NK-cell dysfunction in human renal carcinoma reveals diacylglycerol kinase as key regulator and target for therapeutic intervention.

The relevance of NK cells in tumor control is well established in mouse models and human hematologic malignancies; however, their contribution to the control of human solid tumors remains disputed due to problems with in situ detection and reports of functional inactivity in the tumor milieu. In this study, we established a reliable in situ detection method for NK cells. Moreover, we performed analysis to elucidate mechanisms that impair NK-cell function in the tumor milieu and thereby identify therapeutic targets that allow recovery of NK-cell functionality. It was observed that NK cells from clear cell renal cell carcinoma (ccRCC), compared to NK cells from nontumor kidney and peripheral blood lymphocytes (PBLs), displayed conjoint phenotypic alterations and dysfunction induced by the tumor milieu, which were associated mechanistically with high levels of signaling attenuator diacylglycerol kinase (DGK)-α and blunted mitogen-activated protein kinase pathway activation (ERK1/2, Jun kinase). Reinstating NK-cell functionality was possible by DGK inhibition or brief IL-2 culture, interventions that de-repressed the ERK pathway. The extent of alteration and magnitude of recovery could be linked to NK-cell frequency within ccRCC-infiltrating lymphocytes, possibly explaining the observed survival benefit of patients with NK(high) tumors. In conclusion, DGK-mediated dampening of the ERK pathway ensuing in NK-cell dysfunction was identified as an important escape mechanism in ccRCC. DGK and the ERK pathway thus emerge as promising therapeutic targets to restore suppressed NK-cell activity for the improvement of antitumor immunity.

High DGK-α and disabled MAPK pathways cause dysfunction of human tumor-infiltrating CD8+ T cells that is reversible by pharmacologic intervention.

CD8(+) tumor-infiltrating T cells (CD8-TILs) are found in many types of tumors including human renal cell carcinoma. However, tumor rejection rarely occurs, suggesting limited functional activity in the tumor microenvironment. In this study, we document that CD8-TILs are unresponsive to CD3 stimulation, showing neither lytic activity, nor lytic granule exocytosis, nor IFN-γ production. Mechanistically, no deficits in TCR proximal signaling molecules (lymphocyte-specific protein tyrosine kinase, phospholipase Cγ) were identified. In contrast, distal TCR signaling was suppressed, as T cells of TILs showed strongly reduced steady-state phosphorylation of the MAPK ERK and were unable to increase phosphorylation of ERK and JNK as well as AKT and AKT client proteins (IκB, GSK3) after stimulation. These deficits were tumor-specific as they were not observed in CD8(+) T cells infiltrating non-tumor kidney areas (CD8(+) non-tumor kidney-infiltrating lymphocytes; CD8-NILs). Diacylglycerol kinase-α (DGK-α) was more highly expressed in CD8-TILs compared with that in CD8-NILs, and its inhibition improved ERK phosphorylation and lytic granule exocytosis. Cultivation of TILs in low-dose IL-2 reduced DGK-α protein levels, increased steady-state phosphorylation of ERK, improved stimulation-induced phosphorylation of ERK and AKT, and allowed more CD8-TILs to degranulate and to produce IFN-γ. Additionally, the protein level of the AKT client molecule p27kip, an inhibitory cell cycle protein, was reduced, whereas cyclin E, which promotes G1-S phase transition, was increased. These results indicate that the tumor-inflicted deficits of TILs are reversible. DGK-α inhibition and provision of IL-2 signals could be strategies to recruit the natural CD8(+) T cells to the anti-tumor response and may help prevent inactivation of adoptively transferred T cells thereby improving therapeutic efficacy.

High immune response rates and decreased frequencies of regulatory T cells in metastatic renal cell carcinoma patients after tumor cell vaccination.

Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T(H)1/T(H)2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.

Prognostic relevance of disseminated tumour cells in bone marrow of patients with transitional cell carcinoma.

