Bernd Straub

Dna-based detection of prostate cancer in urine after prostatic massage

OBJECTIVES Promoter hypermethylation of the glutathione-S-transferase P1 (GSTP1) gene is a specific feature of prostate cancer. This epigenetic DNA alteration served as the target for molecular detection of prostate cancer cells in urine sediments after prostatic massage. METHODS Bisulfite treatment followed by methylation-specific polymerase chain reaction was used to detect GSTP1 promoter hypermethylation in DNA isolated from urine sediments obtained after prostatic massage of men with and without prostate cancer. RESULTS GSTP1 promoter hypermethylation was demonstrated in the sediments of 1 (2%) of 45 patients diagnosed with benign prostatic hyperplasia, 2 (29%) of 7 patients with prostatic intraepithelial neoplasia, 15 (68%) of 22 patients with early, intracapsular cancer, and 14 (78%) of 18 patients with locally advanced or systemic prostate cancer, resulting in a specificity of 98% and an overall sensitivity of 73% for the detection of prostate cancer. CONCLUSIONS Urinalysis for GSTP1 promoter hypermethylation constitutes a sensitive and highly specific DNA-based marker for molecular detection of prostate cancer, including early stages.

DNA Alterations in Body Fluids as Molecular Tumor Markers for Urological Malignancies

OBJECTIVES DNA-based tumor markers are characterized by unique specificity rendering them an attractive target for molecular diagnosis of cancer in body fluids like blood serum/plasma and urine. Both cell-free tumor DNA circulating in plasma/serum and cellular tumor DNA are detectable by minimally invasive measures. METHODS Three main detection methods, microsatellite analysis, mutation analysis in genomic or mitochondrial DNA and gene promoter hypermethylation analysis are applied. Detection of gene promoter hypermethylation by methylation-specific PCR enables the best methodical sensitivity requiring a ratio of tumor DNA within normal DNA of less than 1:1000. RESULTS/CONCLUSIONS Tumor DNA derived from renal cell carcinoma, bladder cancer or prostate cancer is detectable in considerably more than 50% of plasma/serum samples and more than 70% of urine samples from these patients. Because the targeted DNA alterations are absent or very rare in controls, the specificity of DNA-based tumor detection methods reaches almost 100%. Although the methodology currently is experimental, automatization will make it easier and less expensive. This review is focused on the potential clinical value of DNA-based analysis of body fluids for the initial diagnosis and the follow-up of urologic cancer patients.

“Onco-tese”: testicular sperm extraction in azoospermic cancer patients before chemotherapy—new guidelines?

OBJECTIVES To examine the usefulness of pretreatment testicular sperm extraction because some patients have tumor-induced azoospermia. In view of the high cure rates for testicular germ cell tumors and malignant lymphomas, increasing clinical importance is attached to protecting fertility. High-dose cytostatic therapy may be expected to cause long-term infertility. Thus, the standard procedure for fertility protection is cryopreservation of ejaculated spermatozoa before therapy. METHODS Contralateral testicular biopsies were taken from 14 azoospermic patients with malignant testicular germ cell tumors. In addition, 17 patients with malignant lymphomas underwent unilateral (n = 6) or bilateral (n = 11) testicular biopsy. The tissue specimens were cryopreserved, and the histologic workup was performed at the same time. RESULTS Of the 14 patients with malignant testicular germ cell tumors, 6 had spermatozoa in their testicular biopsies. Sertoli cell-only syndrome was found in 5 patients, and 3 had maturation arrest without detection of spermatozoa. Successful sperm recovery was possible in 8 of the 17 patients with malignant lymphoma, 4 had Sertoli cell-only syndrome, and 5 had maturation arrest. None of the patients had evidence of secondary wound healing or treatment delay because of the testicular biopsy. CONCLUSIONS Our results show that testicular sperm extraction is a useful technique for obtaining spermatozoa before cytotoxic therapy in azoospermic cancer patients. This procedure should be considered as an option for fertility preservation in azoospermic cancer patients, because high cumulative cytostatic doses can cause irreversible fertility alterations.

