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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Clin Chem. Author manuscript; available in PMC Nov 25, 2009.
Published in final edited form as:
PMCID: PMC2782624
NIHMSID: NIHMS126168

Circulating prostate tumor cells detected by RT-PCR in men with localized or castration-refractory prostate cancer: concordance with CellSearch assay and association with bone metastases and with survival

Abstract

BACKGROUND

Reverse transcription-PCR (RT-PCR) assays for detecting or analyzing expression profiles of circulating tumor cells (CTCs) are currently of uncertain clinical value. We assessed men with localized prostate cancer or castration-refractory prostate cancer (CRPC) for CTCs by real-time RT-PCR for KLK3 (PSA) and KLK2 mRNAs. We also assessed the association of CTCs with disease characteristics and survival.

METHODS

KLK3, KLK2, and prostate stem cell antigen (PSCA) mRNAs were determined by standardized, quantitative real-time RT-PCR assays using blood from 180 localized disease patients, 76 metastatic CRPC patients, and 19 healthy volunteers. CRPC samples were also tested for CTCs by an immunomagnetic separation system (CellSearch™) approved for clinical use.

RESULTS

All healthy volunteers were negative for KLK mRNAs. KLK3 or KLK2 mRNAs were positive (≥80 mRNAs per mL blood) in 37 (49%) patients with CRPC, but in only 15 (8%) patients with localized cancer. RT-PCR and CellSearch CTC results were strongly concordant (80-85%) and correlated (Kendall’s tau 0.60-0.68). Among patients with CRPC, KLK mRNAs and CellSearch CTCs were closely associated with clinical evidence of bone metastases and with survival, but only modestly correlated with serum PSA. PSCA mRNA was detected in only 7 CRPC patients (10%) and was associated with positive KLK mRNA status.

CONCLUSION

Real-time RT-PCR assays of KLK mRNAs are highly concordant with CellSearch CTC results in patients with CRPC. KLK2/3-expressing CTCs are common in men with CRPC and bone metastases, but rare in patients with metastases diagnosed only in soft tissues and patients with localized cancer.

INTRODUCTION

For patients with prostate cancer, there is need for improved predictive markers to facilitate treatment selection and to monitor the effects of treatment. This need is particularly acute for patients with metastatic and/or castration-refractory disease. In these patients, the PSA level is only loosely associated with survival, bone scans offer only limited information on changes in the disease, and biopsy has poor sensitivity and is not clinically practical on a repeated basis. In addition, there is a need for improved preoperative staging modalities for patients with localized prostate cancer.

These problems have created interest in circulating tumor cells (CTCs) as a potential marker. Numerous techniques have been employed to detect CTCs. They can be categorized as techniques for detecting tissue- or disease-specific gene expression, such as RT-PCR, and techniques for detecting CTCs as intact cells, such as flow cytometry and immunomagnetic capture. The one assay approved for clinical use in the United States is CellSearch semi-automated immunomagnetic capture and detection (1). It has been hypothesized that a more sensitive method for detection of CTCs may be real-time RT-PCR of tissue-specific transcripts. End-point RT-PCR for molecular staging of prostate cancer was investigated with high expectations in the early 1990s (2, 3). The hope was that the detection of circulating tumor cells might provide a more accurate way to preoperatively predict the pathologic stage and the risk of disease recurrence. However, after initial promise, results have often been discrepant, and most reports have failed to show clinical value (4-11). Also, the proportion of positive samples in different disease stages remains unclear. Frequencies of blood samples positive for prostate-specific RNAs have ranged from 0% to 81% in clinically localized disease and from 31% to 100% in metastatic disease (12, 13). Widespread inconsistencies in results may stem largely from differences in pre-analytical sample processing, differences in analytical methods, poor standardization, and qualitative or semiquantitative detection of end-point RT-PCR products.

