DNA Microarray Format for Detection and Subtyping of Human Papillomavirus

A new human papillomavirus (HPV) assay using high-density DNA microarrays is described. An HPV DNA fragment from the 3′ end of the E1 gene was amplified and digoxigenin labeled by PCR, and the resulting amplicons were hybridized onto type-specific oligonucleotides immobilized on high-density DNA microarrays. For detection, a simple immunohistochemical staining procedure was used with a substrate that has both colorimetric and fluorescent properties. This detection chemistry enables the rapid identification of reactive spots by regular light microscopy and semiquantification by laser scanning. Both single and multiple HPV infections are recognized by this assay, and the corresponding HPV types are easily identified. With this assay, 53 mucosal HPV types were detected and identified. A total of 45 HPV types were identified by a single type-specific probe, whereas the remaining 8 mucosal HPV types could be identified by a specific combination of probes. The simple assay format allows usage of this assay without expensive equipment, making it accessible to all diagnostic laboratories with PCR facilities

Whole-exome sequencing of a meningeal melanocytic tumour reveals activating CYSLTR2 and EIF1AX hotspot mutations and similarities to uveal melanoma

Contains fulltext : 193505.pdf (publisher's version ) (Closed access

DNA hypermethylation analysis in sputum of asymptomatic subjects at risk for lung cancer participating in the NELSON trial: Argument for maximum screening interval of 2years

Aims Lung cancer is the major contributor to cancer mortality due to metastasised disease at time of presentation. The current study investigated DNA hypermethylation of biomarkers RASSF1A, APC, cytoglobin, 3OST2, FAM19A4, PHACTR3 and PRDM14 in sputum of asymptomatic high-risk individuals from the NELSON lung cancer low-dose spiral CT screening trial to detect lung cancer at preclinical stage. Methods Subjects were selected with (i) lung cancer in follow-up (cases; n=65), (ii) minor cytological aberrations (controls; n=120) and (iii) a random selection of subjects without cytological aberrations (controls; n=99). Median follow-up time for controls was 80months. Cut-off values were based on high specificity to assess diagnostic value of the biomarkers. ResultsRASSF1A may denote presence of invasive cancer because of its high specificity (93% (95% CI 89% to 96%); sensitivity 17% (95% CI 4% to 31%), with best performance in a screening interval of 2 years. The panel of RASSF1A, 3OST2 and PRDM14 detected 28% (95% CI 11% to 44%) of lung cancer cases within 2years, with specificity of 90% (95% CI 86% to 94%). Sputum cytology did not detect any lung cancers. Conclusions In a lung cancer screening setting with maximum screening interval of 2years, DNA hypermethylation analysis in sputum may play a role in the detection of preclinical disease, but complementary diagnostic markers are needed to improve sensitivity

EGFR and KRAS quality assurance schemes in pathology: generating normative data for molecular predictive marker analysis in targeted therapy

Item does not contain fulltextINTRODUCTION: The aim of this study was to compare the reproducibility of epidermal growth factor receptor (EGFR) immunohistochemistry (IHC), EGFR gene amplification analysis, and EGFR and KRAS mutation analysis among different laboratories performing routine diagnostic analyses in pathology in The Netherlands, and to generate normative data. METHODS: In 2008, IHC, in-situ hybridisation (ISH) for EGFR, and mutation analysis for EGFR and KRAS were tested. Tissue microarray sections were distributed for IHC and ISH, and tissue sections and isolated DNA with known mutations were distributed for mutation analysis. In 2009, ISH and mutation analysis were evaluated. False-negative and false-positive results were defined as different from the consensus, and sensitivity and specificity were estimated. RESULTS: In 2008, eight laboratories participated in the IHC ring study. In only 4/17 cases (23%) a consensus score of >/=75% was reached, indicating that this analysis was not sufficiently reliable to be applied in clinical practice. For EGFR ISH, and EGFR and KRAS mutation analysis, an interpretable result (success rate) was obtained in >/=97% of the cases, with mean sensitivity >/=96% and specificity >/=95%. For small sample proficiency testing, a norm was established defining outlier laboratories with unsatisfactory performance. CONCLUSIONS: The result of EGFR IHC is not a suitable criterion for reliably selecting patients for anti-EGFR treatment. In contrast, molecular diagnostic methods for EGFR and KRAS mutation detection and EGFR ISH may be reliably performed with high accuracy, allowing treatment decisions for lung cancer.1 oktober 201