Aflibercept Suppression of Angiopoietin-2 in a Rabbit Retinal Vascular Hyperpermeability Model.

Purpose: Anti-vascular endothelial growth factor (anti-VEGF) therapies, which attenuate the capacity of VEGF to bind to VEGF receptors, are standard-of-care options for various retinal disorders that are characterized by pathologic retinal angiogenesis and vascular permeability. Multiple receptors and ligands have also been reported as being involved in these pathways, including angiopoietin-1 (ANG1) and angiopoietin-2 (ANG2). Methods: Electrochemiluminescence immunoassays were used to detect human VEGF (hVEGF), as well as rabbit ANG2 and basic fibroblast growth factor protein levels in vitreous samples derived from a study evaluating the efficacy of the anti-VEGF agents ranibizumab, aflibercept, and brolucizumab in an hVEGF165-induced rabbit retinal vascular hyperpermeability model. Results: hVEGF was completely suppressed in rabbit vitreous after anti-VEGF treatment for 28 days. ANG2 protein in vitreous and ANGPT2 mRNA in retina tissue were similarly suppressed, although the anti-VEGF agents do not directly bind to ANG2. Aflibercept demonstrated the greatest inhibitory effect in ANG2 levels in vitreous, which correlated with strong, durable suppression of intraocular hVEGF levels. Conclusions: This study explored the effects of anti-VEGF therapies beyond direct binding of VEGF by evaluating protein levels and the expression of target genes involved in angiogenesis and associated molecular mechanisms in the rabbit retina and choroid. Translational Relevance: In vivo data suggest that anti-VEGF agents currently used for the treatment of retinal diseases could provide beneficial effects beyond direct binding of VEGF, including suppression of ANG2 protein and ANGPT2 mRNA.

Validation of the SHOX2/PTGER4 DNA Methylation Marker Panel for Plasma-Based Discrimination between Patients with Malignant and Nonmalignant Lung Disease.

INTRODUCTION: Low-dose computed tomography (LDCT) is used for screening for lung cancer (LC) in high-risk patients in the United States. The definition of high risk and the impact of frequent false-positive results of low-dose computed tomography remains a challenge. DNA methylation biomarkers are valuable noninvasive diagnostic tools for cancer detection. This study reports on the evaluation of methylation markers in plasma DNA for LC detection and discrimination of malignant from nonmalignant lung disease. METHODS: Circulating DNA was extracted from 3.5-mL plasma samples, treated with bisulfite using a commercially available kit, purified, and assayed by real-time polymerase chain reaction for assessment of DNA methylation of short stature homeobox 2 gene (SHOX2), prostaglandin E receptor 4 gene (PTGER4), and forkhead box L2 gene (FOXL2). In three independent case-control studies these assays were evaluated and optimized. The resultant assay, a triplex polymerase chain reaction combining SHOX2, PTGER4, and the reference gene actin, beta gene (ACTB), was validated using plasma from patients with and without malignant disease. RESULTS: A panel of SHOX2 and PTGER4 provided promising results in three independent case-control studies examining a total of 330 plasma specimens (area under the receiver operating characteristic curve = 91%-98%). A validation study with 172 patient samples demonstrated significant discriminatory performance in distinguishing patients with LC from subjects without malignancy (area under the curve = 0.88). At a fixed specificity of 90%, sensitivity for LC was 67%; at a fixed sensitivity of 90%, specificity was 73%. CONCLUSIONS: Measurement of SHOX2 and PTGER4 methylation in plasma DNA allowed detection of LC and differentiation of nonmalignant diseases. Development of a diagnostic test based on this panel may provide clinical utility in combination with current imaging techniques to improve LC risk stratification.

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.

Validation of a real-time PCR-based qualitative assay for the detection of methylated SEPT9 DNA in human plasma.

BACKGROUND: Epi proColon® is a new blood-based colorectal cancer (CRC) screening test designed to determine the methylation status of a promoter region of the SEPT9 (septin 9) gene in cell-free DNA isolated from plasma. We describe the analytical and clinical performance of the test. METHODS: Analytical performance at 4 testing laboratories included determination of limit of detection, precision, and reproducibility of the SEPT9 test. Clinical performance was evaluated in a prospective study by use of samples (n = 1544) from subjects enrolled in the PRESEPT clinical trial. Results were analyzed by comparison with colonoscopy, the reference standard. RESULTS: The limit of detection for methylated SEPT9 DNA was 7.8 pg/mL (95% CI 6-11 pg/mL) corresponding to <2 genome copies of methylated SEPT9 per milliliter of plasma. In the prospective clinical trial, sensitivity for all stages of CRC was 68% (95% CI 53%-80%) and for stage I-III CRC, 64% (48%-77%). Adjusted specificity, on the basis of negative colonoscopy findings, was 80.0% (78%-82%). SIGNIFICANCE: The Epi proColon test is a simple, real-time PCR-based assay for the detection of methylated SEPT9 DNA in blood that may provide a noninvasive CRC screening alternative for people noncompliant with current CRC screening guidelines.

TFAP2E-DKK4 and chemoresistance in colorectal cancer.

