODS: This retrospective study is based on prospectively recorded OYST cases at the Institut Gustave-Roussy. A univariate analysis using the logrank test evaluated possible associations between survival and patient or disease covariates. The multivariate analysis was performed using the Cox proportional hazard regression method. RESULTS: Between 1976 and 2006, 84 patients were registered. Since 1991, most of the patients have undergone fertility-sparing surgery. With a median follow-up of 71 months, the overall 5-year and event-free survival rates are 84% and 79%, respectively. In the multivariate model only the absence of ascites and a favourable serum AFP decline rate were significantly associated with better overall survival. CONCLUSIONS: Patients with a poor prognosis factor such as an unfavourable serum AFP decline may be considered for aggressive treatment whereas those with good prognostic factors could be given less courses of chemotherapy.
Assuntos
Tumor do Seio Endodérmico/mortalidade , Neoplasias Ovarianas/mortalidade , Tumor do Seio Endodérmico/tratamento farmacológico , Tumor do Seio Endodérmico/cirurgia , Feminino , França/epidemiologia , Humanos , Infertilidade Feminina/prevenção & controle , Estimativa de Kaplan-Meier , Recidiva Local de Neoplasia/mortalidade , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/cirurgia , Prognóstico , Estudos Retrospectivos , Adulto Jovem , alfa-Fetoproteínas/metabolismoRESUMO
OBJECTIVE: The progression of medullary thyroid cancer is difficult to assess with imaging modalities; we studied the interest of calcitonin and carcinoembryonic antigen (CEA) doubling times and of Ki-67 labeling and mitotic index (MI). PATIENTS AND METHODS: Fifty-five consecutive medullary thyroid carcinoma (MTC) patients with elevated calcitonin levels underwent repeated imaging studies in order to assess tumor burden and progression status. We looked for relationships between tumor burden and levels of calcitonin and CEA and between progression status according to the response evaluation criteria in solid tumors (RECIST) and calcitonin and CEA doubling times, and Ki-67 labeling and MI. RESULTS: The calcitonin and CEA levels were correlated with tumor burden. Ten patients with calcitonin levels below 816 pg/ml had no imaged tumor foci. Among the 45 patients with imaged tumor foci, 19 had stable disease and 26 had progressive disease, according to the RECIST. The calcitonin and CEA doubling times were strongly related to disease progression, with very few overlaps: 94% of patients with doubling times shorter than 25 months had progressive disease and 86% of patients with doubling times longer than 24 months had stable disease. Ki-67 labeling and MI were not significantly associated with disease progression. CONCLUSION: For MTC patients, the doubling times of both calcitonin and CEA are efficient tools for assessing tumor progression.
Assuntos
Biomarcadores Tumorais/sangue , Calcitonina/sangue , Antígeno Carcinoembrionário/sangue , Carcinoma/sangue , Neoplasias da Glândula Tireoide/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Feminino , Humanos , Antígeno Ki-67/sangue , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
BACKGROUND: Cancer antigen CA125 is known as a valuable marker for the management of ovarian cancer. METHODS: The analytical and clinical performance of the Access OV Monitor Immunoassay System (Beckman Coulter) was evaluated at five different European sites and compared with a reference system, defined as CA125 on the Elecsys System (Roche Diagnostics). RESULTS: Total imprecision (% CV) of the OV Monitor ranged between 3.1% and 8.8%, and inter-laboratory reproducibility between 4.7% and 5.0%. Linearity upon dilution showed a mean recovery of 100% (SD + 8.1%). Endogenous interferents had no influence on OV Monitor levels (mean recoveries: hemoglobin 107%, bilirubin 103%, triglycerides 103%). There was no high-dose hook effect up to 27,193 kU/L. Clinical performance investigated in sera from 1811 individuals showed a good correlation between the Access OV Monitor and Elecsys CA125 (R = 0.982, slope = 0.921, intercept = +1.951). OV Monitor serum levels were low in healthy individuals (n = 267, median = 9.7 kU/L, 95th percentile = 30.8 kU/L), higher in individuals with various benign diseases (n = 549, medians = 10.9-16.4 kU/L, 95th percentiles = 44.2-355 kU/L) and even higher in individuals suffering from various cancers (n = 995, medians = 12.4-445 kU/L; 95th percentiles = 53.4-4664 kU/L). Optimal diagnostic accuracy for cancer detection against the relevant benign control group by the OV Monitor was found for ovarian cancer [area under the curve (AUC) 0.898]. Results for the reference CA125 assay were comparable (AUC 0.899). CONCLUSIONS: The Access OV Monitor provides very good methodological characteristics and demonstrates an excellent analytical and clinical correlation with Elecsys CA125. The best diagnostic accuracy for the OV Monitor was found in ovarian cancer. Our results also suggest a clinical value of the OV Monitor in other cancers.
