Detection and characterization of circulating tumor cells in patients with testicular germ cell tumors and prostate cancer

Abstract

Circulating tumor cells (CTCs) are cells that have detached from primary tumor and are present in blood circulation. Detection of CTCs has been reported in urogenital malignancies including prostate and bladder cancer. In patients with castration-resistant prostate cancer, presence of CTCs has been associated with poor prognosis. This study aimed to investigate the prevalence, clinical relevance and characterize CTCs in testicular germ cell tumors and prostate cancer. Germ cell tumors (GCTs) represent the most frequent malignancies among young men. Histologically, GCTs are divided into two classes: seminomatous and nonseminomatous germ cell tumors (NSGCTs). There is scant information about the presence of CTCs in blood of patients with GCTs. Considering heterogeneity of GCTs, CTCs were investigated using two independent assays targeting germ and epithelial cell-specific markers, and results were correlated with disease stage, histology, and serum tumor markers. CTCs were enriched from peripheral (n=143 patients) and testicular vein blood (n=19 patients) using Ficoll density gradient centrifugation. For CTC detection, a combination of germ (anti-SALL4, anti-OCT3/4) and epithelial cell-specific (anti-keratin, anti-EpCAM) antibodies was used. In parallel, 122 corresponding PB samples were analysed using the CellSearch® system. Additionally, in the cohort of 51 patients with NSGCTs, peripheral blood samples were examined for presence of potential therapeutical targets on level of CTCs: CD30 and Glypican-3. In total, CTCs were detected in 25/143 (17.5%) peripheral blood samples, whereas 11.5% of patients were CTC-positive when considering exclusively the CellSearch® assay. Twelve (23.5%) peripheral blood samples showed membranous expression of CD30, whereas Glypican-3 staining was found in CTCs from 6 (11.8%) patients. Five (9.8%) patients had CTCs positive for both CD30 and GPC3. The presence of CTCs in PB correlated to 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) than from seminomas, with higher percentage of yolk sac (P<0.001) and teratoma (P=0.004) components. Furthermore, CTC detection was associated with elevated AFP (P=0.025), βHCG (P=0.002) and LDH (P=0.002) serum levels. Incidence and numbers of CTCs in TVB were much higher comparing to PB. This is the first study reporting on CTCs in patients with 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 peripheral blood vessels. Evaluation of CTCs in patients with GCTs might serve as potential diagnostic and/or prognostic biomarker as well as lead to therapy improvement.Transrectal ultrasound guided prostate biopsy is a standard diagnostic procedure for prostate cancer diagnosis. However, the possibility of dissemination of cancer cells by biopsy is not negligible. The aim of this study was to investigate if prostate biopsy is associated with hematogenous dissemination of CTCs. Peripheral blood samples were obtained before and after performing prostate biopsy from 75 patients with serum prostate-specific antigen (PSA) levels >2 ng/mL. CTCs were detected with the usage of two methods - the established CellSearch® system and the EPISPOT assay that detects living PSA-secreting tumor cells. Using the CellSearch® system, in 8 (12.1%) patients CTCs were detected before, and 20 (29.7%) samples collected after biopsy were CTC-positive (P=0.017). With the usage of the EPISPOT assay, PSA-secreting CTCs were found in peripheral blood of 20 (28.5%) patients before biopsy, whereas 31 (44.3%) cases collected after performing biopsy were positive (P=0.027). When combining the results of both assays (27/75, 37.0%) patients were positive for CTCs before, and significantly more (42/75, 56.0%) of cases were detected CTC-positive after performing biopsy (P=0.009). This is the first report suggesting that prostate biopsy may cause dissemination of not only apoptotic but also viable prostate cells.