We have developed sensitive, highly reproducible, and fully standardized real-time quantitative RT-PCR assays for mRNAs of prostate-specific antigen (KLK3) and human kallikrein 2 (KLK2) (14-16). In this study, we compared this method to CellSearch for patients with castration-resistant prostate cancer (CRPC), and investigated the association of CTCs with disease characteristics and survival. We also investigated the frequency of KLK3- or KLK2-expressing CTCs in men with clinically localized prostate cancer. Finally, we describe a new real-time quantitative RT-PCR assay for the detection of prostate stem cell antigen (PSCA) mRNA. The PSCA gene is over-expressed in prostate cancer metastases (17), and our objective was to assess the frequency of PSCA-expressing CTCs in the patients with CRPC.

MATERIALS AND METHODS

Participants, sample collection, and CellSearch analysis

The study enrolled 80 patients treated at Memorial Sloan-Kettering Cancer Center for metastatic CRPC with castrate levels of testosterone (<50 ng/dL). Radionuclide bone scans were reviewed for the presence or absence of metastatic bone disease, and CT and/or MRI scans were evaluated for lymph node, liver, or lung soft tissue disease, or epidural and prostatic/pelvic masses. Four patients without evidence of metastatic disease or castrate levels of testosterone were excluded. As controls, the study enrolled 19 healthy volunteers: 12 men under age of 40 without prostate cancer and 7 women.

Three groups of patients with localized disease were enrolled. The first consisted of 42 patients who had undergone radical prostatectomy (RP) at MSKCC at least 6 months before sample collection (median 24 months, IQR 6-80 months). [Eleven patients (26%) had pathologic stage pT3a, 4 patients (10%) pT3b, and 27 patients pT2 cancer with positive surgical margins (3 patients; 7%), capsular invasion (15; 36%), or no adverse pathologic features (9; 21%).] The second group included 87 patients scheduled to undergo radical prostatectomy for clinically localized prostate cancer, whose RP specimen subsequently showed at least one unfavorable feature, defined as seminal vesicle invasion, extracapsular extension or capsular invasion, positive surgical margin, or lymph node involvement. [Thirty-two patients (37%) had pathologic stage pT3a, 9 (10%) pT3b, and 2 (2%) pT4. The remaining patients had pT2 cancer with either lymph node involvement (1 patient; 1%), positive surgical margin (7; 8%), or capsular invasion (36; 41%).] The third group consisted of 51 patients diagnosed with prostate cancer at University Hospital of Hamburg (UKE), Germany, and scheduled to undergo either RP for clinically localized cancer, or radiation therapy of the prostate. [Six of the 26 pre-RP patients (23%) had pathologic stage of pT3a, 6 (23%) pT3b. Fourteen patients had pT2 cancer, two (8%) with positive surgical margins, and 12 (46%) with no adverse pathologic features.] Biochemical recurrence was defined as having at least one serum PSA value >0.4 ng/mL.

Peripheral blood (2.5 mL) was collected in PAXgene Blood RNA tubes (PreAnalytiX). Samples were incubated at room temperature for 24 hours and stored at -20°C and -80°C until RNA isolation. For CRPC patients, a second sample of 7.5 mL was collected at the same visit in a CellSave tube and processed for CTC counts by CellSearch immunomagnetic selection as described (18). All samples were collected under Institutional Review Board approved protocols with informed consent.

RNA isolation and cDNA synthesis

RNA was isolated with PAXgene Blood RNA kit (PreAnalytiX) according to the manufacturer’s instructions, including the optional DNase digestion. An internal standard RNA, m3PSA (described in Supplemental Data), was added in prefixed amounts to each patient sample at the beginning of the RNA isolation (PAXgene RNA isolation protocol step 5), resulting in 10 000 molecules per μl of reverse transcription reaction. The internal standard reflects any variation arising from different steps of the RT-PCR protocol from RNA isolation to signal detection. RNA was quantified by RiboGreen RNA Quantitation Reagent (Invitrogen) and immediately divided into two aliquots and reverse transcribed with High-Capacity cDNA Archive kit (Applied Biosystems). RNA priming and reverse transcription are detailed in Supplemental Data. Briefly, one aliquot of RNA was reverse transcribed with a mixture of sequence-specific RNA primers and anchored oligo(dT)12 primers for KLK mRNA and internal standard RNA detection, and the second aliquot was reverse transcribed with a mixture of random primers and anchored oligo(dT)12 primers for detection of PSCA.

