Disseminated Tumor Cells in Prostate Cancer Patients after Radical Prostatectomy and without Evidence of Disease Predicts Biochemical Recurrence

Although more than 90% of prostate cancer is considered localized at the time of diagnosis, the rate of biochemical [prostate-specific antigens (PSA)] recurrence after radical prostatectomy is 20% to 30%. Additionally, although PSA elevations occur most often in the 5 years following radical prostatectomy, they may occur as long as 10 to 15 years after surgery (1, 2). Furthermore, the mean time from PSA recurrence to clinical metastasis is 8 years (1). These facts suggest the persistence of tumor cells in a state of either complete or near dormancy prior to metastatic progression.

The persistence of cancer cells within the vasculature of breast cancer patients years after treatment and without evidence of disease has been well documented with a variety of techniques, including flow cytometry, immunohistochemistry, and immunomagnetic selection (3, 4). Breast cancer cells seem to disseminate early from the primary tumor and undergo a distinct evolutionary process (5). Some of these cells are capable of remaining dormant and may be responsible for delayed metastatic progression (6).

We hypothesize that a similar process of tumor dormancy occurs in patients with prostate cancer. Numerous studies have shown the ability to detect tumor cells in the blood [circulating tumor cells (CTC)] and bone marrow [disseminated tumor cells (DTC)] of prostate cancer patients, but their significance has not been determined (7-11, reviewed in 12). As in breast cancer, there is evidence that prostate cancer cells disseminate early on from the primary tumor. Using comparative genomic hybridization, Klein et al. (5) found significant heterogeneity in DTC within individual patients with prostate or breast cancer. These genetic disparities may indicate early divergence of the DTC and separate evolution. Unlike breast cancer, however, in which large prospective trials have consistently shown a prognostic impact of these cells (13–15), evidence for a correlation between disease progression and the presence of circulating or disseminated tumor cells in prostate cancer is limited to small pilot studies (7, 16). Furthermore, there is little data on the prognostic significance of these cells after radical prostatectomy (16, 17). Therefore, the importance of these cells in prostate cancer remains unclear, and the possibility that they may represent dormant tumor cells has not been well elucidated.

Prostate cancer metastases occur predominantly in the bone, and we have refined a sensitive assay that enriches and identifies DTC from the bone marrow of men with prostate cancer (8, 10). We measured the presence of DTC before and after radical prostatectomy to determine if these data would be clinically relevant and lend insight into the metastatic process. The purpose of this study was to describe the prevalence of DTC before and after radical prostatectomy, and to determine if the presence of DTC after radical prostatectomy is associated with biochemical recurrence.

Patient selection. From January 1, 2002 to December 31, 2006, bone marrow aspirates from prostate cancer patients were prospectively collected for the purpose of identifying important molecular markers in DTC and evaluating whether these cells are correlated with clinical outcomes. Here, in order to assess the clinical importance of these cells, we retrospectively assembled a cohort of 631 consecutive patients with prostate cancer and no other known prior or simultaneous epithelial malignancies who underwent satisfactory bone marrow aspiration. Aspirates were obtained from 569 patients at the time of radical prostatectomy and 98 patients with no evidence of disease (NED) after radical prostatectomy. Forty-nine patients were present in both groups. All patients who underwent radical prostatectomy at the authors' institution and all those seen in follow-up after radical prostatectomy were asked to participate. Samples from NED patients were drawn at least 3 mo after radical prostatectomy, and in eight men with more than one sample after radical prostatectomy only their first sample was included in order to standardize the analysis.

Men were considered to be NED following radical prostatectomy if they had a serum PSA level <0.4 ng/mL. Patients with PSA ≥0.4 ng/mL and patients who had undergone salvage radiation treatment were considered to have recurrent disease. This PSA cutoff was selected due to the large percentage of patients with PSA between 0.1 and 0.4 ng/mL who never undergo further PSA progression and therefore do not have a definitive recurrence of their prostate cancer (18, 19). For example, in a cohort of 773 men with PSA between 0.20 and 0.29 ng/mL after radical prostatectomy, the rate of further PSA rise or treatment reported by Amling et al. (18) was only 50%. Thirty-four men with PSA <2.5 ng/mL, negative digital rectal exam, and no evidence of any malignancy were studied as controls. All specimens were obtained after informed consent and collected using protocols approved by the Institutional Review Board and Human Subject Division at the University of Washington.

