Maria Jung

Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.

Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

Formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.

The Immune Checkpoint Regulator PD-L1 Is Highly Expressed in Aggressive Primary Prostate Cancer

Purpose: Therapies targeting the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway promote anti-tumor immunity and have shown promising results in various tumors. Preliminary data further indicate that immunohistochemically detected PD-L1 may be predictive for anti-PD-1 therapy. So far, no data are available on PD-L1 expression in primary prostate cancer. Experimental Design: Following validation of a monoclonal antibody, immunohistochemical analysis of PD-L1 expression was performed in two independent, well-characterized cohorts of primary prostate cancer patients following radical prostatectomy (RP), and resulting data were correlated to clinicopathological parameters and outcome. Results: In the training cohort (n = 209), 52.2% of cases expressed moderate to high PD-L1 levels, which positively correlated with proliferation (Ki-67, P < 0.001), Gleason score (P = 0.004), and androgen receptor (AR) expression (P < 0.001). Furthermore, PD-L1 positivity was prognostic for biochemical recurrence [BCR; P = 0.004; HR, 2.37; 95% confidence interval (CI), 1.32–4.25]. In the test cohort (n = 611), moderate to high PD-L1 expression was detected in 61.7% and remained prognostic for BCR in univariate Cox analysis (P = 0.011; HR, 1.49; 95% CI, 1.10–2.02). The correlation of Ki-67 and AR with PD-L1 expression was confirmed in the test cohort (P < 0.001). In multivariate Cox analysis of all patients, PD-L1 was corroborated as independently prognostic for BCR (P = 0.007; HR, 1.46; 95% CI, 1.11–1.92). Conclusions: We provide first evidence that expression of the therapy target PD-L1 is not only highly prevalent in primary prostate cancer cells but is also an independent indicator of BCR, suggesting a biologic relevance in primary tumors. Further studies need to ascertain if PD-1/PD-L1–targeted therapy might be a treatment option for hormone-naïve prostate cancers. Clin Cancer Res; 22(8); 1969–77. ©2015 AACR.

CDO1 promoter methylation is associated with gene silencing and is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients

ABSTRACT Molecular biomarkers may facilitate the distinction between aggressive and clinically insignificant prostate cancer (PCa), thereby potentially aiding individualized treatment. We analyzed cysteine dioxygenase 1 (CDO1) promoter methylation and mRNA expression in order to evaluate its potential as prognostic biomarker. CDO1 methylation and mRNA expression were determined in cell lines and formalin-fixed paraffin-embedded prostatectomy specimens from a first cohort of 300 PCa patients using methylation-specific qPCR and qRT-PCR. Univariate and multivariate Cox proportional hazards and Kaplan-Meier analyses were performed to evaluate biochemical recurrence (BCR)-free survival. Results were confirmed in an independent second cohort comprising 498 PCa cases. Methylation and mRNA expression data from the second cohort were generated by The Cancer Genome Atlas (TCGA) Research Network by means of Infinium HumanMethylation450 BeadChip and RNASeq. CDO1 was hypermethylated in PCa compared to normal adjacent tissues and benign prostatic hyperplasia (P < 0.001) and was associated with reduced gene expression (ρ = −0.91, P = 0.005). Using two different methodologies for methylation quantification, high CDO1 methylation as continuous variable was associated with BCR in univariate analysis (first cohort: HR = 1.02, P = 0.002, 95% CI [1.01–1.03]; second cohort: HR = 1.02, P = 0.032, 95% CI [1.00–1.03]) but failed to reach statistical significance in multivariate analysis. CDO1 promoter methylation is involved in gene regulation and is a potential prognostic biomarker for BCR-free survival in PCa patients following radical prostatectomy. Further studies are needed to validate CDO1 methylation assays and to evaluate the clinical utility of CDO1 methylation for the management of PCa.

Bisulfite Conversion of DNA from Tissues, Cell Lines, Buffy Coat, FFPE Tissues, Microdissected Cells, Swabs, Sputum, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

