Search

Background/aim: Cell-free DNA may serve as a biomarker for patients with cancer; we designed our study to determine its potential in patients with bladder cancer (BCA)., Materials and Methods: Short β-actin (ACTB)-106 and large ACTB-384 fragments were quantified using real time PCR (RT-PCR); the ratio of ACTB-384/ACTB-106 was defined as DNA integrity. We analyzed the serum from 95 patients with and from 132 without BCA., Results: Patients with BCA had increased ACTB-106 levels and lower DNA integrity compared to patients without cancer. However, patients undergoing transurethral bladder resection (TURB) with histological exclusion of BCA had a similar ACTB-106 level and DNA integrity, as patients with BCA. Cell-free DNA was not correlated with smoker status, pT stage, grade or lymph node metastasis, or DNA integrity. There was a weak inverse correlation of age with DNA integrity in patients with BCA., Conclusion: Analysis of serum cell-free DNA levels and fragmentation patterns are of limited value regarding the identification of patients with BCA.

Purpose: Increased levels of cell-free circulating DNA have been described in various malignancies as a diagnostic and prognostic biomarker. We analyzed the significance of cell-free DNA in patients with testicular cancer., Materials and Methods: Cell-free DNA was isolated from the serum of 74 patients with testicular cancer, including 39 with seminoma and 35 with nonseminoma, and 35 healthy individuals. Real-time polymerase chain reaction was used to quantify a 106, a 193 and a 384 actin-beta DNA fragment. DNA integrity is expressed as the ratio of large (193 or 384 bp) to short (106 bp) DNA fragments., Results: Actin-beta-106/193/384 fragment levels were significantly increased in patients with cancer compared to those in healthy individuals (each p <0.001). DNA integrity was significantly decreased in patients with cancer (p <0.001). Cell-free DNA fragment levels were not different when comparing patients with nonseminoma and seminoma (p >0.24). ROC analysis demonstrated that cell-free DNA levels distinguished patients with cancer from healthy individuals with 87% sensitivity and 97% specificity. Even in 31 patients in whom the established serum tumor markers alpha-fetoprotein, human chorionic gonadotropin, placental alkaline phosphatase and lactate dehydrogenase were normal cell-free DNA levels allowed us to distinguish between patients with cancer and healthy individuals with 84% sensitivity and 97% specificity. Cell-free DNA levels were more frequently increased in patients with clinical stage 3 than in patients with stage 1 or 2 disease (p <0.046)., Conclusions: Cell-free DNA levels are increased in patients with testicular cancer and they allow the accurate discrimination of healthy individuals. The high sensitivity of cell-free DNA could facilitate the management of testicular cancer, especially in patients with conventional tumor markers that are not increased.

Our study was designed to evaluate Cell-Free DNA in sera of prostate cancer (PCA) patients as a useful biomarker. Real-time PCR was used to amplify a <200 bp PTGS2 DNA fragment that biochemically characterizes apoptosis and a larger >250 bp Reprimo DNA fragment that defines mostly other cell death entities. The apoptosis index (AI) expresses the ratio of PTGS2 to Reprimo DNA fragments. GSTP1 hypermethylation was assessed to evaluate the amount of tumor-derived DNA. We analyzed serum of 216 patients (168 PCA; 5 incidental PCA; 42 benign prostatic hyperplasia (BPH); 11 healthy individuals). Distinctly elevated concentrations of PTGS2 fragments were detected in PCA compared to BPH and healthy individuals (median: 70.2, 10.5 and 7.1 ng/ml, respectively; p < 0.0001). The AI was significantly increased in PCA vs. BPH patients and healthy individuals (6.01 vs. 1.54 and 0.84 respectively; p = 0.002-0.0001). GSTP1 hypermethylation was only present in a small percentage (mean 1.92%) of circulating DNA. Concentrations of apoptotic PTGS2 fragments discriminated sensitively (88%) and specifically (64%) between BPH and PCA, whereas the AI was more specific (82%) but less sensitive (70%). The AI correlated with histological grading (p = 0.044). Kaplan-Meier analysis for a subset of 124 patients revealed a significant correlation between apoptotic PTGS2 fragments or the AI and PSA recurrence following radical prostatectomy (p = 0.0395-0.0482). In conclusion, circulating PTGS2 fragments of apoptotic origin and the AI are promising serum biomarkers for the diagnosis and prognosis of PCA. We suggest that cancer-induced apoptosis of peripheral noncancerous tissues is relevant in many malignancies., (Copyright 2007 Wiley-Liss, Inc.)

