Detection of circulating tumor cells from renal carcinoma patients: experiences of a two-center study.

Approximately 30-40% of primary and localized renal cell carcinoma (RCC) will eventually become metastatic disease. Therefore, the detection and molecular characterization of circulating tumor cells (CTC) in RCC may have important prognostic and therapeutic implications. Venous blood samples were obtained from a total of 214 RCC patients before and after nephrectomy or during adjuvant immune chemotherapy in two urological centers. After density gradient centrifugation, the CD45-negative cell population was isolated from peripheral blood samples (BS) by a semi-automated immunomagnetic depletion procedure using the MACS technology. Enriched cell populations potentially containing CTC were stained for cytokeratin and evaluated by a trained pathologist. CTC were found in 105 out of 363 BS (29%) originating from 80 out of 214 patients (37%). The median tumor cell number was five (range 1-51) per BS, i.e. approximately one CTC was detectable per 2-3 ml peripheral blood after tumor cell enrichment. For a subpopulation, follow-up data indicate that 62% of the patients with CTC detection in the blood developed progressive disease with single or multiple distant metastases or died because of RCC within two years. Here we show that the standardized immunomagnetic depletion protocol is a powerful tool for detecting and isolating intact RCC-derived CTC. The occurrence and the quantity of CTC in RCC patients is an early disease event. Furthermore, the occurrence of CTC is correlated with an advanced tumor stage and seems to be associated with a more aggressive tumor phenotype.

Isolation and enrichment of urologic tumor cells in blood samples by a semi-automated CD45 depletion autoMACS protocol.

The purpose of this study was to demonstrate the efficacy of an enrichment protocol for the detection of circulating carcinoma cells in the bloodstream of patients with various urologic cancers. Using 16-ml peripheral blood samples (BS) the mononuclear cells were isolated by density gradient centrifugation. The CD45 leukocyte depletion method based on a previous study was slightly modified and semi-automated by an immunomagnetic cell separation unit (autoMACS). Enriched tumor cells were analyzed on a single slide by cytokeratin (CK) immunocytochemistry. The number of recovered DU-145 prostate cancer cells in various spiking experiments was 70-88%. By the optimized tumor cell enrichment protocol 186 BS originated from 128 patients with different urologic cancers (60 prostate carcinoma, 34 bladder cancers, 24 renal cell carcinoma and 10 other tumors) were investigated before, during and after tumor surgery. In 59 BS from 52 patients on average 5 tumor cells were detected in each BS containing tumor cells. The median number of identified tumor cells was 8 cells per BS and patient. Tumor cells were found for the 3 tumor types with representative BS numbers in 29-39% of the investigated BS and in 38-53% of the affected patients. The detection rates increased in the order prostate carcinoma < renal cell carcinoma < bladder cancer. Surprisingly, in four bladder tumor cases with identified disseminated tumor cells in BS, the histopathological examination of the transurethral resection of bladder tumor specimens showed no evidence for tumor cells in situ but the affected patients had clinically known and histologically defined tumor residue or a bladder tumor recurrence during the follow-up. The semi-automated CD45 autoMACS depletion protocol for the enrichment and the detection of disseminated tumor cells in the peripheral bloodstream allows to study up to 20 BS per working day prospectively by one technician. The improved sensitivity and specificity might be of importance when applying the protocol to BS in future clinical studies.

Detection of circulating tumor cells in peripheral blood of patients with renal cell carcinoma correlates with prognosis.

PURPOSE: The aim of this study was to evaluate the clinical relevance of the presence of disseminated tumor cells in peripheral blood (so-called circulating tumor cells) for renal cell carcinoma patients. METHODS: Two hundred thirty-three peripheral blood samples from 154 renal cell carcinoma patients were investigated for the presence of disseminated tumor cells by autoMACS technique and immunocytochemical staining of cytokeratin. The frequency of circulating tumor cells was analyzed statistically for correlation with relevant clinical data. RESULTS: Two kinds of tumor cells were detected: those with expression of cytokeratin 8/18 (CK+) and cells without a detectable cytokeratin expression, which we called large blue-stained cells with a tumorlike morphology. After following the CD45 autoMACS depletion protocol, we identified circulating tumor cells in 96 (41%) of 233 peripheral blood samples, which originated from 81 (53%) of 154 renal cell carcinoma patients. A significant correlation between the detection of circulating tumor cells and positive lymph node status (P < 0.001; chi(2) test) and the presence of synchronous metastases at the time of primary tumor resection (P = 0.014; chi(2) test) was found. In a multivariate Cox's regression hazard model, presence of CK+ circulating tumor cells was significantly correlated with poor overall survival for renal cell carcinoma patients (relative risk, 2.3; P = 0.048). CONCLUSIONS: The presence of circulating tumor cells correlated to lymph node status and presence of synchronous metastases in renal cell carcinoma. It is important to evaluate CK+ and blue-stained tumor cells together to determine the role of circulating tumor cells in tumor behavior and disease progression. Detection of CK+ circulating tumor cells in peripheral blood is a significant and independent prognostic factor for renal cell carcinoma.