Standard curve

As an external standard curve, we used standards containing varying amounts of KLK3 and KLK2 RNA (2.5 to 104 RNA copies per μL of reverse transcription reaction, which would correspond to 160 to 6.4×105 copies per mL of blood) and a fixed amount of m3PSA RNA (10 000 RNA molecules per μl of reverse transcription reaction). Standard RNAs were diluted into 100 ng/μL tRNA (Escherichia coli MRE 600 tRNA, Roche Applied Science) in sterile water. The external standards were analyzed along with all patient samples. In vitro production of RNA standards and data analysis have been described (19-21).

Real-time RT-PCR methodology

The real-time quantitative RT-PCR methodology for KLK3 and KLK2 mRNA has been described in detail (14-16, 19). In this study, 2.5 μL cDNA, representing 39 μL blood, was used as template in 10 μL amplification reactions. All samples were run in duplicate. The mean within-assay variation (maximum difference between duplicate reactions) was <0.5 Ct for the internal standard mRNA and <1 Ct for KLK3 and KLK2 mRNAs.

RT-PCR assay for PSCA mRNA

An internally standardized real-time quantitative RT-PCR assay was developed for PSCA mRNA. The primer and probe sequences were designed to span the exon splice junctions; the oligonucleotide sequences are shown in Supplemental Data Table 1. Reporter probe was ordered from Thermo Electron Corporation with a 5′ aminolinker and 3′ phosphate, and was 5′ terbium-labeled and purified as described (16). The quencher probe was modified by 3′ Dabcyl quencher moiety (Thermo Electron Corporation). PCR reactions were run in duplicate using 5 μL cDNA as template in 25 μL reactions, with reaction compositions and thermal cycling as described for KLK3 mRNA (15). Data were analyzed as described (22). The PSCA mRNA assay was performed on 71 of the 76 samples from patients with CRPC.

Statistical methods

A positive RT-PCR result was defined as ≥80 copies of target RNA per mL blood in both PCR replicates, with at least 20% recovery of the internal standard. A positive CellSearch result was defined as ≥5 CTCs per 7.5 mL blood sample.

We analyzed the relationship between KLK mRNA and CellSearch CTC results in two ways. First, we calculated the concordance: the proportion of patients categorized as either both positive or both negative for KLK mRNA and CellSearch. Second, we considered the KLK mRNA and CTC results as continuous variables and estimated correlation by Kendall’s tau.

We tested the association of positive RT-PCR result to clinical characteristics of the CRPC patients: presence or absence of biochemical progression at the time of research blood draw, location of metastasis, and overall survival. The probability of survival after the time of blood draw was estimated using Kaplan-Meier methods. Univariate associations of PSA, log CTC, log KLK2 mRNA, and log KLK3 mRNA were evaluated using Cox proportional hazards regression models. Predictive accuracy is given by the concordance probability estimate (CPE). The CPE measures the level of concordance between the survival time and the prognostic index as determined by the Cox model. The CPE ranges between 0.5 and 1.0, with 1.0 representing perfect concordance between the prognostic index and survival time, and 0.5 representing absence of relationship between prognostic index and survival time. Due to the limited follow-up, we were unable to compare actual recurrence outcomes of the patients with localized disease; we instead used the postoperative nomogram probability as a surrogate for recurrence outcomes. The postoperative nomogram probability (23) was computed for all patients with locally advanced disease who underwent RP (42 patients with postoperative blood samples and 87 patients with preoperative blood samples from MSKCC; 26 patients with preoperative blood samples from UKE), and was compared for those positive for KLK3 or KLK2 mRNA versus those negative for both KLK3 and KLK2 mRNAs using the Mann-Whitney test.

