Dimo Dietrich

SHOX2 DNA Methylation Is a Biomarker for the Diagnosis of Lung Cancer in Plasma

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.

SHOX2 DNA Methylation is a Biomarker for the diagnosis of lung cancer based on bronchial aspirates

BackgroundThis 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.MethodsMarker 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.ResultsValid 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%]).ConclusionsHypermethylation 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.

DNA methylation markers predict outcome in node-positive, Estrogen receptor-positive breast cancer with adjuvant anthracycline-based chemotherapy

Purpose: We have shown that DNA methylation of the PITX2 gene predicts risk of distant recurrence in steroid hormone receptor-positive, node-negative breast cancer. Here, we present results from a multicenter study investigating whether PITX2 and other candidate DNA methylation markers predict outcome in node-positive, estrogen receptor-positive, HER-2-negative breast cancer patients who received adjuvant anthracycline-based chemotherapy. Experimental Design: Using a microarray platform, we analyzed DNA methylation in regulatory regions of PITX2 and 60 additional candidate genes in 241 breast cancer specimens. Using Cox regression analysis, we assessed the predictive power of the individual marker/marker panel candidates. Clinical endpoints were time to distant metastasis, disease-free survival, and overall survival. A nested bootstrap/cross-validation strategy was applied to identify and validate marker panels. Results: DNA methylation of PITX2 and 14 other genes was correlated with clinical outcome. In multivariate models, each methylation marker added significant information to established clinical factors. A four-marker panel including PITX2, BMP4, FGF4, and C20orf55 was identified that resulted in improvement of outcome prediction compared with PITX2 alone. Conclusions: This study provides further evidence for the PITX2 biomarker, which has now been successfully confirmed to predict outcome among different breast cancer patient populations. We further identify new DNA methylation biomarkers, three of which can be combined into a panel with PITX2 to increase the outcome prediction performance in our anthracycline-treated primary breast cancer population. Our results show that a well-defined panel of DNA methylation markers enables outcome prediction in lymph node-positive, HER-2-negative breast cancer patients treated with anthracycline-based chemotherapy.

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 bio-markers (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. (J Histochem Cytochem 57:477–489, 2009)

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.

DNA methylation of the homeobox genes PITX2 and SHOX2 predicts outcome in non-small-cell lung cancer patients.

Biomarkers that facilitate prediction of disease progression in lung cancer patients might be clinically valuable in optimizing individualized therapy. In this study, the ability of the DNA methylation biomarkers PITX2 and SHOX2 to predict disease outcome in lung cancer patients has been evaluated. Quantitative, methylation-specific (HeavyMethyl), real-time polymerase chain reaction assays were used to measure DNA methylation of PITX2 and SHOX2 in bisulfite-converted DNA from formalin-fixed, paraffin-embedded tissues from 474 non–small-cell lung cancer patients. In univariate Cox Proportional Hazard analysis, high methylation of SHOX2 and PITX2 was a significant predictor of progression-free survival [SHOX2: n=465, hazard ratio (HR)=1.395 (1.130 to 1.721), P=0.002; PITX2: n=445, HR=1.312 (1.059 to 1.625), P=0.013]. Patients with low methylation of either PITX2 and/or SHOX2 (n=319) showed a significantly higher risk of disease progression as compared with patients with higher methylation of both genes [n=126; HR=1.555 (1.210 to 1.999), P=0.001]. This was particularly true for the subgroup of patients receiving no adjuvant radiotherapy or chemotherapy [n=258, HR=1.838 (1.252 to 2.698), P=0.002]. In multivariate analysis, both biomarkers added significant independent prognostic information to pT, pN, pM, and grade. Another interesting finding of this study was that SHOX2 and PITX2 DNA methylation was shown to be inversely correlated with TTF1 (also known as NKX2-1) expression (PITX2: P=0.018, SHOX2: P<0.001). TFF1 expression was previously found to be associated with improved survival in the same patient cohort. DNA methylation of PITX2 and SHOX2 is an independent prognostic biomarker for disease progression in non–small-cell lung cancer patients.

