RESUMO
Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.
Assuntos
Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/genética , DNA/análise , Fezes/química , Adulto , Análise de Dados , Fluorescência , Humanos , Pessoa de Meia-Idade , Sangue Oculto , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Fatores de Risco , TemperaturaRESUMO
Fluorescence long DNA (FL-DNA) is a non-invasive and simple-to-perform stool DNA test. This assay consists of a qualitative and quantitative real-time PCR (RT PCR) analysis. FL-DNA has great potential in colorectal cancer (CRC) lesions detection used alone or in combination with the standard CRC screening tool: immunochemical fecal occult blood test (iFOBT).
Assuntos
Neoplasias Colorretais/diagnóstico , DNA/análise , Detecção Precoce de Câncer/métodos , Fezes/química , Reação em Cadeia da Polimerase em Tempo Real/métodos , Detecção Precoce de Câncer/instrumentação , Humanos , Sangue Oculto , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Fatores de TempoRESUMO
CONTEXT: - Patients with non-small cell lung cancer harboring ALK receptor tyrosine kinase ( ALK), ROS proto-oncogene 1 ( ROS1), and ret proto-oncogene ( RET) gene rearrangements can benefit from specific kinase inhibitors. Detection of fusion genes is critical for determining the best treatment. Assessing rearrangements in non-small cell lung cancer remains challenging, particularly for lung cytology. OBJECTIVE: - To examine the possible application of the multiplex, transcript-based NanoString system (NanoString Technologies, Seattle, Washington) in the evaluation of fusion genes in lung adenocarcinoma samples. DATA SOURCES: - This study is a narrative literature review. Studies about NanoString, gene fusions, and lung adenocarcinoma were collected from PubMed (National Center for Biotechnology Information, Bethesda, Maryland). We found 7 articles about the application of the NanoString system to detect fusion genes on formalin-fixed, paraffin-embedded tumor tissues and one article evaluating the adequacy of lung cytologic specimens for NanoString gene expression analysis. CONCLUSIONS: - To maximize the yield of molecular tests on small lung biopsies, the NanoString nCounter system has been suggested to detect fusion genes. NanoString fusion gene assays have been successfully applied on formalin-fixed, paraffin-embedded tissues. Although there are only a few studies available, the application of NanoString assays may also be feasible in lung cytology. According to available data, the NanoString system could strengthen the routine molecular characterization of lung adenocarcinoma.
Assuntos
Adenocarcinoma de Pulmão/genética , Biomarcadores Tumorais/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nanotecnologia/métodos , Fusão Oncogênica , Proteínas de Fusão Oncogênica/análise , Biomarcadores Tumorais/genética , Humanos , Proto-Oncogene MasRESUMO
The World Health Organization recently revised the diagnosis of glioma, to integrate molecular parameters, including IDH mutations and codeletion (loss of heterozygosity; LOH) of chromosome arms 1p/19q, into the definitions of adult glioma histological subtypes. Mutations in the TERT promoter may also be useful for glioma diagnosis and prognosis. The integration of molecular markers into routine diagnosis requires their rapid and reliable assessment. We propose a MassARRAY (MS)-based test that can identify 1p/19q codeletion using quantitative SNP genotyping and, simultaneously, characterize hotspot mutations in the IDH1, IDH2, and TERT genes in tumor DNA. We determined the reliability of the MS approach testing 50 gliomas and comparing the MS results with those obtained by standard methods, such as short tandem repeat genotyping, array comparative genomic hybridization (array-CGH) and Fluorescence In Situ Hybridization (FISH) for 1p/19q codeletion and Sanger sequencing for hotspots mutations. The results indicate that MS is suitable for the accurate, rapid, and cost-effective evaluation of chromosome deletions combined with hotspot mutation detection. This MS approach could be similarly exploited in evaluation of LOH in other situations of clinical and/or research importance.
RESUMO
BACKGROUND: DNA integrity analysis could represent an alternative approach to the early detection of colorectal cancer. Previously, fluorescence long DNA (FL-DNA) in stools was extracted using a manual approach and analyzed by capillary electrophoresis assay (CE FL-DNA). We aimed to improve diagnostic accuracy using a simpler and more standardized method [Real Time PCR FL-DNA (RT FL-DNA)] for the detection of early malignant lesions in a population undergoing colorectal cancer screening. METHODS: From 241 stool samples, DNA was extracted using manual and semiautomatic extraction systems and analyzed using FL-DNA tests by CE and RT assays. The RT FL-DNA approach showed slightly higher sensitivity and specificity compared with the CE FL-DNA method. Furthermore, we compared the RT FL-DNA approach with the iFOBT report. RESULTS: Nonparametric ranking statistics were used to analyze the relationship between the median values of RT FL-DNA and the clinicohistopathologic characteristics. The median values of both variables were significantly higher in patients with cancer than in patients with noncancerous lesions. According to the Fagan nomogram results, the iFOBT and FL-DNA methods provided more accurate diagnostic information and were able to identify subgroups at varying risks of cancer. CONCLUSIONS: The combination of the semiautomatic extraction system and RT FL-DNA analysis improved the quality of DNA extracted from stool samples. IMPACT: RT FL-DNA shows great potential for colorectal cancer diagnosis as it is a reliable and relatively easy analysis to perform on routinely processed stool samples in combination with iFOBT.