Role of the funding organizations

The funding organizations had no role in study design; data collection, analysis, or interpretation; manuscript preparation; or the decision to publish.

RESULTS

Characteristics of the patients with CRPC

The clinical characteristics of the 76 CRPC patients included in the study are detailed in Supplemental Data Table 2. At the time of research blood draw, 60 of the patients (79%) had rising PSA under androgen depletion therapy. Metastases were limited to soft tissue in 9 patients (12%), limited to bone in 26 patients (34%), and in both soft tissue and bone in the remaining 41 patients (54%). The median serum PSA at the time of research blood draw was 111 ng/mL (IQR 31, 433 ng/mL).

Concordance and correlation of CellSearch CTC counts with KLK and PSCA mRNAs

KLK3 RNA status was positive in 27 (36%) of the 76 patients with CRPC, and KLK2 mRNA in 32 patients (42%). One or both KLK mRNAs were positive in 37 patients (49%). In contrast, both KLKs were negative in all 19 healthy volunteers. CellSearch CTC status was positive in 31 of the 65 CRPC patients with evaluable results (48%). Median CTC count was 4 cells per 7.5 mL blood (interquartile range, 1-31). Thirty-one (48%) had ≥5 CTCs, and 24 (37%) had ≥10 CTCs. PSCA mRNA-positive status was found at a much lower frequency (7 of 71 patients tested; 10%). Six of the 7 patients positive for PSCA mRNA were also positive for one or both KLK mRNAs.

Concordance between KLK RNAs and CellSearch CTCs is shown in Table 1. The status for either KLK3 or KLK2 mRNA was concordant with CellSearch CTCs in 82% of CRPC patients. Among 34 patients with negative CTC status (≤5 cells), 7 (21%) had detectable KLK3 or KLK2 mRNA. To assess the level of confidence that KLK mRNA detects CTCs, we computed the confidence interval that a CTC-positive patient was also positive for KLK mRNA (i.e. sensitivity of KLK mRNA for detecting CTCs). The sensitivity of both KLK3 and KLK2 mRNA was 74% (95% CI, 55%-88%). Sensitivity of either KLK3 or KLK2 mRNA was 84% (95% CI, 66%-95%). As CTCs are given per 7.5 mL blood, and therefore 5 CTCs correspond to only 0.67 cells per mL blood, we also looked at the level of confidence that KLK mRNA detected 2 CTCs per mL (i.e. ≥15 CTCs per 7.5 mL blood). Sensitivity was 82% (18/22, 95% CI 60%-95%) for KLK3 mRNA, 91% (20/22, 95% CI 71%-99%) for KLK2 mRNA, and 95% (21/22, 95% CI 77%-100%) when either KLK3 or KLK2 mRNA was considered. We note that this sensitivity was achieved using a much smaller volume of blood than that used for the CellSearch assay (39 μL blood per PCR reaction). The above is also consistent with prior data showing that the KLK assays are sufficiently sensitive to detect <2 LNCaP cells per mL blood in the background of 107 nucleated cells (or 160 copies of mRNA per mL blood) (15).

Table 1
Concordance and correlation between CellSearch CTC status and KLK3 or KLK2 mRNA status in CRPC patients.

Kendall’s tau correlation between CTC counts and mRNA copy numbers was 0.68 for KLK3 and 0.60 for KLK2 (Table 1). Scatter plots for CTCs vs KLK3 mRNA and vs KLK2 mRNA are shown in Figure 1.

Figure 1
Scatter plots showing the relationship of KLK3 mRNA and KLK2 mRNA with CTC counts determined by CellSearch. Each point represents one patient with CRPC; patients experiencing disease progression at the time of analysis are indicated in blue and non-progressing ...