The impact of chemotherapy on male fertility: a survey of the biologic basis and clinical aspects

The introduction of cisplatin-based polychemotherapy has led to cure rates of up to 90% for the most frequent malignant diseases seen in young men. In view of these high cure rates, increasing clinical importance is now being attached to chemotherapy-induced fertility disorders. Comparative studies examining the impact of cytotoxic chemotherapy on gametogenesis demonstrate significant cytostatic- and dose-specific differences. The extensive literature on possible teratogenic effects of chemotherapy provides no evidence suggesting that offspring of patients with a history of chemotherapy have an increased risk of malformations. However, these studies, the scope and follow-up of which may still be inadequate, have failed to eliminate the fear of such risk. Hormonal protection from chemotherapy-induced testicular damage has thus far succeeded only in animal models pretreated by application of gonadotropin-releasing hormone agonists combined with nonsteroidal antiandrogens or testosterone plus 17 beta-estradiol. The same holds true for hormone therapy aimed at stimulating the recovery of spermatogenesis after chemotherapy-induced testicular damage. Cryopreservation of germ cells can be suggested to patients undergoing cytostatic therapy. In some cases, testicular extraction of spermatozoa can also be offered as a novel approach.

Methylation‐specific PCR for detection of neoplastic DNA in biopsy washings

Epigenetic DNA alterations such as promoter hypermethylation of glutathione S‐transferase P1 (GSTP1) in prostatic adenocarcinoma frequently constitute tumour biomarkers. Neoplastic transformation was identified in washings of prostate biopsies by GSTP1 promoter hypermethylation, using methylation‐specific PCR (MSP). Twenty‐six patients undergoing sextant transrectal prostate biopsies for clinically suspected prostate cancer were enrolled. All ten patients diagnosed with adenocarcinoma (100%) and four of six patients with prostatic intraepithelial neoplasia (67%), but none of ten patients with benign hyperplasia (0%), exhibited GSTP1 promoter hypermethylation in at least one out of six biopsy washings. Since this approach is transferable to various cancer entities, a sensitive and specific DNA‐based analysis of biopsy material seems generally feasible without impeding routine histopathological examination. Copyright

Detection of prostate-specific antigen RNA before and after radical retropubic prostatectomy and transurethral resection of the prostate using “Light-Cycler”-based quantitative real-time polymerase chain reaction

OBJECTIVES To report our initial experience gained in establishing real-time reverse transcriptase-polymerase chain reaction (RT-PCR) detection of prostate-specific antigen (PSA) mRNA using the quantitative online PCR system LightCycler. Many studies have thus far failed to provide the desired proof that the detection of circulating PSA-expressing tumor cells with RT-PCR in the blood samples of patients with prostate cancer (PCa) is a highly sensitive prognostic and course marker. One of the possible reasons is the lack of reliable quantification methods. METHODS Blood samples before and after surgery were obtained from 87 patients who underwent radical prostatectomy for locally confined PCa and 27 patients who underwent transurethral resection of the prostate for benign prostatic hyperplasia. Eight days postoperatively, additional blood samples were obtained from the patients with PCa. Quantitative no-nested RT-PCR for PSA mRNA (291 bp) was performed using the LightCycler system applying the SYBR Green protocol. The number of circulating LNCaP tumor cell-equivalents per sample was estimated from the mean amplification value measured in a given number of LNCaP cells. RESULTS PSA mRNA was detected preoperatively in 19 patients with Stage pT2 tumor (40%) and in 28 patients with tumor greater than Stage pT2 (72%), but in only 2 patients with benign prostatic hyperplasia (8%; analysis of variance, P <0.001). Significant quantitative differences were observed among Stage pT2 disease (1034 LNCaP tumor cell-equivalents/mL), greater than Stage pT2 disease (7830 cells/mL), and benign prostatic hyperplasia (58 cells/mL; analysis of variance for all groups, P <0.001). The correlation between the detection of PSA expression by RT-PCR and the Gleason score and serum PSA value was statistically significant. CONCLUSIONS Our results show that the initial experience with the LightCycler system for PSA-assisted detection of circulating PSA mRNA in PCa by RT-PCR may be a promising preoperative prognostic marker for organ-confined or locally advanced PCa. Long-term follow-up of these patients with PCa must demonstrate the clinical value of molecular diagnostics with quantitative RT-PCR systems.

Impact of chemotherapy on male fertility.

Testicular tumors and malignant lymphomas are, with increasing incidence, the most frequent malignant diseases in men between the age of 15 and 34. With the introduction of cisplatin-based polychemotherapy, cure rates rose to over 90% in patients with germ cell tumors and were comparably favorable at around 80% in those with malignant lymphomas. In view of these high cure rates, increasing clinical importance is attached to chemotherapyinduced fertility disorders. One problem involved in assessing the influence of chemotherapy on fertility is the fact that the malignant disease itself strongly alters spermatogenesis. This complicates an evaluation of the effect of cytostatic therapy on fertility disorders. There are significant cytostatic- and dose-specific differences. Longterm infertility due to cytostatic therapy may be expected in more than 50% of the patients at a cumulative dose of cisplatin > 0.6 g/m2, cyclophosphamide > 6 g/m2, and procarbazine ≧ 4 g/m2. However, it takes up to 3 years or more for spermatogenesis to recover after the termination of chemotherapy. An individual assessment of the post-therapeutic fertility status is extremely limited, since variance of the pretherapeutic fertility status causes interindividual differences, and the numerical data mentioned above only permit a vague estimation. Before patients undergo cytostatic therapy, cryopreservation of germ cells should thus be suggested or, in some cases, testicular extraction of spermatozoa.