Assuntos
Neoplasias Colorretais/genética , DNA/genética , Fezes/química , Detecção Precoce de Câncer , Feminino , Humanos , Masculino , Programas de RastreamentoRESUMO
CONTEXT: Echinoderm microtubule associated proteinlike 4-anaplastic lymphoma receptor tyrosine kinase (EML4-ALK) translocation has been described in a subset of patients with non-small cell lung cancer (NSCLC) and has been shown to have oncogenic activity. Fluorescence in situ hybridization (FISH) is used to detect ALK-positive NSCLC, but it is expensive, time-consuming, and difficult for routine application. OBJECTIVE: To evaluate the potential role of immunohistochemistry (IHC) as a screening tool to identify candidate cases for FISH analysis and for ALK inhibitor therapy in NSCLC. DESIGN: We performed FISH and IHC for ALK and mutational analysis for epidermal growth factor receptor (EGFR) and KRAS in 523 NSCLC specimens. We conducted IHC analysis with the monoclonal antibody D5F3 (Ventana Medical Systems, Tucson, Arizona) and a highly sensitive detection system. We also performed a MassARRAY-based analysis (Sequenom, San Diego, California) in a small subset of 11 samples to detect EML4-ALK rearrangement. RESULTS: Of the 523 NSCLC specimens, 20 (3.8%) were positive for ALK rearrangement by FISH analysis. EGFR and KRAS mutations were identified in 70 (13.4%) and 124 (23.7%) of the 523 tumor samples, respectively. ALK rearrangement and EGFR and KRAS mutations were mutually exclusive. Of 523 tumor samples analyzed, 18 (3.4%) were ALK(+) by IHC, 18 samples (3.4%) had concordant IHC and FISH results, and 2 ALK(+) cases (0.3%) by FISH failed to show ALK protein expression. In the 2 discrepant cases, we did not detect any mass peaks for the EML4-ALK variants by MassARRAY. CONCLUSIONS: Our results show that IHC may be a useful technique for selecting NSCLC cases to undergo ALK FISH analysis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Rearranjo Gênico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Proteínas de Ciclo Celular/genética , Crizotinibe , Feminino , Genes erbB-1 , Testes Genéticos , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/enzimologia , Masculino , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Serina Endopeptidases/genética , Proteínas ras/genéticaRESUMO
Previous studies showed that prostaglandin F2alpha (PGF2alpha) stimulated fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 2 (FGFR2) cytosolic and nuclear accumulation, however, the endocytic pathway has not been elucidated. This study demonstrates that although PGF2alpha increased the formation of clathrin-coated structures in Py1a rat osteoblasts, they were not involved in FGF-2 and FGFR2 trafficking. PGF2alpha increased binding of FGF-2 and FGFR2 and co-localization of reactive sites in addition to nuclear translocation at the nuclear pore complex level. FGF-2 and FGFR2 were in close spatial correlation with importin beta, further supporting nuclear import of the FGF-2/FGFR2 complex. Immunogold and immunofluorescence techniques as well as Western blotting demonstrated increased importin beta protein labeling in response to PGF2alpha. Similar to PGF2alpha, phorbol 12-myristate 13-acetate (PMA) also increased importin beta protein. These data strongly suggest that prostaglandins may regulate osteoblast metabolism via FGF-2/FGFR2/importin beta nuclear trafficking.
Assuntos
Clatrina/metabolismo , Dinoprosta/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Osteoblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , beta Carioferinas/metabolismo , Animais , Células Cultivadas , Dinoprosta/metabolismo , Imuno-Histoquímica , Microscopia Imunoeletrônica , Osteoblastos/ultraestrutura , Transporte Proteico , Ratos , Transdução de SinaisRESUMO
We have previously reported that prostaglandin F(2alpha) (PGF(2alpha)) and its selective agonist fluprostenol increase basic fibroblast growth factor (FGF-2) mRNA and protein production in osteoblastic Py1a cells. The present report extends our previous studies by showing that Py1a cells express FGF receptor-2 (FGFR2) and that treatment with PGF(2alpha) or fluprostenol decreases FGFR2 mRNA. We have used confocal and electron microscopy to show that, under PGF(2alpha) stimulation, FGF-2 and FGFR2 proteins accumulate near the nuclear envelope and colocalize in the nucleus of Py1a cells. Pre-treatment with cycloheximide blocks nuclear labelling for FGF-2 in response to PGF(2alpha). Treatment with SU5402 does not block prostaglandin-mediated nuclear internalization of FGF-2 or FGFR2. Various effectors have been used to investigate the signal transduction pathway. In particular, pre-treatment with phorbol 12-myristate 13-acetate (PMA) prevents the nuclear accumulation of FGF-2 and FGFR2 in response to PGF(2alpha). Similar results are obtained by pre-treatment with the protein kinase C (PKC) inhibitor H-7. In addition, cells treated with PGF(2alpha) exhibit increased nuclear labelling for the mitogen-activated protein kinase (MAPK), p44/ERK2. Pre-treatment with PMA blocks prostaglandin-induced ERK2 nuclear labelling, as confirmed by Western blot analysis. We conclude that PGF(2alpha) stimulates nuclear translocation of FGF-2 and FGFR2 by a PKC-dependent pathway; we also suggest an involvement of MAPK/ERK2 in this process.