Evaluation of Different Blood Collection Tubes and Blood Storage Conditions for the Preservation and Stability of Cell-Free Circulating DNA for the Analysis of the Methylated mSEPT9 Colorectal Cancer Screening Marker.

For the subsequent analysis of the methylated mSEPT9 colorectal cancer screening marker in plasma, different blood collection tubes and blood storage conditions were investigated. The study demonstrated that methylated Septin 9 (mSEPT9) can be consistently detected in plasma samples derived from whole blood samples collected with S-Monovette® K3E and BD Vacutainer ® K2EDTA tubes stored at 2-8 °C for a maximum of 24 h and for samples collected in S-Monovette CPDA tubes stored at 18-25 °C for up to 48 h.

SHOX2 DNA methylation is a biomarker for the diagnosis of lung cancer based on bronchial aspirates.

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.

Circulating methylated SEPT9 DNA in plasma is a biomarker for colorectal cancer.

BACKGROUND: The presence of aberrantly methylated SEPT9 DNA in plasma is highly correlated with the occurrence of colorectal cancer. We report the development of a new SEPT9 biomarker assay and its validation in case-control studies. The development of such a minimally invasive blood-based test may help to reduce the current gap in screening coverage. METHODS: A new SEPT9 DNA methylation assay was developed for plasma. The assay comprised plasma DNA extraction, bisulfite conversion of DNA, purification of bisulfite-converted DNA, quantification of converted DNA by real-time PCR, and measurement of SEPT9 methylation by real-time PCR. Performance of the SEPT9 assay was established in a study of 97 cases with verified colorectal cancer and 172 healthy controls as verified by colonoscopy. Performance based on predetermined algorithms was validated in an independent blinded study with 90 cases and 155 controls. RESULTS: The SEPT9 assay workflow yielded 1.9 microg/L (CI 1.3-3.0) circulating plasma DNA following bisulfite conversion, a recovery of 45%-50% of genomic DNA, similar to yields in previous studies. The SEPT9 assay successfully identified 72% of cancers at a specificity of 93% in the training study and 68% of cancers at a specificity of 89% in the testing study. CONCLUSIONS: Circulating methylated SEPT9 DNA, as measured in the new (m)SEPT9 assay, is a valuable biomarker for minimally invasive detection of colorectal cancer. The new assay is amenable to automation and standardized use in the clinical laboratory.

DNA methylation of the PITX2 gene promoter region is a strong independent prognostic marker of biochemical recurrence in patients with prostate cancer after radical prostatectomy.

PURPOSE: Approximately 35% of patients with prostate cancer who undergo radical prostatectomy experience prostate specific antigen recurrence within 10 years of surgery. Current prognostic indicators cannot sufficiently detect who is at risk for biochemical recurrence. We evaluated DNA methylation markers for prostate cancer prognosis. MATERIALS AND METHODS: We assessed the DNA methylation of 6 marker candidates that were identified in previous studies. Formalin fixed, paraffin embedded tissue sections from a cohort of 605 patients who underwent radical prostatectomy were analyzed using real-time polymerase chain reaction assays. Using a Cox proportional hazard model we determined which markers were significant predictors of biochemical recurrence. RESULTS: ABHD9, Chr3-EST, GPR7, HIST2H2BF and PITX2 were significantly associated with biochemical recurrence. PITX2 methylation was the strongest predictor of biochemical recurrence, providing additional prognostic information to established clinical factors in patients treated with radical prostatectomy and especially in patients at intermediate risk (Gleason 7). Patients with greater than median PITX2 methylation in the tumors were 4 times more likely to experience biochemical recurrence within 8 years after surgery than patients with less than average methylation. CONCLUSIONS: The prognostic information provided by PITX2 methylation adds significantly to currently used clinical variables such as Gleason grade and stage. Therefore, it could contribute to better counseling in patients with prostate cancer.

Quantification of methylated DNA by HeavyMethyl duplex PCR.

The HeavyMethyl (HM) assay is a real-time PCR assay suitable for the qualitative and quantitative DNA methylation analysis of fresh, frozen, or formalin-fixed paraffin-embedded tissues and remote samples, such as serum, plasma, and urine. The HM uses a methylation-specific oligonucleotide blocker and a methylation-specific probe to achieve methylation-specific amplification and detection. A protocol for a duplex real-time PCR for the analysis of the methylation status of the GSTP1 exon1 in prostate tissue samples is presented. This chapter describes the preparation and analysis of a combined HM GSTP1 Exon1 and GSTP1 reference assay.

Novel method for high throughput DNA methylation marker evaluation using PNA-probe library hybridization and MALDI-TOF detection.