Collection of samples. Ten milliliters of bone marrow were aspirated from the iliac crest into a 30-mL syringe containing 10 mL of 6% sodium citrate. In samples obtained from patients just prior to radical prostatectomy, bilateral aspirates were obtained and combined for a total of 20 mL of bone marrow. Samples from patients prior to radical prostatectomy and from controls were obtained under general anesthesia and from the anterior iliac crest. Postprostatectomy samples were obtained using local anesthesia and taken from the posterior iliac crest. Processing of samples commenced within 1 h and was completed within 4 h.

Cell enrichment. Cell enrichment and isolation was done as previously described (10). Briefly, bone marrow aspirates were placed over a 15-mL volume of Ficoll-Isopaque 1.077 g/mL (Accurate Chemical). Centrifugation subsequently yielded a mononuclear cell layer containing DTC, if present. The MACS system for immunomagnetic selection (Miltenyi Biotec) was then employed. Anti-CD45 and anti-CD61 antibodies were used for negative selection, targeting leukocytes, megakaryocytes, and platelets. Positive selection was then done with immunomagnetic beads coated with antihuman epithelial antigen antibodies.

Identification of disseminated tumor cells. For identification and isolation of DTC, the enriched population was subjected to immunostaining with FITC-labeled anti-BerEP4 antibodies (Dako) which bind a different epitope on human epithelial antigen than the antihuman epithelial antigen antibody used for positive selection. A phycoerythrin-conjugated anti-CD45 antibody was also added for identification of leukocytes. The cells were kept on ice and viewed under fluorescent light using an inverted microscope. Patients were considered DTC-positive if they had one or more cells staining BerEP4 positive and CD-45 negative. This cut-point of one cell was defined a priori as we hypothesized that a single detectable cell may carry significance for tumor recurrence.

Statistical methods. Descriptive statistics were used to compare demographic and disease characteristics of patients with and without DTC. Univariate comparisons were tested using the Pearson χ² test. Kaplan-Meier methods were used to compare the unadjusted survival of patients with and without DTC. Cox proportional hazards regression was used to compare survival of patients with and without DTC after adjusting for demographic and disease characteristics. Age (categorized), race (White, African American), preoperative PSA (continuous), pathologic stage (organ confined, nonorgan confined), pathologic grade (Gleason 5-6, Gleason 7, Gleason 8-10), and margin status (positive, negative) were selected a priori as covariates for the multivariate regression model.

Because the time between radical prostatectomy and bone marrow aspiration was not standardized, two separate models were considered. In the first model, the time under observation started at the date of radical prostatectomy. In the second model, the time under observation started at the date of bone marrow aspiration postsurgery. Patients who did not exhibit a PSA recurrence were censored at the date of last follow-up. Proportional hazards assumptions were tested with Schoenfeld residuals. Stata version 9.2 (StataCorp) was used for the statistical analyses.

DTC prior to prostatectomy. Prior to radical prostatectomy, 408 of 569 patients (72%) had ≥1 DTC present (Table 1). The median age of this group was 63 years (range, 40-81 years). In the control group of men with PSA <2.5 ng/mL, 3 of 34 (8.8%) had cells present in their bone marrow (χ², P < 0.01). The median age of the controls was 50 years (range, 24-81 years). Of the subset of controls >40 years old (median, 62 years), 1 of 20 (5%) was positive. The three positive controls were followed for a median of 48 months (range, 13-48 months), and none have developed any evidence of malignancy. Pathologic characteristics of the cohort before radical prostatectomy are shown in Table 1. The median age at radical prostatectomy for this group was 60 years (range, 35-78 years). There was no correlation between the presence of DTC and any of the following variables: preoperative PSA, Gleason grade, pathologic stage, or tumor volume. The absence of correlation persisted even when patients were stratified by number of DTC detected (i.e., ≥5 or ≥100 cells per sample; data not shown).

DTC in men NED after radical prostatectomy. Follow-up bone marrow aspirates were done in 98 patients at a median of 20 months after radical prostatectomy (range, 3-216 months). Median PSA follow-up was 42 months (range, 13-228 months) from the time of radical prostatectomy and 13 months (range, 1-58 months) from the time of bone marrow aspiration. Table 2 shows the characteristics of NED patients after radical prostatectomy stratified by presence of DTC. Overall, 57% of patients classified as NED after radical prostatectomy had DTC present, which was significantly different than the rate of detection prior to radical prostatectomy (P = 0.004). The rate of detection of DTC was similar at <1 year from radical prostatectomy (9 of 14, or 64%) and 1 to 5 years (32 of 51, or 63%; Fig. 1). At >5 years, detection was slightly lower (15 of 33, or 45%), but this difference was not statistically significant compared with detection at ≤5 years (χ², P = 0.10). Additionally, of the 83 men with PSA <0.1 ng/mL at the time of bone marrow aspiration, 48 (58%) had DTC present. DTC were detected in two men 12 years after radical prostatectomy, neither of whom have recurred to date.