Assuntos
Anticorpos/sangue , Antígeno Ca-125/análise , Imunoensaio/métodos , Neoplasias Ovarianas/diagnóstico , Europa (Continente) , Feminino , Humanos , Imunoensaio/instrumentação , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Gastrointestinal cancer antigen CA19-9 is known as a valuable marker for the management of patients with pancreatic cancer. METHODS: The analytical and clinical performance of the Access GI Monitor assay (Beckman Coulter) was evaluated on the UniCel Dxl 800 Immunoassay System at five different European sites and compared with a reference method, defined as CA19-9 on the Elecsys System (Roche Diagnostics). RESULTS: Total imprecision (%CV) of the GI Monitor ranged between 3.4% and 7.7%, and inter-laboratory reproducibility between 3.6% and 4.0%. Linearity upon dilution showed a mean recovery of 97.4% (SD + 7.2%). Endogenous interferents had no influence on GI Monitor levels (mean recoveries: hemoglobin 103%, bilirubin 106%, triglycerides 106%). There was no high-dose hook effect up to 115,000 kU/L. Clinical performance investigated in sera from 1811 individuals showed a good correlation between the Access GI Monitor and Elecsys CA19-9 (R = 0.959, slope = 1.004, intercept = +0.17). GI Monitor serum levels were low in healthy individuals (n = 267, median = 6.0 kU/L, 95th percentile=23.1 kU/L), higher in individuals with various benign diseases (n = 550, medians = 5.8-13.4 kU/L, 95th percentiles = 30.1-195.5 kU/L) and even higher in individuals suffering from various cancers (n = 995, medians = 8.4-233.8 kU/L, 95th percentiles = 53.7-13,902 kU/L). Optimal diagnostic accuracy for cancer detection against the relevant benign control group by the GI Monitor was found for pancreatic cancer [area under the curve (AUC) 0.83]. Results for the reference CA19-9 assay were comparable (AUC 0.85). CONCLUSIONS: The Access GI Monitor provides very good methodological characteristics and demonstrates an excellent analytical and clinical correlation with the Elecsys CA19-9. The GI Monitor shows the best diagnostic accuracy in pancreatic cancer. Our results also suggest a clinical value of the GI Monitor in other cancers.
Assuntos
Anticorpos/química , Antígeno CA-19-9/sangue , Imunoensaio/métodos , Neoplasias Pancreáticas/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Europa (Continente) , Feminino , Humanos , Imunoensaio/instrumentação , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Cancer antigen CA15-3 antigen is known as a valuable marker for the management of breast cancer. METHODS: The analytical and clinical performance of the Access BR Monitor Immunoassay System (Beckman Coulter) was evaluated at five different European sites and compared with a reference system, defined as CA15-3 on the Elecsys System (Roche Diagnostics). RESULTS: Total imprecision (% CV) of the BR Monitor ranged between 5.5% and 11.7%, and inter-laboratory reproducibility between 3.4% and 5.1%. Linearity upon dilution showed a mean recovery of 98.5% (SD + 9.1%). Endogenous interferents had no influence on BR Monitor levels (mean recoveries: hemoglobin 112%, bilirubin 111%, triglycerides 108%). There was no high-dose hook effect up to 13,540 kU/L. Clinical performance investigated in sera from individuals showed a general correlation between the Access BR Monitor and Elecsys CA15-3 (R = 0.797), with a slope of 1.383. CA15-3 serum levels, as measured by the BR Monitor, were low in healthy individuals (n = 267, median = 11.9 kU/L, 95th percentile = 23.5 kU/L), higher in individuals with various benign diseases (n = 549, medians = 11.3-15.6 kU/L, 95th percentiles = 21.6-54.6 kU/L) and even higher in individuals suffering from various cancers (n = 995, medians = 11.2-22.8 kU/L, 95th percentiles = 30.0-429.7 kU/L). Best diagnostic accuracy for cancer detection against the relevant benign control group by the BR Monitor was found for locoregional and metastatic breast cancer, as well as for ovarian cancer [area under the curve (AUC) 0.619, 0.897 and 0.774]. Results for the reference CA15-3 assay were comparable (AUC 0.611, 0.887 and 0.818). CONCLUSIONS: The Access BR Monitor provides accurate methodological characteristics and demonstrates an analytical and clinical correlation with Elecsys CA15-3. Best diagnostic accuracy for the BR Monitor was found in breast and ovarian cancer. Our results also suggest a clinical value of the BR Monitor in other cancers.