The methylation of CpG dinucleotides has become a topic of great interest in cancer research, and the methylation of promoter regions of several tumor suppressor genes has been identified as a marker of tumorigenesis. Evaluation of DNA methylation markers in tumor tissue requires hundreds of samples, which must be analyzed quantitatively due to the heterogeneous composition of biological material. Therefore novel, fast and inexpensive methods for high throughput analysis are needed. Here we introduce a new assay based on peptide nucleic acid (PNA)-library hybridization and subsequent MALDI-TOF analysis. This method is multiplexable, allows the use of standard 384 well automated pipetting, and is more specific and flexible than established methods, such as microarrays and MS-SNuPE. The approach was used to evaluate three candidate colon cancer methylation markers previously identified in a microarray study. The methylation of the genes Ade-nomatous polyposis coli (APC), glycogen synthase kinase-beta-3 (GSK3beta) and eyes absent 4 (EYA4) was analyzed in 12 colon cancer and 12 normal tissues. APC and EYA4 were confirmed as being differentially methylated in colon cancer patients whereas GSK3beta did not show differential methylation.

A real-time PCR assay for DNA-methylation using methylation-specific blockers.

DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.

Tumour class prediction and discovery by microarray-based DNA methylation analysis.

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers.

Performance evaluation of the DNA methylation biomarker SHOX2 for the aid in diagnosis of lung cancer based on the analysis of bronchial aspirates.

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.

Quantitative DNA methylation profiling on a high-density oligonucleotide microarray.

Recently, the analysis and functional elucidation of CpG island methylation has become a focus area of genomic research. Deviations from the normal parental imprinting pattern have been shown to cause developmental defects associated with serious symptoms. Aberrant DNA methylation of tumor suppressor and other functional genes, especially when found in 5' untranslated regions and early exons, has been associated with tumorigenesis. In the context of applying DNA methylation analysis for the molecular characterization of cancer and other diseases, standardized protocols enabling parallel genome-wide methylation profiling of numerous samples are required. DNA methylation profiling is described using a CpG island microarray representing more than 50,000 CpG-rich DNA fragments. Fragments were selected to represent the vast majority of known 5'-untranslated regions as well as the first exons of thousands of genes. Measurement probes were designed to represent these fragments were displayed on an Affymetrix custom array. A modified procedure for differential methylation hybridization (DMH) is described for methylation enrichment. Application of a novel signal normalization concept enables accurate and reproducible measurements using a single fluorescence channel. The use of defined calibrator material allows quantification of DNA methylation patterns by DMH in a massively parallel fashion.

Performance of epigenetic markers SEPT9 and ALX4 in plasma for detection of colorectal precancerous lesions.

BACKGROUND: Screening for colorectal cancer (CRC) has shown to reduce cancer-related mortality, however, acceptance and compliance to current programmes are poor. Developing new, more acceptable non-invasive tests for the detection of cancerous and precancerous colorectal lesions would not only allow preselection of individuals for colonoscopy, but may also prevent cancer by removal of precancerous lesions. METHODS: Plasma from 128 individuals (cohort I - exploratory study: 73 cases / 55 controls) was used to test the performance of a single marker, SEPT9, using a real-time quantitative PCR assay. To validate performance of SEPT9, plasma of 76 individuals (cohort II - validation study: 54 cases / 22 controls) was assessed. Additionally, improvement of predictive capability considering SEPT9 and additionally ALX4 methylation was investigated within these patients. RESULTS: In both cohorts combined, methylation of SEPT9 was observed in 9% of controls (3/33), 29% of patients with colorectal precancerous lesions (27/94) and 73% of colorectal cancer patients (24/33). The presence of both SEPT9 and ALX4 markers was analysed in cohort II and was observed in 5% of controls (1/22) and 37% of patients with polyps (18/49). Interestingly, also 3/5 (60%) patients with colorectal cancer were tested positive by the two marker panel in plasma. CONCLUSIONS: While these data confirm the detection rate of SEPT9 as a biomarker for colorectal cancer, they also show that methylated DNA from advanced precancerous colorectal lesions can be detected using a panel of two DNA methylation markers, ALX4 and SEPT9. If confirmed in larger studies these data indicate that screening for colorectal precancerous lesions with a blood-based test may be as feasible as screening for invasive cancer.

A spectinomycin resistance determinant from the spectinomycin producer Streptomyces flavopersicus.

The spectinomycin (sp) resistance determinant from Streptomyces flavopersicus was cloned into Streptomyces lividans using the plasmid vector pIJ699. A plasmid, pDGL15, with a 3.65 kb insert from S. flavopersicus conferring resistance to Sp was isolated. DNA sequence analysis of the 3651 1 bp DNA insert revealed four open reading frames (ORFs). The amino acid sequence deduced from one ORF (SpcN) showed a high degree of similarity to an aminoglycoside phosphotransferase (StrN) and from a second one (SpcR) to a regulatory protein (StrR) of the streptomycin biosynthesis gene cluster from S. griseus. The two other ORFs were incomplete and the deduced amino acid sequences showed similarities to an amidinotransferase encoded in the streptomycin biosynthesis gene cluster of S. griseus and to the transposase of IS112, respectively. Expression of the spcN gene in E. coli under the control of tac promoter conferred Sp resistance to the cells. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent aminoglycoside phosphotransferase which phosphorylates Sp and actinamine, the aminocyclitol moiety of Sp.