Purpose: CpG island hypermethylation is a frequent event in bladder carcinogenesis and progression. We investigated the diagnostic and prognostic value of hypermethylation in cell-free serum DNA of patients with bladder cancer., Materials and Methods: The study cohort consisted of 45 patients with bladder cancer undergoing cystectomy and 45 with histologically confirmed benign prostatic hyperplasia serving as controls. Hypermethylation at APC, DAPK, GSTP1, PTGS2, TIG1 and Reprimo was analyzed using real-time polymerase chain reaction following methylation sensitive restriction endonuclease treatment., Results: Hypermethylation at the APC and GSTP1 promoter was detected in 59% of cases, whereas TIG1 (32%), PTGS2 (24%) and DAPK (2%) were less frequently hypermethylated. In the benign prostatic hyperplasia group 3 patients also harbored methylated GSTP1 DNA, whereas none of the other gene sites was methylated. Hypermethylation at APC, GSTP1 or TIG1 distinguished patients with bladder cancer and controls most accurately with 80% sensitivity and 93% specificity. Hypermethylation significantly correlated with prognostic unfavorable clinicopathological parameters, including APC with pT stage, GSTP1, or GSTP1 or TIG1 with multifocal bladder cancer and APC, or APC or TIG1 with surgical margin positivity. Bladder cancer specific mortality was significantly increased in patients with APC hypermethylation., Conclusions: The detection of hypermethylation in cell-free serum DNA provides valuable diagnostic and prognostic information that can still be improved by combining the results of 3 gene sites (APC, GSTP1 and TIG1). The presence of hypermethylated DNA in the serum of patients with bladder cancer is associated with a worse outcome. Our results suggest that measuring hypermethylation in the serum of patients with bladder cancer is a useful biomarker.

Purpose: DNA hypermethylation is a common cancer associated alteration. We analyzed methylation patterns of cell-free serum DNA in patients with testicular cancer. Materials and Methods: Hypermethylation at APC, GSTP1, PTGS2, p14(ARF), p16(INK) and RASSF1A was analyzed using real-time polymerase chain reaction following methylation sensitive restriction endonuclease treatment in 73 patients with testicular cancer and 35 healthy individuals. Results: Hypermethylation was more common in patients with testicular cancer than in healthy individuals, including APC 57% and 6%, p16(INK) 53% and 17%, p14(ARF) 53% and 0%, RASSF1A 47% and 0%, PTGS2 45% and 0%, and GSTP1 25% and 0%, respectively (each p <0.01). Methylation frequencies at the investigated gene sites were similar in nonseminoma and seminoma cases (p >0.05). Diagnostic information was increased when multiple gene sites were analyzed in combination (ROC AUC 0.834, 67% sensitivity and 97% specificity). Diagnostic information was superior to the analysis of AFP/HCG/PLAP/LDH (combined sensitivity 58% and AUC 0.791). The sensitivity of hypermethylation in patients with unsuspicious conventional tumor markers was 71% (AUC 0.871, 97% specificity). Hypermethylation at PTGS2 was more common in patients with pT1 stage tumors (p = 0.011). Conclusions: The detection of hypermethylated cell-free serum DNA has the potential of a useful additional diagnostic parameter in patients with testicular germ cell cancer. Furthermore, in cases without conventional tumor marker increases testing CpG island hypermethylation in cell-free circulation DNA may improve the ability to detect early and/or recurrent testicular cancer. [Copyright &y& Elsevier]