Assuntos
Anticorpos/química , Neoplasias da Mama/diagnóstico , Imunoensaio/métodos , Mucina-1/sangue , Neoplasias Ovarianas/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Neoplasias da Mama/sangue , Europa (Continente) , Feminino , Humanos , Imunoensaio/instrumentação , Pessoa de Meia-Idade , Neoplasias Ovarianas/sangue , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
The HIV-1 integrase (IN) catalyzes the integration of viral DNA in the human genome. In vitro the enzyme displays an equilibrium of monomers, dimers, tetramers and larger oligomers. However, its functional oligomeric form in vivo is not known. We report a study of the auto-associative properties of three peptides denoted K156, E156 and E159. These derive from the alpha4 helix of the IN catalytic core. The alpha4 helix is an amphipatic helix exposed at the surface of the protein and could be involved in the oligomerization process through its hydrophobic face. The peptides were obtained from the replacement of several amino acid residues by more helicogenic ones in the alpha4 helix peptide. K156 carries the basic residues Lys156 and Lys159, which have been shown important for the binding of IN to viral DNA. In E156 and E159 they are replaced with the acidic residue Glu. A fourth peptide K(E)156 obtained from the replacement of hydrophobic residues with Glu in K156 in order to abolish the auto-associative properties is used as a negative control. The capacity shown by peptides for alpha-helical formation is demonstrated by circular dichroism (CD) analysis performed in aqueous solution and in aqueous trifluoroethanol (TFE) mixtures. Both electrospray ionization mass spectrometry (ESI-MS) and glutaraldehyde chemical cross-linking show that peptides adopt different solvent-dependent equilibriums of monomers, dimers, trimers and tetramers. Oligomerization of peptides in aqueous solution is related to their ability to form helical structures. Addition of a small amount of TFE (<10%) stimulates helix stabilization and the interhelical hydrophobic contacts. Higher amounts of TFE alter the hydrophobic contacts and disrupt the oligomeric species. In addition to hydrophobic interactions, the patterns indicate that the biologically important Lys156 and Lys159 residues also participate in helix association. K(E)156 despite its ability to adopt a helical structure is unable to associate into oligomers, demonstrating the importance of hydrophobic contacts for oligomerization. Thus, the designed peptides provide us information on the functional properties of the alpha4 IN that seems to hold a dual role in DNA recognition and protein oligomerization.
Assuntos
Integrase de HIV/química , HIV-1/enzimologia , Peptídeos/química , Dicroísmo Circular , Modelos Químicos , Estrutura Molecular , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The last decade has contributed to our understanding of the three-dimensional structure of the human immunodeficiency virus, type 1 (HIV-1) integrase (IN) and to the description of how the enzyme catalyzes the viral DNA integration into the host DNA. Recognition of the viral DNA termini by IN is sequence-specific, and that of the host DNA does not require particular sequence, although in physicochemical studies IN fails to discriminate between the two interactions. Here, such discrimination was allowed thanks to a model system using designed oligonucleotides and peptides as binding structures. Spectroscopic (circular dichroism, NMR, and fluorescence anisotropy) techniques and biochemical (enzymatic and filter binding) assays clearly indicated that the amphipathic helix alpha4, located at the catalytic domain surface, is responsible for the specific high affinity binding of the enzyme to viral DNA. Analogues of the alpha4 peptide having increased helicity and still bearing the biologically relevant lysines 156 and 159 on the DNA binding face, and oligonucleotides conserving an intact attachment site, are required to achieve high affinity complexes (Kd of 1.5 nm). Data corroborate previous in vivo results obtained with mutated viruses.