µ-Acetylide and µ-alkenylidene ligands in “click” triazole syntheses

In a computational investigation, dinuclear and tetranuclear copper acetylide complexes display both superior stability and higher reactivity in the CuAAC reaction than do mononuclear complexes due to favored dicopper(I,III) µ-alkenylidene fragments, instead of ring strain in a CuCC intermediate.

Real-time quantitative reverse transcriptase-polymerase chain reaction for luteinizing hormone-releasing hormone receptor gene mRNA expression in human prostate cancer.

OBJECTIVES To determine quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) detection of luteinizing hormone-releasing hormone (LHRH) receptor mRNA expressing prostate cancer (CaP) and benign prostatic hyperplasia (BPH) tissue by sequence-specific detection with hybridization probes using the LightCycler. Various in vitro studies have indicated that LHRH agonists and antagonists may have a direct inhibitory effect on CaP mediated by specific LHRH receptors. The incidence of LHRH receptor mRNA expression was demonstrated in 50% to 100% of clinical CaP and BPH tissue by current RT-PCR methods. However, qualitative RT-PCR can only demonstrate the existence of a gene; an exact quantification of LHRH-receptor mRNA expression has not yet been done. METHODS Quantitative real-time RT-PCR for LHRH receptor mRNA was performed using the LightCycler system and the RNA Amplification Kit hybridization probes in 35 patients with CaP and 38 patients with BPH. RESULTS Thirty-one (88.6%) of the 35 patients with CaP and 36 (94.7%) of the 38 patients with BPH had positive RT-PCR for LHRH receptor mRNA. The number of positive amplifications per 1 microg total RNA was not significantly lower in CaP at 695,428 +/- 350,860 than in BPH at 1,617,654 +/- 787,874. Patients with CaP evidenced a significant negative correlation between the amplification rate for LHRH receptor mRNA and Gleason score (r = -0.476; P = 0.004). CONCLUSIONS In the past, the potential mechanisms acting on LHRH receptors in CaP have been identified as possible antitumor strategies for this type of cancer. Our study is the first to show that CaP does not have a higher expression of LHRH receptor mRNA expression than BPH when using quantitative sequence-specific oligonucleotide hybridization probes that only detect certain PCR products.

Quantitative Real-Time RT-PCR for Detection of Circulating Prostate-Specific Antigen mRNA Using Sequence-Specific Oligonucleotide Hybridization Probes in Prostate Cancer Patients

Objective: A great number of studies have failed thus far to demonstrate that the presence of PSA-expressing tumor cells in the blood of prostate cancer (PC) patients is a highly sensitive prognostic marker, particularly after radical prostatectomy (RPX). These studies have only relied on qualitative or semiquantitative detection techniques, however. We report our initial experience in testing real-time RT-PCR for the detection of PSA mRNA using a quantitative online PCR system, the LightCyclerTM, and sequence-specific oligonucleotide hybridization probes. Methods: Blood samples were obtained before and after surgery from 129 patients undergoing RPX for localized PC and from 19 patients undergoing transurethral resection of the prostate for benign prostatic hyperplasia (BPH). Quantitative RT-PCR for the detection PSA mRNA was performed using the LightCycler system with RNA Amplification Kit Hybridization Probes and sequence-specific oligonucleotide hybridization probes. Results: PSA mRNA was detected by the LightCycler in 28 patients (39%) with pT2 tumors, in 22 patients (38%) with >pT2 tumors, but in only 3 patients (16%) with BPH. The mean values of the LNCaP cell equivalents were higher in >pT2 patients than in pT2 patients (37 × 103 and 104 × 103) or BPH patients (7.1 × 103 and 4.8 × 103) both preoperatively (333 × 103/ml blood) and postoperatively (545 × 103). Conclusion: Real-time RT-PCR with the LightCycler appears to be a promising method for the preoperative detection of circulating LNCaP tumor cells in PC as reflected by PSA mRNA. Considering the low detection rates in BPH patients, the method may also be suitable for patient monitoring after RPX and could thus play an important role in deciding on early radiotherapy or even hormone ablation therapy. Additional long-term follow-up will have to show whether patients with high expression of PSA mRNA actually have an increased risk or recurrence and whether the method is suitable as well to detect progression.