Fourteen patients (14%) in the NED cohort have recurred after bone marrow aspiration to date, and 12 of these 14 patients (86%) had DTC present after radical prostatectomy. In comparison, DTC were detected in 44 of 84 (52%) of patients who have not recurred to date (χ², P = 0.02). As seen in Table 3, in the multivariate analysis and in the model using time from radical prostatectomy, DTC was an independent predictor for recurrence: men with DTC present after radical prostatectomy had a significantly greater risk of recurrence than men without these cells present (hazard ratio 6.87; 95% confidence interval, 1.03-45.87). Figure 2A shows the Kaplan-Meier recurrence-free survival curve from the time of radical prostatectomy for patients with and without DTC (log rank, P = 0.01). Similarly, in the second model, when time under observation started as the time of bone marrow aspiration (Table 3), detection of DTC was associated with a hazard ratio of 4.20 (95% confidence interval, 0.79-22.39). The Kaplan-Meier curve for this analysis is shown in Fig. 2B (log rank, P = 0.03).

We also looked separately at the 49 patients who had bone marrow aspirates both at the time of radical prostatectomy and in follow-up when NED. Two patients were negative in both samples, 3 patients were negative at radical prostatectomy and positive in follow-up, 17 patients had DTC detected at radical prostatectomy and none when NED, and 27 patients had DTC in both samples. Recurrences have occurred in 1 of 3 (33%) who changed from negative to positive and in 4 of 27 (15%) with DTC in both samples. No patients in the other groups have recurred.

The objective of this study was to describe the prevalence of DTC in patients with prostate cancer prior to radical prostatectomy and to assess the relationship between risk of prostate cancer recurrence and presence of disseminated prostate cancer cells after radical prostatectomy. We presented three major findings: First, DTC were present in >70% of patients with prostate cancer prior to radical prostatectomy – a significantly larger proportion than most previous reports (20–23). The high rate of dissemination prior to treatment may explain the apparent lack of correlation between disseminated prostate cells and tumor pathology, and is suggestive of early shedding of prostate cancer cells that have the capacity to reside within the bone marrow. Second, DTC were detected in 57% of patients NED after radical prostatectomy, including 45% (15 of 33) of patients >5 years removed from surgery. This has implications in terms of tumor dormancy and delayed recurrence. Finally, NED patients with DTC present after radical prostatectomy had a nearly 7-fold increased risk of recurrence compared with those patients without DTC. This is the first report of an association between DTC and biochemical recurrence in men without evidence of disease after radical prostatectomy.

Early dissemination of cancer cells regardless of stage, grade, or tumor volume is supported by molecular evidence showing significant heterogeneity among DTC and between DTC and their primary tumors (4, 5). Furthermore, a comparison of genetic patterns in patients with multifocal prostate cancer and CTC suggested that a single focus – often the smallest focus and as small as 0.2 cm³ – may have been the most likely source of CTC in these patients (24). This concurs with our data showing a high rate of DTC detection prior to radical prostatectomy even in low-risk patients.

We also have completed an initial comprehensive study on genomic aberrations in the DTC population using array comparative genomic hybridization (25). Using as few as 10 pooled DTC from 48 patients prior to radical prostatectomy and 11 patients with advanced disease, we made several notable observations. Epithelial cells derived from the bone marrow aspirates of patients prior to radical prostatectomy had significantly more genomic deviations than normal cells (14 versus 4, P = 0.005), implying again that these are tumor cells. These genomic alterations were similar, but not identical, to those of the primary tumor. There was also a progressive evolution in genomic changes between those DTC analyzed from patients before radical prostatectomy and those from patients with advanced disease. Additionally, the genomic array comparative genomic hybridization profile of the DTC from advanced stage patients were more similar to those of metastases than to those of primary tumors, with commonly expressed gains in 8q and losses in 8p12-23, 10q26, 13q, and 16q21. These molecular investigations provide strong justification for the further characterization of these shed tumor cells with the expectation that such studies will provide insight to help stratify patients prior to treatment, select the most appropriate treatment, and facilitate novel therapeutic strategies.

To date, there are few published studies evaluating the significance of CTC and DTC in prostate cancer patients after radical prostatectomy. One report evaluated 50 postprostatectomy patients by peripheral blood reverse transcription-PCR, detecting CTC in 47% of patients with a rising PSA versus only 3% without a rising PSA at the time of blood draw (26). Tombal et al. (27) evaluated 55 men with biochemical recurrence after radical prostatectomy and detected a trend towards a shorter doubling time in men with CTC compared with those without CTC. Only one recent study has tested for tumor cells in the bone marrow after therapy, finding that the presence of DTC ≥2 years after definitive radiotherapy was associated with some decrease in progression-free survival (28).

