ABSTRACT
Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).
Subject(s)
Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Plasmids/genetics , Real-Time Polymerase Chain Reaction/standards , Calibration , Cloning, Molecular , DNA , Escherichia coli Proteins/genetics , Gene Dosage , Humans , Membrane Transport Proteins/genetics , Proto-Oncogene Proteins c-bcr/genetics , RNA, Messenger/metabolism , Reference StandardsABSTRACT
OBJECTIVE: The PINCH-2 gene was previously shown to be overexpressed in malignant mesothelioma compared with ovarian/peritoneal serous carcinoma in Affymetrix array analysis. The objective of the present study was to validate this finding at the mRNA and protein level. METHODS: Effusions (n = 91; 71 ovarian and 10 breast carcinomas, 10 malignant mesotheliomas) were assayed for PINCH-2 mRNA expression using quantitative PCR. PINCH-2 protein expression was analysed in 37 effusions using flow cytometry. RESULTS: Quantitative PCR analysis showed significantly higher PINCH-2 mRNA levels in mesotheliomas compared with carcinomas (P = 0.004). Values of <10 copies were found exclusively in carcinoma effusions (25.4% of ovarian and 50% of breast carcinomas). However, PINCH-2 protein expression by flow cytometry did not differ significantly between the three cancer types. No association was observed between PINCH-2 levels and patient survival or expression of previously-studied molecules related to adhesion, metastasis and apoptosis inhibition in ovarian carcinoma. CONCLUSIONS: Our data suggest that PINCH-2 mRNA is overexpressed in malignant mesothelioma compared with carcinomas involving serosal cavities, and that low levels of this gene argue against the diagnosis of mesothelioma. The frequent PINCH-2 protein expression in all three studied cancers suggests a role for this molecule in cancer cell biology in effusions and merits further research.
Subject(s)
Adenocarcinoma/genetics , Breast Neoplasms/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Mesothelioma/genetics , Neoplasms, Cystic, Mucinous, and Serous/genetics , Ovarian Neoplasms/genetics , Adaptor Proteins, Signal Transducing , Adenocarcinoma/diagnosis , Adenocarcinoma/pathology , Biomarkers, Tumor/analysis , Breast Neoplasms/diagnosis , Breast Neoplasms/pathology , DNA Primers , DNA-Binding Proteins/metabolism , Diagnosis, Differential , Exudates and Transudates , Female , Flow Cytometry , Gene Expression Profiling , Humans , LIM Domain Proteins , Male , Membrane Proteins , Mesothelioma/diagnosis , Mesothelioma/pathology , Neoplasms, Cystic, Mucinous, and Serous/diagnosis , Neoplasms, Cystic, Mucinous, and Serous/pathology , Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/pathology , Polymerase Chain Reaction , RNA, Messenger/metabolismABSTRACT
BACKGROUND: Enhanced cyclooxygenase-2 (COX-2) expression is associated with carcinogenesis, ischemia, angiogenesis, inflammation, and neurodegeneration. The preventing effect of aspirin and nonsteroidal anti-inflammatory drugs is partly due to inhibition of the COX-2 enzyme. Fruit and vegetables (FVs) contain numerous compounds that may decrease disease risk by several different mechanisms, for example through the inhibition of COX-2 activity. OBJECTIVE: We tested the hypothesis that an increased intake of FVs would modulate the COX-2 expression in peripheral blood cells. DESIGN: A strictly controlled dietary crossover study (n = 39). After 1 week run-in period with no FVs in the diet, one group was given two portions of FVs (2 FV), while another group was given five portions (5 FV) daily for 14 days. Following a 2 weeks washout period and 1 week run-in, the regimens were switched between the groups. Gene expression analysis of COX-2 mRNA in blood samples was performed by quantitative real-time-PCR. RESULTS: No significant treatment effect of diet intervention was found in the crossover analyses (P = 0.74). However, the individual variation in response may seem large. CONCLUSIONS: These data does not contradict the recommendations for an increased intake of FVs. Further studies on expression directly and indirectly, through analysis of factors regulating and being regulated by COX-2, should be carried out. A first step would be to evaluate the correspondence between COX-2 mRNA expression and products of the COX pathway, like prostaglandins. Naturally occurring polymorphisms of COX-2 promoters and coding regions might contribute to functional variations and response to different diets. SPONSORSHIP: Norwegian Research Council, National Nutrition Council, Throne Holst Foundation for Nutrition Research, Freia Chokoladefabriks Medisinske Fond and the Norwegian Cancer Society.