OBJECTIVE: This prospective study is the first immunocytochemical investigation of the frequency and prognostic value of CK+ tumour cells in the bone marrow of patients with transitional cell carcinoma (TCC). METHODS: Bone marrow aspirates from 228 TCC patients were taken preoperatively. Cytospins were made and stained by immunocytochemistry using the monoclonal antibodies CK2 and A45-B/B3. 27 patients with no evidence of any malignant disease served as control group. RESULTS: CK+ tumour cells were detected in 28% (63/228) of the TCC patients. No CK+ cells (0/27) were detected in the control group. In multivariate analysis the detection of > or =3 CK+ cells in bone marrow was an independent prognostic factor (hazard ratio=2.7, p<0.05) in patients with T2-4 tumour classification. CONCLUSION: Disseminated CK+ cells play a role in the biology of tumour spread of TCC, and their immunocytochemical detection can be useful in assessing the prognosis of TCC patients with an invasive tumour.

Influence of CD80, interleukin-2, and interleukin-7 expression in human renal cell carcinoma on the expansion, function, and survival of tumor-specific CTLs.

PURPOSE: A renal cell carcinoma (RCC) line, RCC-26, has been identified as a suitable candidate for development of an allogeneic tumor cell vaccine based on its expression of a variety of tumor-associated antigens (TAA). To improve immunogenicity, RCC-26 cells were genetically engineered to express CD80 alone or in combination with interleukin (IL)-2 or IL-7. The effect of these modifications on proliferation, function, and survival of autologous and allogeneic tumor-specific CTLs was assessed. EXPERIMENTAL DESIGN: RCC-26 sublines expressing different transgenes were tested for their capacity to reactivate cytokine secretion and cytotoxicity in autologous tumor-infiltrating lymphocytes, to improve proliferation and survival of tumor-associated T cells present in autologous peripheral blood, and to induce tumor-associated responses in naive allogeneic lymphocytes. The expression of several common TAA was quantitated in the RCC-26 sublines using reverse transcription-PCR to identify surrogate markers for immune monitoring in clinical trials. RESULTS: Gene-modified RCC-26 cells showed enhanced immunogenicity. CD80 expression was necessary to induce RCC-associated CTL in blood of healthy allogeneic donors. It also improved proliferation of autologous effector-memory T cells. Further enhancement was achieved with IL-2 through induction of the antiapoptosis protein Bcl-x(L). The candidate vaccine lines overexpressed several common TAA that are suitable markers for immune monitoring. CONCLUSIONS: RCC-26 cells coexpressing CD80 and cytokine transgenes display improved immunogenic characteristics, supporting their use as allogeneic tumor cell vaccines for HLA-A2-matched patients with metastatic RCC.

Local recurrence in patients after radical cystectomy and orthotopic ileal neobladder: impact on function.

Whether an orthotopic bladder substitute should be constructed in patients with locally advanced or lymph node positive bladder cancer remains a subject of debate. These patients are at risk that local recurrence may impair reservoir function in orthotopic neobladders. We retrospectively assessed reservoir function in 68 consecutive patients with locally advanced bladder cancer. Tumor stage was multifocal carcinoma in situ, multifocal pT1 disease, pT2, pT3a, pT3b, and pT4a in 3, 4, 19, 11, 25, and 6 patients respectively. Lymph nodes were positive for carcinoma in 17 patients. Out of the 68 patients, 65 could be followed for at least three months. Within a median follow-up of 26 months (range three to 87 months), recurrence developed in 16 of the 65 patients (25%). 7 patients (11%) had distant failure. 9 patients (14%) showed local and distant recurrence. In the six out of the nine patients with local recurrence located lateral, dorsal, or cranial of the neobladder, adequate neobladder function was retained until the last follow-up visit or until death. Only those three patients with local recurrence involving the pelvic floor or urethra needed a suprapubic catheter due to urinary retention caused by tumor obstruction. Excluding pelvic floor and anterior urethral disease, we recommend orthotopic bladder substitution even in locally advanced but resectable disease as far as the patient is in a good performance status.

Survival benefit of radical prostatectomy in lymph node-positive patients with prostate cancer.