BACKGROUND: Chemotherapy for advanced colorectal cancer leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential downstream target of TFAP2E and has been implicated in chemotherapy resistance. We aimed to further evaluate the role of TFAP2E and DKK4 as predictors of the response of colorectal cancer to chemotherapy. METHODS: We analyzed the expression, methylation, and function of TFAP2E in colorectal-cancer cell lines in vitro and in patients with colorectal cancer. We examined an initial cohort of 74 patients, followed by four cohorts of patients (total, 220) undergoing chemotherapy or chemoradiation. RESULTS: TFAP2E was hypermethylated in 38 of 74 patients (51%) in the initial cohort. Hypermethylation was associated with decreased expression of TFAP2E in primary and metastatic colorectal-cancer specimens and cell lines. Colorectal-cancer cell lines overexpressing DKK4 showed increased chemoresistance to fluorouracil but not irinotecan or oxaliplatin. In the four other patient cohorts, TFAP2E hypermethylation was significantly associated with nonresponse to chemotherapy (P<0.001). Conversely, the probability of response among patients with hypomethylation was approximately six times that in the entire population (overall estimated risk ratio, 5.74; 95% confidence interval, 3.36 to 9.79). Epigenetic alterations of TFAP2E were independent of mutations in key regulatory cancer genes, microsatellite instability, and other genes that affect fluorouracil metabolism. CONCLUSIONS: TFAP2E hypermethylation is associated with clinical nonresponsiveness to chemotherapy in colorectal cancer. Functional assays confirm that TFAP2E-dependent resistance is mediated through DKK4. In patients who have colorectal cancer with TFAP2E hypermethylation, targeting of DKK4 may be an option to overcome TFAP2E-mediated drug resistance. (Funded by Deutsche Forschungsgemeinschaft and others.).

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.

A combined HM-PCR/SNuPE method for high sensitive detection of rare DNA methylation.

BACKGROUND: DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation. RESULTS: This combined method allows detection of 14 pg (that is, four to five genomic copies) of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng) of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2) and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer. CONCLUSION: The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.

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.

Analysis of DNA methylation of multiple genes in microdissected cells from formalin-fixed and paraffin-embedded tissues.

A procedure for simultaneous quantification of DNA methylation of several genes in minute amounts of sample material was developed and applied to microdissected formalin-fixed and paraffin-embedded breast tissues. The procedure is comprised of an optimized bisulfite treatment protocol suitable for samples containing only few cells, a multiplex preamplification and subsequent locus specific reamplification, and a novel quantitative bisulfite sequencing method based on the incorporation of a normalization domain into the PCR product. A real-time PCR assay amplifying repetitive elements was established to quantify low amounts of bisulfite-treated DNA. Ten prognostic and diagnostic epigenetic breast cancer biomarkers (PITX2, RASSF1A, PLAU, LHX3, PITX3, LIMK1, SLITRK1, SLIT2, HS3ST2, and TFF1) were analyzed in tissue samples obtained from two patients with invasive ductal carcinoma of the breast. The microdissected samples were obtained from several areas within the tumor tissue, including intraductal and invasive carcinoma, adenosis, and normal ductal epithelia of adjacent normal tissue, as well as stroma, tumor infiltrating lymphocytes, and adipose tissue. Overall, reliable quantification was possible for all genes. For most genes, increased DNA methylation in invasive and intraductal carcinoma cells compared with other tissue components was observed. For TFF1, decreased methylation levels were observed in tumor cells.

Prevention of PCR cross-contamination by UNG treatment of bisulfite-treated DNA.

Amplification of sodium bisulfite-treated DNA is widely used to study DNA methylation. The proportion of methylated sequences of a specific DNA region in a sample can be determined by the analysis of PCR products or directly calculated from real-time PCR amplification of bisulfite-treated DNA. At the same time, PCR based methods always involve the risk of false positive or incorrect quantitative results due to the unintended reamplification of contaminating PCR products. The incubation of PCR reactions with Uracil-DNA Glycosylase (UNG) prior to the thermal cycling in combination with the use of dUTP in the PCR amplification is a commonly used technology to prevent such cross-contamination. Since sodium bisulfite treatment converts unmethylated cytosine bases into uracil residues, not only contaminating PCR products but also the converted template DNAs would be degraded as well. This chapter describes a modified bisulfite treatment procedure to generate sulfonated DNA enabling the application of UNG-based carryover prevention to DNA methylation analysis. The high efficiency of the decontamination procedure as well as the universal applicability of this simple method is shown.

DNA methylation biomarkers for blood-based colorectal cancer screening.

BACKGROUND: Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma. METHODS: We first used restriction enzyme-based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range. RESULTS: Restriction enzyme-based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%-73%] of plasma samples from CRC patients and not detected in 69% (62%-76%) of the controls. The corresponding results for NGFR were 51% (42%-60%) and 84% (77%-89%); for SEPT9, the values were 69% (60%-77%) and 86% (80%-91%). CONCLUSIONS: The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.

Control of carry-over contamination for PCR-based DNA methylation quantification using bisulfite treated DNA.

In this study, we adapted the well known uracil DNA glycosylase (UNG) carry-over prevention system for PCR, and applied it to the analysis of DNA methylation based on sodium bisulfite conversion. As sodium bisulfite treatment converts unmethylated cytosine bases into uracil residues, bisulfite treated DNA is sensitive to UNG treatment. Therefore, UNG cannot be used for carry-over prevention of PCR using bisulfite treated template DNA, as not only contaminating products of previous PCR, but also the actual template will be degraded. We modified the bisulfite treatment procedure and generated DNA containing sulfonated uracil residues. Surprisingly, and in contrast to uracil, 6-sulfonyl uracil containing DNA (SafeBis DNA) is resistant to UNG. We showed that the new procedure removes up to 10,000 copies of contaminating PCR product in a closed PCR vessel without significant loss of analytical or clinical sensitivity of the DNA methylation analysis.

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.