Gene silencing by promoter DNA hypermethylation is common in PCA. The role of HXKy methylation as an up-/downstream or parallel silencing mechanism is not yet clear. We analyzed HXKy methylation at gene sites that are aberrantly DNA-methylated in PCA (i.e. GSTP1, RARβ, APC and PTGS2). Cancerous (PC3, DU145, LNCaP) and benign prostate hyperplasia (BPH1) cell lines were studied using chromatin immunoprecipitation and methylation specific PCR. Gene expression was analyzed using quantitative RT-PCR. In PCA, histone modifications characteristic of gene repression (di-/tri-methylated H3K9, H3K27, H4K20) were predominantly observed at the studied gene sites and paralleled aberrant DNA hypermethylation. In BPH, no aberrant DNA hypermethylation and mostly histone modifications supporting active gene expression (H3K4, H3K79 di-/tri methylation) were seen at the respective gene sites. Mono-methylated H3K4 and H3K9 modifications that have moderate effects on gene expression were frequent in both PCA and BPH. PC3 cells grown in the presence of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (aza) resulted in DNA demethylation and moderate gene reexpression whereas repressive HXKy di-methylation increased 1.5-3 fold and tri-methylation was mostly unchanged. Removal of aza reverses gene expression. These findings suggest that HXKy methylation is upstream to DNA hypermethylation in PCA. [ABSTRACT FROM AUTHOR]

Cancer patients have increased levels of serum DNA fragments (SDF) compared to most patients with non-malignant disease and healthy individuals. In PCA, DNA hypermethylation is also frequently observed (>80%) at promoter CpG islands of GSTP1, PTGS2 and TIG1. Therefore we analysed hypermethylation of these genes in SDF from patients with PCA (n=168) and benign prostate hyperplasia (BPH; n=42) and controls (n=11) for information about the cellular origin of DNA. SDF concentrations and the hypermethylated fractions were analysed using a methylation-sensitive restriction enzyme based real-time PCR. SDF levels were significantly increased in PCA vs BPH vs controls (65 vs 20 vs 7 ng/mL; p<0.001). Hypermethylation of GSTP1, TIG1 or PTGS2 was more frequently detected in PCA than in BPH patients and controls (overall 47% vs 8% vs 0%; p<0.001), but did not exceed 10% of total DNA. SDF levels distinguished PCA from BPH patients with a sensitivity of 83% and specificity of 69%. The diagnostic significance of SDF was greater than that of hypermethylation data. In conjunction with SDF, hypermethylation numbers did not contribute any further information. Both SDF levels and the hypermethylation status were not correlated to advanced tumor stage or grade (p>0.05). In summary SDF is a promising diagnostic marker in PCA. It does not primarily originate from tumor cells but from apoptotic and necrotic cells surrounding the tumor. [ABSTRACT FROM AUTHOR]

Abstract: The presence of small amounts of circulating DNA in plasma was demonstrated 60 years ago. Since then, cell-free DNA has been tested for quantity, fragmentation pattern, and tumor-specific sequences in patients with various malignancies. Recent studies have shown that cell-free DNA levels are distinctly increased in most patients with prostate cancer (PCA) and that the DNA fragmentation pattern is different from healthy individuals and patients with benign prostate disease. The origin of this circulating DNA remains largely unknown, but it is established that a small fraction of the DNA is derived from the tumor itself, and genetic (allelic imbalances) and epigenetic (DNA methylation) alterations are regularly detected in patients with PCA. The detection of increased DNA levels and tumor-specific DNA sequences may provide diagnostic and prognostic information. The recent findings in the emerging field of cell-free DNA will be discussed. [Copyright &y& Elsevier]