BACKGROUND: Positive lymph node (LN) status is considered a systemic disease state. In prostate cancer, LN-positive diagnosis during pelvic LN dissection (PLND) potentially leads to the abandonment of radical prostatectomy (RP). OBJECTIVE: To compare the overall survival (OS) and relative survival (RS; as an estimate for cancer-specific survival) in LN-positive patients with or without RP. DESIGN, SETTING, AND PARTICIPANTS: Between 1988 and 2007, a total of 35 629 men with prostate cancer were identified at the Munich Cancer Registry; of those, 1413 patients had positive LNs. INTERVENTION: Of these 1413 LN-positive patients, prostatectomy was abandoned in 456 LN-positive patients, whereas 957 underwent RP despite the LN-positive finding. MEASUREMENTS: Crucial analyses are based on 938 LN-positive patients (688 with RP and 250 without RP) with complete data regarding age, grade, and prostate-specific antigen (PSA). OS (Kaplan-Meier estimates) and RS are presented, and Cox regression analysis was used to show the influence of predictors such as clinical stage, age at surgery, number of positive LNs, PSA level, grade, and extent of surgery. RESULTS: Median follow-up was 5.6 yr. OS of patients at 5 yr and 10 yr was 84% and 64%, respectively, with RP and was 60% and 28%, respectively, with aborted RP. The RS of patients at 5 yr and 10 yr was 95% and 86%, respectively, with RP and was 70% and 40%, respectively, with abandoned surgery. There was an imbalance, however, in the number of positive LNs: 17.2% with RP had four or more positive nodes versus 28% in the patient group without RP. In the multivariate model, RP was a strong independent predictor of survival (hazard ratio: 2.04 [95% confidence interval, 1.59-2.63; p<0.0001]). CONCLUSION: LN-positive patients with complete RP had improved survival compared to patients with abandoned RP. These results suggest that RP may have a survival benefit and the abandonment of RP in node-positive cases may not be justified.

Phase 1 trial of allogeneic gene-modified tumor cell vaccine RCC-26/CD80/IL-2 in patients with metastatic renal cell carcinoma.

Preclinical studies showed that the allogeneic tumor cell line RCC-26 displayed natural immunogenic potential that was enhanced through expression of CD80 costimulatory molecules and secretion of interleukin-2. Here we report the study of RCC-26/CD80/IL-2 cells in a phase 1 vaccine trial of renal cell carcinoma patients with metastatic disease (mRCC). Fifteen patients of the HLA-A*0201 allotype, with at least one metastatic lesion, were included. Irradiated vaccine cells were applied in increasing doses of 2.5, 10, and 40 x 10(6) cells over 22 weeks. Primary study parameters included safety and toxicity. Sequential blood samples were analyzed by interferon-gamma enzyme-linked immunospot assays to detect tumor antigen-associated (TAA) effector cells. The vaccine was well tolerated and the designated vaccination course was completed in 9 of 15 patients. Neither vaccine-induced autoimmunity nor systemic side effects were observed. Delayed-type hypersensitivity skin reactions were detected in 11 of 12 evaluated patients and were particularly strong in patients with prolonged survival. In parallel, vaccine-induced immune responses against vaccine or overexpressed TAA were detected in 9 of 12 evaluated patients. No tumor regressions occurred according to RECIST (Response Evaluation Criteria in Solid Tumors) criteria; however, median time to progression was 5.3 months and median survival was 15.6 months, indicating substantial disease stabilization. We conclude that vaccine use was safe and feasible in mRCC. Clinical benefits were limited in these patients with advanced disease; however, immune monitoring revealed vaccine-induced responses against multiple TAAs in the majority of study participants. These results suggest that this vaccine could be useful in combination therapies and/or minimal residual disease.

Efficacy and safety of TachoSil as haemostatic treatment versus standard suturing in kidney tumour resection: a randomised prospective study.