Nucleic acid-based biomarkers in body fluids of patients with urologic malignancies

Abstract This review focuses on the promising potential of nucleic acids in body fluids such as blood and urine as diagnostic, prognostic, predictive and monitoring biomarkers in urologic malignancies. The tremendous progress in the basic knowledge of molecular processes in cancer, as shown in the companion review on nucleic acid-based biomarkers in tissue of urologic tumors, provides a strong rationale for using these molecular changes as non-invasive markers in body fluids. The changes observed in body fluids are an integrative result, reflecting both tissue changes and processes occurring in the body fluids. The availability of sensitive methods has only recently made possible detailed studies of DNA- and RNA-based markers in body fluids. In addition to these biological aspects, methodological aspects of the determination of nucleic acids in body fluids, i.e. pre-analytical, analytical and post-analytical issues, are particularly emphasized. The characteristic changes of RNA (differential mRNA and miRNA expression) and DNA (concentrations, integrity index, mutations, microsatellite and methylation alterations) in serum/plasma and urine samples of patients suffering from the essential urologic cancers of the prostate, bladder, kidney and testis are summarized and critically discussed below. To translate the promising results into clinical practice, laboratory scientists and clinicians have to collaborate to resolve the challenges of harmonized and feasible pre-analytical and analytical conditions for the selected markers and to validate these markers in well-designed and sufficiently powered multi-center studies.

CDO1 Promoter Methylation is a Biomarker for Outcome Prediction of Anthracycline Treated, Estrogen Receptor-Positive, Lymph Node-Positive Breast Cancer Patients

BackgroundVarious biomarkers for prediction of distant metastasis in lymph-node negative breast cancer have been described; however, predictive biomarkers for patients with lymph-node positive (LNP) disease in the context of distinct systemic therapies are still very much needed. DNA methylation is aberrant in breast cancer and is likely to play a major role in disease progression. In this study, the DNA methylation status of 202 candidate loci was screened to identify those loci that may predict outcome in LNP/estrogen receptor-positive (ER+) breast cancer patients with adjuvant anthracycline-based chemotherapy.MethodsQuantitative bisulfite sequencing was used to analyze DNA methylation biomarker candidates in a retrospective cohort of 162 LNP/ER+ breast cancer patients, who received adjuvant anthracycline-based chemotherapy. First, twelve breast cancer specimens were analyzed for all 202 candidate loci to exclude genes that showed no differential methylation. To identify genes that predict distant metastasis, the remaining loci were analyzed in 84 selected cases, including the 12 initial ones. Significant loci were analyzed in the remaining 78 independent cases. Metastasis-free survival analysis was conducted by using Cox regression, time-dependent ROC analysis, and the Kaplan-Meier method. Pairwise multivariate regression analysis was performed by linear Cox Proportional Hazard models, testing the association between methylation scores and clinical parameters with respect to metastasis-free survival.ResultsOf the 202 loci analysed, 37 showed some indication of differential DNA methylation among the initial 12 patient samples tested. Of those, 6 loci were associated with outcome in the initial cohort (n = 84, log rank test, p < 0.05).Promoter DNA methylation of cysteine dioxygenase 1 (CDO1) was confirmed in univariate and in pairwise multivariate analysis adjusting for age at surgery, pathological T stage, progesterone receptor status, grade, and endocrine therapy as a strong and independent biomarker for outcome prediction in the independent validation set (log rank test p-value = 0.0010).ConclusionsCDO1 methylation was shown to be a strong predictor for distant metastasis in retrospective cohorts of LNP/ER+ breast cancer patients, who had received adjuvant anthracycline-based chemotherapy.

Development and clinical validation of a real-time PCR assay for PITX2 DNA methylation to predict prostate-specific antigen recurrence in prostate cancer patients following radical prostatectomy

Prostate cancer is the most common cancer among men. The prospective discrimination of aggressive and clinically insignificant tumors still poses a significant and, as yet, unsolved problem. PITX2 DNA methylation is a strong prognostic biomarker in prostate cancer. Recently, a diagnostic microarray for prostate cancer prognosis based on PITX2 methylation has been developed and validated. Because this microarray requires nonstandard laboratory equipment, its use in a diagnostic setting is limited. This study aimed to develop and validate an alternative quantitative real-time PCR assay for measuring PITX2 methylation that can easily be established in clinical laboratories, thereby facilitating the implementation of this biomarker in clinical practice. A methylation cut-off for patient stratification was established in a training cohort (n = 157) and validated in an independent test set (n = 523) of men treated with radical prostatectomy. In univariate Cox proportional hazards analysis, PITX2 hypermethylation was a significant predictor for biochemical recurrence (P < 0.001, hazard ratio = 2.614). Moreover, PITX2 hypermethylation added significant prognostic information (P = 0.003, hazard ratio = 1.814) to the Gleason score, pathological T stage, prostate-specific antigen, and surgical margins in a multivariate analysis. The clinical performance was particularly high in patients at intermediate risk (Gleason score of 7) and in samples containing high tumor cell content. This assay might aid in risk stratification and support the decision-making process when determining whether a patient might benefit from adjuvant treatment after radical prostatectomy.

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.