Comparing KLK2 to KLK3 also yielded high concordance (52/65 or 80%) and correlation (Kendall’s tau 0.66) (Table 1).

Association with disease characteristics

Positive status for KLK3 and KLK2 mRNAs was found only in patients who had clinical evidence of bone metastases; one or both KLK mRNAs was positive in 13 patients (50%) with bone metastases alone, in 24 patients (59%) with bone and soft tissue metastases, and in none of the patients with soft tissue metastases alone (Fig. 2; Supplemental Data Table 3). Similarly, a positive CellSearch result was more frequent in patients with bone metastases. PSA levels in serum (measured within 3 days of collecting blood for CTC analyses) were modestly correlated with KLK mRNA copy numbers and CellSearch CTC counts. Kendall’s tau correlations were 0.43 for serum PSA and KLK3 mRNA, 0.35 for serum PSA and KLK2 mRNA, and 0.42 for serum PSA and CTC counts (all p<0.001).

Figure 2
CTC and KLK and PSCA mRNA status among CRPC patients with metastases in bone (n=26), bone and soft tissue (n=41), and soft tissue only (n=9). Five patients were not tested for PSCA mRNA, and CellSearch data were not available for 11 patients.

Positive status for KLK mRNAs and for CTCs also varied with the number of systemic therapies undergone by the patients. The frequency of patients positive for either KLK mRNA was higher in patients after treatment failure with multiple chemotherapeutic regimens (11/18 patients; 61%) compared to those with first-line chemotherapy (9/20 patients; 45%) or only hormonal therapy (16/38 patients; 42%). Interestingly, this effect was even more pronounced for CellSearch CTCs, which were positive for 10/14 (71%) patients with failure of multiple regimens, 10/19 (53%) patients who received first-line chemotherapy, and 11/32 (34%) patients who received only hormonal therapy.

Association with survival

Among the 76 CRPC patients, 34 died during follow-up; median time to death was 17 months. Median follow-up for survivors was 14 months. Kaplan-Meier survival estimates show that survival was shorter among patients positive for KLK mRNA (Fig. 3). The corresponding plot constructed for the 60 patients with evidence of disease progression at the time of blood draw was essentially identical (not shown). On univariate analysis, each KLK mRNA, CTC count, and serum PSA were all highly associated with survival (all p<0.001; Table 2). Multivariable models containing these variables in various combinations were constructed (Table 2); to facilitate comparisons between models, we included only patients with available PSA levels and CTC counts (n=60). The predictive accuracy of serum PSA alone was 0.728; it was similar with the inclusion of KLK3 mRNA (0.726), but it increased to 0.749 and 0.765 with the inclusion of KLK2 mRNA or CellSearch CTC counts, respectively. The full model (PSA + CTCs + KLK2 + KLK3) had a predictive accuracy of 0.759, which was similar to the model including PSA and CTCs only.

Figure 3
Kaplan-Meier survival probability according to KLK mRNA status for the 76 patients with CRPC.
Table 2
Univariate analysis of associations with survival by Cox proportional hazards regression and predictiveaccuracy of univariate and multivariable models predicting survival

KLK mRNAs in localized disease

Either or both KLK3 and KLK2 mRNAs were detected in only a small proportion of the patients with localized disease. Patient characteristics according to KLK mRNA status are shown in Supplemental Data Tables 4 and 5. Among the patients treated at MSKCC, one or both KLK mRNAs was detected in 6 (14%) of the 42 with samples collected after RP, and in 6 (7%) of the 87 with samples collected before RP. Results were similar in the 51 pretreatment samples from UKE; only three (6%) were positive for KLK mRNA.