OBJECTIVE: Elective nephron-sparing surgery (NSS) for renal cell carcinoma (RCC) has gained general acceptance as an alternative to radical nephrectomy. To achieve haemostasis without risk of local ischaemia and necrosis of kidney parenchyma after standard haemostatic suturing, we investigated TachoSil's efficacy and safety as atraumatic haemostatic treatment after kidney tumour resection. METHODS: A total of 185 patients scheduled for NSS for small, superficial kidney tumours were included in an open, randomised, prospective, multicentre, parallel-group trial. Primary objectives were to test haemostatic efficacy and safety of TachoSil versus standard suturing. Efficacy was tested by comparing intraoperative time to haemostasis (primary end point). Secondary objectives included proportion of subjects with haemostasis after 10 min of trial treatment, occurrence of haematoma on day 2 after surgery, volume and haemoglobin concentration of postoperative drainage fluid, and surgeon's rating of usefulness of trial treatments. Safety was evaluated by occurrence of adverse events. RESULTS: In the intent-to-treat population, time to haemostasis was significantly shorter with TachoSil versus standard suturing (mean: 5.3 vs. 9.5 min [p<0.0001]). Haemostasis was obtained within 10 min in 92% of patients in the TachoSil group and in 67% in the standard treatment group (p<0.0001). Differences in other secondary end points were not statistically significant. Both treatments were well tolerated. Surgeons rated TachoSil higher in terms of convenience to prepare and apply, and impression of efficacy. CONCLUSION: TachoSil was superior to standard suturing in obtaining intraoperative control of haemorrhage and was as well tolerated as standard haemostatic treatment during NSS.

Frequency and prognostic relevance of disseminated tumor cells in bone marrow of patients with metastatic renal cell carcinoma.

BACKGROUND: The prognostic relevance of disseminated cytokeratin-positive (CK+) tumor cells in the bone marrow of patients with different types of carcinoma has been demonstrated in several studies. In this prospective study, the frequency and prognostic value of CK+ tumor cells was investigated in the bone marrow of 55 consecutive patients with metastatic renal cell carcinoma (M1 RCC) in comparison with 256 M0 RCC patients from a previous study. METHODS: Aspiration of bone marrow from the anterior iliac crest was performed immediately before tumor resection in RCC patients. Cytospins were made and stained by immunocytochemistry using the APAAP (alkaline phosphatase-antialkaline phosphatase) protocol and monoclonal antibodies CK2 and A45-B/B3. Twenty-seven patients with no evidence of any malignant disease served as a control group. RESULTS: CK+ tumor cells were detected in 42% (23 of 55 patients) of the M1 patients and 25% (63 of 256 patients) of the M0 patients (P <.01). No CK+ cells (0 of 27 patients) were detected in the control group. In the M1 group, CK- patients demonstrated a trend toward a better outcome compared with CK+ patients (log-rank test, P = .19). This difference was significant when applying a higher threshold (0-2 CK+ cells vs. > or = 3 CK+ cells; P <.05). On multivariate analysis, the detection of > or = 3 CK+ cells in the bone marrow was found to be an independent prognostic factor (P <.001). CONCLUSIONS: The results of the current study indicate that disseminated CK+ cells play a role in the biology of tumor spread of RCC, and that their immunocytochemical detection can be useful in assessing the prognosis of patients with M1 disease.

Cell-based vaccines for renal cell carcinoma: genetically-engineered tumor cells and monocyte-derived dendritic cells.

Initial vaccine developments for renal cell carcinoma (RCC) have concentrated on cell-based approaches in which tumor cells themselves provide mixtures of unknown tumor-associated antigens as immunizing agents. Antigens derived from autologous tumors can direct responses to molecular composites characteristic of individual tumors, whereas antigens derived from allogeneic tumor cells must be commonly shared by RCC. Three types of cell-based vaccine for RCC have been investigated: isolated tumor cell suspensions, gene modified tumor cells and dendritic cells (DCs) expressing RCC-associated antigens. Approaches using genetic modification of autologous RCC have included ex vivo modification of tumor cells or modification of tumors in vivo. We have used gene-modification of allogeneic tumor cell lines to create generic RCC vaccines. More recently, emphasis has shifted to the use of DCs as cell-based vaccines for RCC. DCs have moved to a position of central interest because of their excellent stimulatory capacity, combined with their ability to process and present antigens to both naive CD4 and CD8 cells. The long impasse in identifying molecular targets for specific immunotherapy of RCC is now rapidly being overcome through the use of tools and information emerging from human genome research. Identification of candidate molecules expressed by RCC using cDNA arrays, combined with protein arrays and identification of peptides presented by MHC molecules, allow specific vaccines to be tailored to the antigenic profile of individual tumors, providing the basis for development of patient-specific vaccines.

