PDF file - 209KB, Positivity for SALL4, OCT3/4, keratins, EpCAM and Cellsearch(registered trademark) assay in control cell lines. A, Double fluorescence staining for SALL4 (green) / keratins (orange) and OCT3/4 (green) / EpCAM (orange) counterstained with DAPI (blue) to visualize cells' nuclei in 4 different germ cell tumor cell lines (magnification: 400x). Scale bar, 50 microm. B, Representation of the staining of tumor cells from 4 different germ cell tumor cell lines spiked into blood of healthy donors using the Cellsearch(registered trademark) assay. C, Representative images of primary testicular germ cell tumor samples positive for SALL4, OCT3/4, keratins and EpCAM immunohistochemical staining counterstained with Hematoxylin to visualize cells' nuclei (magnification - 400x, scale bar - 50 microm). (CK, cytokeratin. PE, phycoerythrin. APC, allophycocyanin. DAPI, 4′,6-diamidino-2-phenylindole). D, Dot-like pattern keratin (orange) staining in TCam-2 and NT2 cell lines, counterstained with DAPI (blue) to visualize cells' nuclei (magnification: 1000x). Scale bar, 20 microm.

PDF file - 65KB, Characterization of relapsed, treatment-refractory germ cell tumor patients.

Abstract Purpose: Germ cell tumors (GCTs) represent the most frequent malignancies among young men, but little is known about circulating tumor cells (CTCs) in these tumors. Considering their heterogeneity, CTCs were investigated using two independent assays targeting germ cell tumor and epithelial cell–specific markers, and results were correlated with disease stage, histology, and serum tumor markers. Experimental Design: CTCs were enriched from peripheral blood (n = 143 patients) and testicular vein blood (TVB, n = 19 patients) using Ficoll density gradient centrifugation. For CTC detection, a combination of germ cell tumor (anti-SALL4, anti-OCT3/4) and epithelial cell–specific (anti-keratin, anti-EpCAM) antibodies was used. In parallel, 122 corresponding peripheral blood samples were analyzed using the CellSearch system. Results: In total, CTCs were detected in 25 of 143 (17.5%) peripheral blood samples, whereas only 11.5% of patients were CTC-positive when considering exclusively the CellSearch assay. The presence of CTCs in peripheral blood correlated with clinical stage (P < 0.001) with 41% of CTC positivity in patients with metastasized tumors and 100% in patients with relapsed and chemotherapy-refractory disease. Histologically, CTC-positive patients suffered more frequently from nonseminomatous primary tumors (P < 0.001), with higher percentage of yolk sac (P < 0.001) and teratoma (P = 0.004) components. Furthermore, CTC detection was associated with elevated serum levels of α-fetoprotein (AFP; P = 0.025), β-human chorionic gonadotropin (βHCG; P = 0.002), and lactate dehydrogenase (LDH; P = 0.002). Incidence and numbers of CTCs in TVB were much higher than in peripheral blood. Conclusions: The inclusion of germ cell tumor–specific markers improves CTC detection in GCTs. CTCs occur frequently in patients with more aggressive disease, and there is a gradient of CTCs with decreasing numbers from the tumor-draining vein to the periphery. Clin Cancer Res; 20(14); 3830–41. ©2014 AACR.

BACKGROUND:Clinical management of germ cell tumours (GCTs) relies on monitoring of serum tumour markers. However, the markers α-fetoprotein (AFP), the β-subunit of human chorionic gonadotropin (bHCG), and lactate dehydrogenase (LDH) are expressed in <60% of GCT cases. OBJECTIVE:To test the utility of the microRNAs (miRNAs) miR-371a-3p, miR-372-3p, miR-373-3p, and miR-367-3p as sensitive and specific GCT serum biomarkers. DESIGN, SETTING, AND PARTICIPANTS:Serum levels of miRNAs were measured in 166 consecutive patients with GCT before and after treatment and in 106 male controls. In the first 50 consecutive patients, all four miRNAs were measured. In the main study, only the most sensitive miRNA was further analysed. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS:The specificity and sensitivity of the four miRNAs were studied using receiver operating characteristic curves. miRNA sensitivities were compared to those of classical markers. Statistical cross-comparisons of miRNA levels for GCT subgroups and controls were performed at various time points during treatment. RESULTS AND LIMITATIONS:Overall, miR-371a-3p performed best, with 88.7% sensitivity (95% confidence interval [CI] 82.5-93.3%) and 93.4% specificity (95% CI 86.9-97.3%) and an area under the curve of 0.94, outperforming AFP, bHCG, and LDH (combined sensitivity 50%). According to Kernel density estimation, the sensitivity and specificity were 86.3% and 92.5%, respectively. miR-371a-3p levels dropped to normal after completion of treatment. The miRNA levels correlated with treatment failure and relapse. Teratoma did not express miR-371a-3p. CONCLUSIONS:The miRNA miR-371a-3p is a specific and sensitive novel serum GCT biomarker that accurately correlates with disease activity. Validation of this test in a large-scale prospective study is needed. PATIENT SUMMARY: miR-371a-3p is a novel serum marker for germ cell tumours that is expressed by 88.7% of patients and thus is far more sensitive and specific than classical serum markers. It correlates with tumour burden and treatment results. Validation in a large patient cohort is needed.