No association was apparent between KLK mRNA status and unfavorable localized disease features (Supplemental Data Tables 4 and 5). There was no significant difference in postoperative nomogram probability of local recurrence between patients positive for KLK3 or KLK2 mRNA versus those negative for both mRNAs (p=0.3 for MSKCC preoperative; p=0.6 for MSKCC postoperative; and p=0.6 for UKE preoperative). Also, there was no association between positive status for KLK mRNAs and the time interval from last biopsy or other prostatic manipulation (such as TURP) to research blood draw (data not shown).

DISCUSSION

RT-PCR has been extensively used as a means of detecting circulating prostate tumor cells, but, in part because of discrepant results, it has yet to become accepted. Our approach has been to use internally and externally standardized quantitative real-time RT-PCR, extensively optimized assay conditions, as well as sample collection procedures that help to preserve the true RNA profile. Using this method, we have shown a high correlation and concordance of results of our real-time RT-PCR assays with results of an independent assay for CTCs, CellSearch, in patients with metastatic CRPC. Real-time RT-PCR assays were able to detect KLK3 or KLK2 mRNAs in ≥95% of samples that had 15 or more CTCs per 7.5 mL blood by CellSearch assay. CellSearch has been approved by the US Food and Drug Administration for predicting progression-free and overall survival in metastatic breast cancer (1, 24-26), and was recently also approved for use in advanced prostate cancer. The cells isolated by CellSearch technology from patients with progressive CRPC have molecular features of malignant prostate epithelial cells (18). The high concordance between KLK mRNA and CellSearch CTC results suggests that both methods target the same cell population, and that real-time RT-PCR assays targeting KLK3 and KLK2 mRNAs reliably detect circulating tumor cells in the majority of men with metastatic prostate cancer.

The proportion of patients with metastatic CRPC positive for KLK3 or KLK2 mRNA was approximately 50%. In contrast, few patients were positive for another androgen receptor responsive gene, PSCA, although PSCA-positive status was highly associated with positive status for KLK mRNAs.

Although overall correlation between KLK and CTC results is high, the scatter plots suggest the possibility of a distinct subpopulation of patients who shed CTCs with high KLK mRNA copy numbers that escape detection by the CellSearch assay. Approximately 20% of patients with <5 CTCs per 7.5 mL blood had detectable KLK3 or KLK2 mRNA. Among these patients, two had 0 CTCs, two had 1 CTC, and three had 2-4 CTCs per 7.5 mL blood. No healthy subjects had KLK mRNA signal, implying that these transcripts are limited to CTCs and the detection of such prostate-specific transcripts in blood implies tumor cell dissemination. In addition, very few patients with localized prostate cancer were positive for KLK3 or KLK2 mRNA. Hence, samples positive for KLK mRNA but negative by CellSearch cannot be explained by compromised specificity; instead, we hypothesize that these CTCs were too few or lacked cell surface markers for detection by CellSearch. Conversely, some CRPC patients shed CTCs with very little or no detectable KLK mRNAs. This may reflect therapeutic repression of androgen receptor function. From the scatter plots it is evident that the numbers of KLK2 and KLK3 RNAs per CTC vary among CRPC patients. These numbers, derived from combining RT-PCR data with CellSearch CTC data, might prove informative for androgen receptor function, which in turn may relate to critical disease characteristics, treatment options, and outcome. Determining the clinical value of this information will require further studies of KLK mRNA status in CTCs in a large cohort of patients with advanced cancer.

CTCs were more frequent in patients who had received two or more chemotherapy regimens than in those with fewer systemic treatments. This effect was stronger for CellSearch CTCs than for KLK mRNAs. This may possibly result from the tendency of androgen receptor function, and therefore also KLK2 and KLK3 expression, to diminish in more advanced disease. In patients with only hormonal therapy, KLK mRNAs seemed to be the more sensitive assay. Higher CTC numbers have earlier been reported in patients receiving second-line therapy (27).