Disseminated tumor cells in bone marrow of patients with transitional cell carcinoma: immunocytochemical detection and correlation with established prognostic indicators.

PURPOSE: Previous investigations have demonstrated the prognostic value of disseminated cytokeratin positive cells in bone marrow of patients with breast, gastric, colon and prostate cancer. We evaluated the potential of an immunocytochemical assay, using a monoclonal antibody against cytokeratin 18 (CK 18), for the detection of disseminated tumor cells in bone marrow aspirates of patients with transitional cell carcinoma. MATERIALS AND METHODS: Bone marrow aspiration was performed preoperatively on 128 patients with transitional cell carcinoma of various stages and on 27 controls with nonmalignant disease. Cytospin preparations of mononuclear bone marrow cells were incubated with a monoclonal anti-CK 18 antibody and stained using the alkaline phosphatase anti-alkaline phosphatase technique. RESULTS: Of the patients with transitional cell carcinoma 29.7% and none of the controls had a CK 18 positive bone marrow result. A significant correlation between the incidence of CK 18 positive cells in bone marrow and invasive transitional cell carcinoma (p <0.01), lymph node involvement (p <0.01), medium/high grade transitional cell carcinoma (p <0.01) and tumor progression in recurrent transitional cell carcinoma (p <0.05) was demonstrated. Furthermore, the mean number of CK 18 positive cells in bone marrow aspirates of patients with stage M+ and/or N+ disease was nearly 3 times as high as that of patients without clinically evident metastatic disease (10.4 versus 3.8 CK 18 positive cells per patient). CONCLUSIONS: A significant correlation between the incidence of CK 18 positive bone marrow results in patients with transitional cell carcinoma and established risk factors could be demonstrated in our study. Further prospective followup studies should be performed to determine the prognostic value of these findings.

Isolation of circulating cancer cells from whole blood by immunomagnetic cell enrichment and unenriched immunocytochemistry in vitro.

PURPOSE: We improved tumor cell detection compared with currently available immunocytochemical methods by immunomagnetic cell enrichment. MATERIALS AND METHODS: Two methods of immunomagnetic enrichment using antibody coated magnetic beads were tested and compared with unenriched immunocytochemistry, including positive selection of epithelial cells with the antiepithelial antibody BER-EP4 and depletion of mononuclear cells with the anti-leukocyte antibody CD45. Various numbers of tumor cells from the 4 tissue culture cell lines DU 145, RT-4, KTCTL-2 and KTCTL-30 obtained from urological tumors were added to whole blood and mononuclear cells were isolated by density centrifugation. After incubation of the cell suspensions with beads cell separation was done in a magnetic field. After centrifugation on glass slides immunocytochemical staining for cytokeratin was performed. A total of 96 experiments were completed and negative controls were obtained. RESULTS: The number of tumor cells detected by positive selection and depletion was significantly higher compared with immunocytochemistry (Wilcoxon test p <0.01). Mean enrichment factor and tumor cell recovery rates were 12.9% and 43.5% for positive selection, and 9.4% and 32.6% for depletion, respectively (p <0.05). With 1 tumor cell suspended in up to 30 ml. full blood unenriched immunocytochemistry failed to detect cancer cells, whereas positive selection revealed epithelial cells in 12 of 14 cases (85.5%) and depletion in all 14 (p <0.05). No false-positive results were observed. CONCLUSIONS: Compared with unenriched immunocytochemistry immunomagnetic enrichment significantly improves the detection of epithelial cells added to blood. A significant advantage was observed for positive selection. Immunomagnetic enrichment may be important for clinical practice in the future.

Detection and prognostic value of cytokeratin positive tumor cells in bone marrow of patients with renal cell carcinoma.