Purpose: Germ cell tumors (GCTs) represent the most frequent malignancies among young men, but little is known about circulating tumor cells (CTCs) in these tumors. Considering their heterogeneity, CTCs were investigated using two independent assays targeting germ cell tumor and epithelial cell–specific markers, and results were correlated with disease stage, histology, and serum tumor markers. Experimental Design: CTCs were enriched from peripheral blood (n = 143 patients) and testicular vein blood (TVB, n = 19 patients) using Ficoll density gradient centrifugation. For CTC detection, a combination of germ cell tumor (anti-SALL4, anti-OCT3/4) and epithelial cell–specific (anti-keratin, anti-EpCAM) antibodies was used. In parallel, 122 corresponding peripheral blood samples were analyzed using the CellSearch system. Results: In total, CTCs were detected in 25 of 143 (17.5%) peripheral blood samples, whereas only 11.5% of patients were CTC-positive when considering exclusively the CellSearch assay. The presence of CTCs in peripheral blood correlated with clinical stage (P < 0.001) with 41% of CTC positivity in patients with metastasized tumors and 100% in patients with relapsed and chemotherapy-refractory disease. Histologically, CTC-positive patients suffered more frequently from nonseminomatous primary tumors (P < 0.001), with higher percentage of yolk sac (P < 0.001) and teratoma (P = 0.004) components. Furthermore, CTC detection was associated with elevated serum levels of α-fetoprotein (AFP; P = 0.025), β-human chorionic gonadotropin (βHCG; P = 0.002), and lactate dehydrogenase (LDH; P = 0.002). Incidence and numbers of CTCs in TVB were much higher than in peripheral blood. Conclusions: The inclusion of germ cell tumor–specific markers improves CTC detection in GCTs. CTCs occur frequently in patients with more aggressive disease, and there is a gradient of CTCs with decreasing numbers from the tumor-draining vein to the periphery. Clin Cancer Res; 20(14); 3830–41. ©2014 AACR.

Purpose: Germ cell tumors (GCTs) represent the most frequent malignancies among young men, but little is known about circulating tumor cells (CTCs) in these tumors. Considering their heterogeneity, CTCs were investigated using two independent assays targeting germ cell tumor and epithelial cell–specific markers, and results were correlated with disease stage, histology, and serum tumor markers. Experimental Design: CTCs were enriched from peripheral blood (n 1⁄4 143 patients) and testicular vein blood (TVB, n1⁄4 19 patients) using Ficoll density gradient centrifugation. ForCTCdetection, a combinationof germ cell tumor (anti-SALL4, anti-OCT3/4) and epithelial cell–specific (anti-keratin, anti-EpCAM) antibodies was used. Inparallel, 122 correspondingperipheral blood sampleswere analyzedusing theCellSearch system. Results: In total, CTCsweredetected in25of 143 (17.5%)peripheral blood samples,whereas only 11.5% of patients were CTC-positive when considering exclusively the CellSearch assay. The presence of CTCs in peripheral blood correlated with clinical stage (P < 0.001) with 41% of CTC positivity in patients with metastasized tumors and 100% in patients with relapsed and chemotherapy-refractory disease. Histologically, CTC-positive patients sufferedmore frequently fromnonseminomatous primary tumors (P < 0.001), with higher percentage of yolk sac (P < 0.001) and teratoma (P 1⁄4 0.004) components. Furthermore, CTC detection was associated with elevated serum levels of a-fetoprotein (AFP; P 1⁄4 0.025), b-human chorionic gonadotropin (bHCG; P1⁄4 0.002), and lactate dehydrogenase (LDH; P1⁄4 0.002). Incidence and numbers of CTCs in TVB were much higher than in peripheral blood. Conclusions: The inclusion of germ cell tumor–specificmarkers improves CTC detection in GCTs. CTCs occur frequently in patients with more aggressive disease, and there is a gradient of CTCs with decreasing numbers from the tumor-draining vein to the periphery. Clin Cancer Res; 20(14); 1–12. 2014 AACR.

