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Mainz, Germany

Kneip C.,Theracode GmbH | Schmidt B.,Universitatsklinikum Halle Saale | Seegebarth A.,Bavarian Nordic | Weickmann S.,Charite - Medical University of Berlin | And 4 more authors.
Journal of Thoracic Oncology | Year: 2011

Introduction: Recently, analysis of DNA methylation of the SHOX2 locus was shown to reliably identify lung cancer in bronchial aspirates of patients with disease. As a plasma-based assay would expand the possible applications of the SHOX2 biomarker, this study aimed to develop a modified SHOX2 assay for use in a blood-based test and to analyze the performance of this optimized SHOX2 methylation assay in plasma. Methods: Quantitative real-time polymerase chain reaction was used to analyze DNA methylation of SHOX2 in plasma samples from 411 individuals. A training study (20 stage IV patients with lung cancer and 20 controls) was performed to show the feasibility of detecting the SHOX2 biomarker in blood and to determine a methylation cutoff for patient classification. The resulting cutoff was verified in a testing study composed of 371 plasma samples from patients with lung cancer and controls. Results: DNA methylation of SHOX2 could be used as a biomarker to distinguish between malignant lung disease and controls at a sensitivity of 60% (95% confidence interval: 53-67%) and a specificity of 90% (95% confidence interval: 84-94%). Cancer in patients with stages II (72%), III (55%), and IV (83%) was detected at a higher sensitivity when compared with stage I patients. Small cell lung cancer (80%) and squamous cell carcinoma (63%) were detected at the highest sensitivity when compared with adenocarcinomas. Conclusions: SHOX2 DNA methylation is a biomarker for detecting the presence of malignant lung disease in blood plasma from patients with lung cancer. © 2011 by the International Association for the Study of Lung Cancer. Source

Schneider K.U.,University of Heidelberg | Dietrich D.,Epigenomics AG | Fleischhacker M.,Charite - Medical University of Berlin | Leschber G.,ELK Berlin Chest Hospital | And 11 more authors.
BMC Cancer | Year: 2011

Background: DNA methylation in the SHOX2 locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with SHOX2 gene expression and/or copy number alterations. An amplification of the SHOX2 gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples.Methods: SHOX2 expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect SHOX2 DNA methylation levels. SHOX2 expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH.Results: A hypermethylation of the SHOX2 locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the SHOX2 gene showed no difference.Conclusions: Frequent gene amplification correlated with hypermethylation of the SHOX2 gene locus. This concerted effect qualifies SHOX2 DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples. © 2011 Schneider et al; licensee BioMed Central Ltd. Source

Lower M.,Johannes Gutenberg University Mainz | Renard B.Y.,Johannes Gutenberg University Mainz | Renard B.Y.,Robert Koch Institute | de Graaf J.,Johannes Gutenberg University Mainz | And 10 more authors.
PLoS Computational Biology | Year: 2012

Next generation sequencing (NGS) has enabled high throughput discovery of somatic mutations. Detection depends on experimental design, lab platforms, parameters and analysis algorithms. However, NGS-based somatic mutation detection is prone to erroneous calls, with reported validation rates near 54% and congruence between algorithms less than 50%. Here, we developed an algorithm to assign a single statistic, a false discovery rate (FDR), to each somatic mutation identified by NGS. This FDR confidence value accurately discriminates true mutations from erroneous calls. Using sequencing data generated from triplicate exome profiling of C57BL/6 mice and B16-F10 melanoma cells, we used the existing algorithms GATK, SAMtools and SomaticSNiPer to identify somatic mutations. For each identified mutation, our algorithm assigned an FDR. We selected 139 mutations for validation, including 50 somatic mutations assigned a low FDR (high confidence) and 44 mutations assigned a high FDR (low confidence). All of the high confidence somatic mutations validated (50 of 50), none of the 44 low confidence somatic mutations validated, and 15 of 45 mutations with an intermediate FDR validated. Furthermore, the assignment of a single FDR to individual mutations enables statistical comparisons of lab and computation methodologies, including ROC curves and AUC metrics. Using the HiSeq 2000, single end 50 nt reads from replicates generate the highest confidence somatic mutation call set. © 2012 Löwer et al. Source

Dietrich D.,Epigenomics AG | Kneip C.,Theracode GmbH | Raji O.,University of Liverpool | Liloglou T.,University of Liverpool | And 12 more authors.
International Journal of Oncology | Year: 2012

In the identification of subjects with lung cancer, increased DNA methylation of the SHOX2 gene locus in bronchial aspirates has previously been proven to be a clinically valuable biomarker. This is particularly true in cases where the cytological and histological results following bronchoscopy are undetermined. This previous case control study was conducted using research assay components and a complex work flow. To facilitate the use in a diagnostic setting, a CE marked in vitro diagnostic test kit to quantify SHOX2 DNA methylation in bronchial aspirates was developed and characterized. The presented assay for measuring SHOX2 DNA methylation in bronchial aspirates is based on two major steps: generation of bisulfite converted template DNA from patient samples followed by subsequent determination of SHOX2 biomarker methylation by real-time PCR. Individual kits for DNA preparation, real-time PCR analysis and work flow control were developed. This study describes the analytical performance (reproducibility, accuracy, interfering substances, cross-reactivity) of the in vitro diagnostic (IVD) test kit 'Epi proLung BL Reflex Assay'. In addition, the intended use of the test was validated in a clinical performance evaluation (case control) study comprised of 250 patients (125 cases, 125 controls). The results describe the test as a robust and reliable diagnostic tool for identifying patients with lung cancer using Saccomanno-fixed bronchial lavage specimens (AUC [95% confidence intervals] = 0.94 [0.91-0.98], sensitivity 78% [69-86]/specificity 96% [90-99]). This test may be used as a diagnostic adjunct to existing clinical and pathological investigations in lung cancer. Source

Boisguerin V.,Johannes Gutenberg University Mainz | Boisguerin V.,BioNTech AG | Castle J.C.,Johannes Gutenberg University Mainz | Loewer M.,Johannes Gutenberg University Mainz | And 11 more authors.
British Journal of Cancer | Year: 2014

Cancer is a disease caused by DNA mutations. Cancer therapies targeting defined functional mutations have shown clinical benefit. However, as 95% of the mutations in a tumour are unique to that single patient and only a small number of mutations are shared between patients, the addressed medical need is modest. A rapidly determined patient-specific tumour mutation pattern combined with a flexible mutation-targeting drug platform could generate a mutation-targeting individualised therapy, which would benefit each single patient. Next-generation sequencing enables the rapid identification of somatic mutations in individual tumours (the mutanome). Immunoinformatics enables predictions of mutation immunogenicity. Mutation-targeting RNA-based vaccines can be rapidly and affordably synthesised as custom GMP drug products. Integration of these cutting-edge technologies into a clinically applicable process holds the promise of a disruptive innovation benefiting cancer patients. Here, we describe our translation of the individualised RNA-based cancer vaccine concept into clinic trials. © 2014 Cancer Research UK. All rights reserved. Source

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