Locus-specific analyses of DNA methylation patterns usually require a bisulfite conversion of the DNA, where cytosines are deaminated to uracils, while methylated and hydroxymethylated cytosines remain unaffected. The specific discrimination of hydroxymethylation and methylation can be achieved by introducing an oxidation of 5-hydroxymethylcytosines to 5-formylcytosines and subsequent bisulfite-mediated deamination of 5-formylcytosines.DNA methylation analysis of cell-free circulating DNA in liquid biopsies, i.e., blood samples (serum and plasma), urine, aspirates, bronchial lavages, pleural effusions, and ascites, is of great interest in clinical research. However, due to the generally low concentration of circulating cell-free DNA in body fluids, high volumes need to be analyzed. A reduction of this volume, e.g., by means of a polymer-mediated enrichment, is required in order to facilitate the bisulfite conversion. Further, these sample types usually contain a cellular fraction which is of additional interest and requires specific protocols for the sample preparation.Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used source for tissue-based clinical research. Due to degradation and covalent modifications of DNA in FFPE tissue samples, optimized protocols for the DNA preparation and bisulfite conversion are required.This chapter describes methods and protocols for the sample preparation and subsequent high-speed bisulfite conversion and DNA clean-up for several types of relevant samples, i.e., serum, plasma, urine, buffy coat, aspirates, sputum, lavages, effusions, ascites, swabs, fresh tissues, cell lines, FFPE tissues, and laser microdissected cells.Additionally, two real-time PCR assays for DNA quantification and quality control are described. The cytosine-free fragment (CFF) assay allows for the simultaneous quantification of bisulfite converted and total DNA and thus the determination of bisulfite conversion efficiency. The Mer9 real-time PCR assay amplifies the bisulfite converted sequence of the repetitive element Mer9 and enables the accurate quantification of minute DNA amounts, as present in microdissected cells and body fluids.

CXCL12 promoter methylation and PD-L1 expression as prognostic biomarkers in prostate cancer patients.

Background The CXCR4/CXCL12 axis plays a central role in systemic metastasis of prostate carcinoma (PCa), thereby representing a promising target for future therapies. Recent data suggest that the CXCR4/CXCL12 axis is functionally linked to the PD-1/PD-L1 immune checkpoint. We evaluated the prognostic value of aberrant CXCL12 DNA methylation with respect to PD-L1 expression in primary PCa. Results CXCL12 methylation showed a consistent significant correlation with Gleason grading groups in both cohorts (p < 0.001 for training and p = 0.034 for testing cohort). Short BCR-free survival was significantly associated with aberrant CXCL12 methylation in both cohorts and served as an independent prognostic factor in the testing cohort (hazard ratio = 1.92 [95%CI: 1.12–3.27], p = 0.049). Concomitant aberrant CXCL12 methylation and high PD-L1 expression was significantly associated with shorter BCR-free survival (p = 0.005). In comparative analysis, the CXCL12 methylation assay was able to provide approximately equivalent results in biopsy and prostatectomy specimens. Materials and Methods CXCL12 methylation was determined by means of a methylation specific quantitative PCR analysis in a radical prostatectomy patient cohort (n = 247, training cohort). Data published by The Cancer Genome Atlas served as a testing cohort (n = 498). CXCL12 methylation results were correlated to clinicopathological parameters including biochemical recurrence (BCR)-free survival. Conclusions CXCL12 methylation is a powerful prognostic biomarker for BCR in PCa patients after radical prostatectomy. Further studies need to ascertain if CXCL12 methylation may aid in planning active surveillance strategies.

DNA Methylation Analysis of Free-Circulating DNA in Body Fluids

Circulating cell-free DNA in body fluids is an analyte of great interest in basic and clinical research. The analyses of DNA methylation and hydroxymethylation patterns in body fluids might allow one to determine the certain state of a disease, in particular of cancer. DNA methylation biomarkers in liquid biopsies, i.e. blood plasma samples, may help optimizing personalized therapy for individual patients. DNA methylation analyses of specific loci usually require a bisulfite conversion of the DNA, which requires a sufficiently high amount of DNA at the appropriate concentration. However, free-circulating DNA is generally low concentrated. Therefore, high volumes of body fluids need to be analyzed. This high volume needs to be reduced in order to facilitate the bisulfite conversion. In addition, disease-related free-circulating DNA is even less abundant than normal DNA in the total amount of free-circulating DNA. Accordingly, analytical and pre-analytical methods are needed, which permit an accurate and sensitive quantification of single methylated DNA copies in the presence of unmethylated DNA in abundance.This protocol describes two methods for DNA enrichment from body fluids: DNA extraction by means of magnetic beads and polymer-mediated enrichment of DNA. Subsequent bisulfite conversion is achieved by means of a high-speed conversion protocol. Adaptions of the workflow required for the analysis of hydroxymethylation via oxidation 5-hydroxymethylcytosines to 5-formylcytosines prior to the bisulfite conversion are introduced. A quantitative real-time PCR based on the methylation-specific and HeavyMethyl PCR methodologies is introduced. This qPCR assay allows for an accurate and sensitive quantification of single copies of the DNA methylation biomarkers SHOX2 and SEPT9 in blood plasma. Specific issues regarding the analysis of body fluids and respective trouble shooting approaches are discussed.