RESUMO
BACKGROUND: This study aimed to show that SHOX2 DNA methylation is a tumor marker in patients with suspected lung cancer by using bronchial fluid aspirated during bronchoscopy. Such a biomarker would be clinically valuable, especially when, following the first bronchoscopy, a final diagnosis cannot be established by histology or cytology. A test with a low false positive rate can reduce the need for further invasive and costly procedures and ensure early treatment. METHODS: Marker discovery was carried out by differential methylation hybridization (DMH) and real-time PCR. The real-time PCR based HeavyMethyl technology was used for quantitative analysis of DNA methylation of SHOX2 using bronchial aspirates from two clinical centres in a case-control study. Fresh-frozen and Saccomanno-fixed samples were used to show the tumor marker performance in different sample types of clinical relevance. RESULTS: Valid measurements were obtained from a total of 523 patient samples (242 controls, 281 cases). DNA methylation of SHOX2 allowed to distinguish between malignant and benign lung disease, i.e. abscesses, infections, obstructive lung diseases, sarcoidosis, scleroderma, stenoses, at high specificity (68% sensitivity [95% CI 62-73%], 95% specificity [95% CI 91-97%]). CONCLUSIONS: Hypermethylation of SHOX2 in bronchial aspirates appears to be a clinically useful tumor marker for identifying subjects with lung carcinoma, especially if histological and cytological findings after bronchoscopy are ambiguous.
Assuntos
Biomarcadores Tumorais/metabolismo , Brônquios/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Neoplasias Pulmonares/metabolismo , Adulto , Idoso , Broncoscopia/métodos , Carcinoma/metabolismo , Estudos de Casos e Controles , Metilação de DNA , Reações Falso-Positivas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , Sensibilidade e EspecificidadeRESUMO
DNA methylation is an important epigenetic mechanism involved in fundamental biological processes such as development, imprinting, and carcino-genesis. For these reasons, DNA methylation represents a valuable source for cancer biomarkers. Methods for the sensitive and specific detection of methylated DNA are a prerequisite for the implementation of DNA biomarkers into clinical routine when early detection based on the analysis of body fluids is desired. Here, a novel technique is presented for the detection of DNA methylation biomarkers, based on real-time PCR of bisulfite-treated template with enzymatic digestion of background DNA during amplification using the heat-stable enzyme Tsp509I. An assay for the lung cancer methylation biomarker BARHL2 was used to show clinical and analytical performance of the method in comparison with methylation-specific PCR technology. Both technologies showed comparable performance when analyzing technical DNA mixtures and bronchial lavage samples from 75 patients suspected of having lung cancer. The results demonstrate that the approach is useful for sensitive and specific detection of a few copies of methylated DNA in samples with a high background of unmethylated DNA, such as in clinical samples from body fluids.
Assuntos
Biomarcadores Tumorais/análise , Líquido da Lavagem Broncoalveolar/química , Metilação de DNA , DNA de Neoplasias/análise , Proteínas de Homeodomínio/análise , Neoplasias Pulmonares/química , Proteínas do Tecido Nervoso/análise , Reação em Cadeia da Polimerase/métodos , Biomarcadores Tumorais/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA de Neoplasias/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Sensibilidade e EspecificidadeRESUMO
PURPOSE: To evaluate the feasibility of gene delivery mediated with diagnostic ultrasound and plasmid DNA (pDNA) encapsulated in gas-filled microparticles (GFMP) in rodent tumor models. MATERIALS AND METHODS: This study was performed according to a protocol approved by the regional animal research committee. The model plasmid UT651 (pUT651) that contained the Escherichia coli LacZ gene for beta-galactosidase was used to demonstrate the feasibility of ultrasound-mediated gene delivery in CC531 liver tumors in rats. In preliminary experiments, a single injection of pUT651-containing GFMP was administered intraarterially (n=4) or intravenously (n=6) with simultaneous sonication (color Doppler mode, maximum mechanical index) of the GFMP passing through the capillaries of the tumors. All animals were sacrificed 2-5 days later, and liver tumors were examined for beta-galactosidase expression histochemically. Subsequently, potential medical usefulness of this delivery system was tested in nude mice bearing Capan-1 tumors (adenocarcinoma of the human pancreas) by using the plasmid RC/CMV-p16 (pRC/CMV-p16), which contains tumor suppressor gene p16. The tumor suppressor gene p16 is deleted in Capan-1 cells. Twenty-five tumor-bearing mice were classified into five groups (four to six mice per group, one treatment group, four control groups) at random. All mice were treated once weekly for 5 weeks with intravenous infusion of p16-containing GFMP or control substances with simultaneous tumor sonication with color Doppler mode ultrasound and maximum mechanical index or without ultrasound treatment. The therapeutic effect of p16 was measured as an increase in tumor volume doubling time. Data were analyzed with analysis of variance. Results were considered significant at the 5% critical level (P < .05). RESULTS: A clear expression of pDNA was found in tumors in rats treated with a combination of pUT651-containing GFMP and ultrasound; relevant controls showed a significantly lower expression of marker gene. The controlled ultrasound-triggered release of pRC/CMV-p16 from GFMP leads to a strong tumor growth inhibition, which is significant (P < .002), compared with that in controls. CONCLUSION: A combination of GFMP and ultrasound provides an effective approach for nonviral gene therapy-based cancer treatment.
Assuntos
Terapia Genética/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Animais , Modelos Animais de Doenças , Estudos de Viabilidade , Gases , Masculino , Ratos , UltrassonografiaRESUMO
PURPOSE: To produce and characterize a nonviral ultrasound-controlled release system of plasmid DNA (pDNA) encapsulated in gas-filled poly(D,L-lactide-co-glycolide) microparticles (PLGA-MPs). METHODS: Different cationic polymers were used to form pDNA/polymer complexes to enhance the stability of pDNA during microparticle preparation. The physico-acoustical properties of the microparticles, particle size, pDNA integrity, encapsulation efficiency and pDNA release behavior were studied in vitro. RESULTS: The microparticles had an average particle size of around 5 microm. More than 50% of all microparticles contained a gas core, and when exposed to pulsed ultrasound as used for color Doppler imaging create a signal that yields typical color patterns (stimulated acoustic emission) as a result of the ultrasound-induced destruction of the microparticles. Thirty percent of the pDNA used was successfully encapsulated and approximately 10% of the encapsulated pDNA was released by ultrasound within 10 min. CONCLUSIONS: Plasmid DNA can be encapsulated in biodegradable gas-filled PLGA-MPs without hints for a structural disintegration. A pDNA release by ultrasound-induced microparticle-destruction could be shown in vitro.