You have accessJournal of UrologyPenis/Testis/Urethra: Benign & Malignant Disaese (I)1 Apr 2013703 CIRCULATING TUMOR CELLS CAN BE DETECTED IN PATIENTS WITH TESTICULAR GERM CELL TUMORS Christian Ruf, Paulina Nastaly, Pascal Becker, Hendrik Isbarn, Friedemann Honecker, Klaus Pantel, Sabine Riethdorf, Dirk Hoeppner, Margit Fisch, Walter Wagner, and Sascha Ahyai Christian RufChristian Ruf Hamburg, Germany , Paulina NastalyPaulina Nastaly Hamburg, Germany , Pascal BeckerPascal Becker Hamburg, Germany , Hendrik IsbarnHendrik Isbarn Hamburg, Germany , Friedemann HoneckerFriedemann Honecker Hamburg, Germany , Klaus PantelKlaus Pantel Hamburg, Germany , Sabine RiethdorfSabine Riethdorf Hamburg, Germany , Dirk HoeppnerDirk Hoeppner Berlin, Germany , Margit FischMargit Fisch Hamburg, Germany , Walter WagnerWalter Wagner Hamburg, Germany , and Sascha AhyaiSascha Ahyai Hamburg, Germany View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.261AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The available serum tumor markers (AFP, β-HCG, LDH) are increased in only approximately 60% of testicular germ cell tumors (TGCT) patients. Therefore, additional markers would facilitate clinical diagnosis, staging and treatment of these patients. Detection of circulating tumor cells (CTC) has been associated with poor prognosis in other malignancies (e.g. Prostate-, Colon- and Breastcancer), however, little is known about the presence of CTC in blood of TGCT patients. METHODS In order to select the most appropriate markers for CTCs detection, the expression of different epithelial and germ cell-specific markers was examined in 4 different TGCT cell lines (TCam-2/2102Ep/NCCIT/NT2) as well as 12 histologically different primary testicular tumors. Peripheral and testicular vein blood samples from 73 and 12 patients, respectively, were collected and examined for CTCs. All samples were enriched for mononuclear cells using Ficoll density gradient centrifugation and CTCs were detected by alkaline phosphatase (AP) enzymatic activity and immunocytochemistry using anti-keratin, anti-EpCAM and anti-SALL4-antibodies. Additionally, peripheral blood samples were tested for the presence of CTCs by the automated CellSearch® system. Patients with ≥1 CTC/sample were classified as CTC-positive. To compare results for testicular and peripheral blood, CTC yields were calculated as number of CTC per 1 ml of blood. RESULTS Based on the previous analyses, double immunofluorescent staining for SALL4 and keratin, as well as analysis of AP activity were performed to detect CTCs. According to both detection systems û the one based on selected markers and automated CellSearch®, 7 (58.3%) testicular vein and 13 (17.8%) peripheral blood samples showed presence of at least one CTC. The CTC number ranged from 5 to 108/ml and 0.13 to 2.18/ml of testicular vein and peripheral blood, respectively. CTC were detected in seminoma and non-seminoma, in clinically metastasized and non-metastasized stages. CONCLUSIONS This is the first study to demonstrate CTC detection in TGCT patients. The proposed detection systems seem to be specific and sensitive for the identification of CTC. An association between CTC and histological subtype as well as metastatic stage is under investigation and will be presented at the meeting. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e289 Peer Review Report Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christian Ruf Hamburg, Germany More articles by this author Paulina Nastaly Hamburg, Germany More articles by this author Pascal Becker Hamburg, Germany More articles by this author Hendrik Isbarn Hamburg, Germany More articles by this author Friedemann Honecker Hamburg, Germany More articles by this author Klaus Pantel Hamburg, Germany More articles by this author Sabine Riethdorf Hamburg, Germany More articles by this author Dirk Hoeppner Berlin, Germany More articles by this author Margit Fisch Hamburg, Germany More articles by this author Walter Wagner Hamburg, Germany More articles by this author Sascha Ahyai Hamburg, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...