PURPOSE: Several investigations indicate the prognostic value of disseminated cytokeratin positive tumor cells in bone marrow of patients with carcinoma of different origin. In this study we evaluated the prognostic significance of epithelial cells in bone marrow of patients with renal cell carcinoma (RCC). MATERIALS AND METHODS: Aspiration of bone marrow was performed preoperatively in 335 patients with RCC between 1990 and 1998. A total of 287 patients fulfilled all study inclusion (eg M0 R0 tumor stage) and exclusion (eg second malignancy during followup) criteria for the final analysis. Cytospin preparations were made after density gradient centrifugation of bone marrow samples and incubated with monoclonal antibodies directed against cytokeratin 18 (CK2) and pan-cytokeratin. Staining was performed using the alkaline phosphatase-anti-alkaline phosphatase method and 256 samples were evaluated. RESULTS In 25% (63 of 256) of the patients cytokeratin positive (CK+) cells were detected in bone marrow. Tumor progression (defined as tumor associated death, local recurrence or new metastases) was present in 12% (31 of 256) during the followup period (median 40 months), and 14% (9 of 63) with CK+ cells and 11% (22 of 193) with negative bone marrow status exhibited tumor progression. Survival analysis (log-rank test) showed no significant difference between the CK+ and cytokeratin negative group. The detection of CK+ cells was not an independent prognostic parameter in multivariate analysis (Cox regression model). CONCLUSIONS: These results indicate that the immunocytochemical detection of disseminated cytokeratin positive tumor cells in the bone marrow of patients with RCC has no prognostic significance.

In vitro comparison of transurethral vaporization of the prostate (TUVP), resection of the prostate (TURP), and vaporization-resection of the prostate (TUVRP).

PROBLEM: Transurethral vaporization of the prostate (TUVP) and vaporization-resection of the prostate (TUVRP) ("vapor cut, band electrode") seem to be alternatives to conventional resection of the prostate (TURP). For TUVP and TUVRP, little in vitro data has yet been published. The aim of this study was to determine settings for optimal performance with TUVP and TUVRP and to investigate electrosurgical parameters relevant to safety. METHODS: Standardized experiments were performed on porcine muscle. Mass loss and coagulation zones were measured optically. Additionally, electrical parameters were recorded. RESULTS: The maximum tissue ablation rates were 3.8 cm3 per min and 6.1 cm3 per min for TUVP and TUVRP, respectively, compared to 6.5 cm3 per min for TURP. The maximum coagulation depths reached 2.1 mm (TUVP), 1.4 mm (TUVRP), and 0.9 mm (TURP). Optimal in vitro settings for TUVP/TUVRP/TURP were as follows: generator power of 250/120/90 W, drag speed of 5/15/20 mm/s, and pressure of 0.40/0.15/0.05 N. Different power generators and electrodes showed considerably varying performance. The energy to remove 1 g of tissue averaged 7.500 J (TUVP), 620 J (TUVRP). and 400 J (TURP). CONCLUSIONS: These results allow quantification of the influence of different variables on TUVP, TUVRP, and TURP in vitro. The TUVP proves to be an effective ablation alternative. Nevertheless, a 15 to 20 times higher energy demand has to be considered. TUVRP combines excellent ablation features with greater coagulation volumes, indicating better hemostasis.

Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors.

Bone marrow is a major homing site for circulating epithelial tumor cells. The present study was aimed to assess the proliferative capacity of occult metastatic cells in bone marrow of patients with operable solid tumors especially with regard to their clinical outcome. We obtained bone marrow aspirates from 153 patients with carcinomas of the prostate (n = 46), breast (n = 45), colon (n = 33), and kidney (n = 29). Most of the patients (87%) had primary disease with no clinical signs of overt metastases [tumor-node-metastasis (TNM)-stage UICC (Union Internationale Contre le Cancer) I-III]. After bone marrow was cultured for 21-102 days under special cell culture conditions, viable epithelial cells were detected by cytokeratin staining in 124 patients (81%). The cultured epithelial cells harbored Ki-ras2 mutations and numerical chromosomal aberrations. The highest median number of expanded tumor cells was observed in prostate cancer (2,619 per flask). There was a significant positive correlation between the number of expanded tumor cells and the UICC-stage of the patients (P = 0.03) or the presence of overt metastases (P = 0.04). Moreover, a strong expansion of tumor cells was correlated to an increased rate of cancer-related deaths (P = 0.007) and a reduced survival of the patients (P = 0.006). In conclusion, the majority of cancer patients have viable tumor cells in their bone marrow at primary tumor diagnosis, and the proliferative potential of these cells determines the clinical outcome.