Given that studies after primary therapy in breast cancer have shown an independent prognostic value for CTC and DTC (15, 29, 30), there is a clear need to identify their role in prostate cancer. It is also important to determine, on a biological level, what mechanisms enable prostate cancer to recur after many years without detection. Our data indicate that, although many patients with DTC prior to radical prostatectomy no longer have detectable DTC after surgery, a large proportion of NED patients harbor tumor cells in their bone marrow long after radical prostatectomy. Additionally, those patients with persistent DTC after radical prostatectomy have a significantly higher risk of biochemical recurrence than those without DTC. These results suggest that tumor dormancy plays a prominent role in prostate cancer recurrence after definitive therapy. Murine models of dormancy have been induced through immunization with tumor markers, and the existence of dormant states due to a lack of angiogenesis has been well documented (31, 32). We suggest that tumor cells in the bone marrow of patients who are clinically disease-free several years after radical prostatectomy are, in fact, dormant tumor cells that may eventually lead to recurrence (33). It remains unknown whether all DTC or a subset of these cells with specific phenotypic characteristics are capable of forming metastases.

The observations from our NED cohort must be tempered by the study size of 98 patients as well as the need for longer follow-up given the delayed nature of biochemical recurrence after radical prostatectomy. There have been only 14 instances of biochemical failure in this cohort, and more events will be required in order to confirm the findings observed to date. Furthermore, it is possible that the low number of events resulted in an imprecise estimate of the influence of stage, grade, preoperative PSA, and margin status as predictors of recurrence in the multivariate analysis. Another limitation of this analysis is the varying time from surgery to bone marrow aspiration; ideally, bone marrow aspirates would have been acquired at a standard interval, although we found that the detection of DTC in these specimens changed very little with time. Additionally, in order to help account for the varying intervals, we employed both a time from radical prostatectomy model and a time from bone marrow aspirate model for the multivariate analysis with similar estimates of risk. Differences in the statistical significance between these two models are likely related to the differences in median follow-up time (42 versus 13 months).

There are significant technical barriers to identifying individual tumor cells from a bone marrow aspirate containing approximately 10⁷ cells. We utilize a multistep enrichment technique with both positive and negative selection, using both epithelial and leukocyte staining markers in order to identify only those cells likely to be DTC. Whereas a semiautomated method of CTC detection has been successfully developed (34), the quantity and variety of cells in the bone marrow pose a greater technical challenge for the detection of DTC. Additionally, the significance of the three “false positives” in the control group remains unclear. Some have suggested that EpCAM may be detected on normal hematopoetic cells (35), but we identified no morphologic features that distinguished positive-staining cells in controls from those seen in prostate cancer patients. Whether the detection of DTC in controls accurately reflects the error rate of the DTC identification technique or signals an undetected epithelial malignancy will take further study and molecular characterization. Furthermore, whereas others have used cutoffs of ≥5 cells to define samples as positive, we sought to maximize sensitivity of the assay by utilizing a single DTC cutoff. A higher cell cutoff would have decreased the false positive rate, but the correlation between DTC and biochemical recurrence seen here supports the single cell definition. As other investigators have concluded, because identification of a single DTC may have clinical importance, this is the ideal definition of a positive sample (36).

The present data indicate that at least some DTC may be dormant tumor cells capable of eventually proliferating and leading to tumor recurrence. Although some DTC-positive patients have not recurred, all but two patients who recurred had DTC detected while still NED in our definition. Those NED patients with DTC seemed to be at a significantly increased risk of recurrence. This investigation was done as a retrospective analysis of prospectively collected data and therefore a separate prospective study will be necessary before any recommendations can be made regarding routine analysis of bone marrow after prostatectomy. A full understanding of these cells will require molecular techniques such as cDNA microarrays and comparative genomic hybridization, and these analyses are ongoing. Further research may help elucidate potential intrinsic and extrinsic factors responsible for enabling a dormant state in these cells and allowing some to proliferate successfully after years of dormancy. Finally, identification of clinically relevant molecular markers on these cells may allow more sensitive cell detection, better prognostication, and, potentially, targets for future therapies.

No potential conflicts of interest were disclosed.

We thank Martin Kinnunen and Bryce Lakely who have dedicated several years to perfecting the techniques discussed here as well as to gathering laboratory data.