CTCs, whether detected by KLK mRNAs or by CellSearch, were very strongly associated with diagnosis of bone metastasis in the CRPC patients. The metastatic disease was confirmed by bone scans and soft tissue imaging, which, although not perfectly accurate, are the standard methods for diagnosing prostate cancer metastases. Notably, none of the patients who had soft tissue metastasis alone had detectable KLK mRNA signal, although the number of patients was small. A similar trend was observed in a study using CellSearch technology on a larger patient population (27). This association with the specific site of metastases, along with the weakness of the correlation of KLK RT-PCR and CellSearch results with serum PSA, indicates that these assays can provide distinct information that is not simply related to increased tumor burden.

KLK mRNA and CellSearch CTC results were both strongly associated with survival. The shorter survival for KLK-positive patients also held among the patients whose disease was progressing, suggesting that analysis of the KLK mRNAs will have utility in this subset. The accuracy of serum PSA in predicting survival was enhanced with the addition of KLK mRNAs and CellSearch results, and the full model all these variables had a predictive accuracy of 0.759. This was similar to a model that included only PSA and CellSearch results (0.765), and similar to a published model that includes PSA, CellSearch results, and albumin (27).

A limitation of the survival analysis is that, because of the cohort size, we were unable to test whether KLK mRNA and CellSearch results were associated with survival independent of potential confounding factors. One such factor could be patients’ disease state as reflected by history of chemotherapy. CTCs were more frequently found in patients who had had treatment failure on chemotherapy, a group that might be expected to have shorter survival than chemotherapy-naive patients. However, in another study, CellSearch CTC results were associated with survival independent of other prognostic factors, including number of prior chemotherapies (28). Testing whether this is also the case for KLK mRNA results will require a larger study.

Our RT-PCR results imply a very low frequency of CTCs in patients with clinically localized disease, even among those with adverse pathologic features. Davis et al, using CellSearch, also reported a low frequency in men with early prostate cancer (3 of 97 patients with 3 or more CTCs per 22.5 mL blood) (29). In that study, however, a similar frequency of CTCs was detected in men without prostate cancer, whereas no CTCs were detected in healthy individuals by our RT-PCR methodology. The very low frequency of CTCs in patients with localized disease appears consistent with the close association of CTCs with skeletal metastases using our RT-PCR assays. This low frequency of CTCs also suggests that large studies with extended follow-up will be required to reliably assess whether detection of CTCs before treatment is associated with systemic disease, e.g. bone metastases, or more generally with worse outcome. Due to the limited follow-up in this study, we were unable to analyze the actual recurrence outcomes of the patients with localized disease.

The Gleason score still provides the gold standard for assessing prostate cancer aggressiveness at diagnosis. Gleason score, however, cannot be easily assessed repeatedly over the course of the disease, and, because prostate cancer has a highly variable natural history, current information on the patient’s disease is required for optimal targeting of therapies. Hence, there is need for predictive markers that can be easily assessed repeatedly. In this study, KLK mRNA assays and CellSearch CTC assays were shown to provide prognostic information and to enhance the predictive accuracy of serum PSA alone in patients with CRPC. The sample material for CTC assays is readily obtainable by standard venipuncture, and real-time quantitative RT-PCR is currently one of the most sensitive methods for detecting CTCs. The concordance between RT-PCR and CellSearch methods, albeit in a small patient population, shows proof of concept that these may be equally valid approaches in detecting disseminated prostate tumor cells. Moreover, the approaches are complementary; CellSearch enables intact CTCs to be counted and characterized by FISH and immunohistochemistry, and KLK mRNA assays provide sensitive and quantitative detection of CTC-specific gene expression. However, the pathobiologic mechanisms that contribute to the shedding of these cells remain to be defined, and so investigation continues in order to define patient groups where detecton of CTCs has the most potential.

Supplementary Material

Acknowledgments

H.I. Scher, Veridex; H. Lilja, National Cancer Institute. The work was supported by NIH Prostate SPORE Grant (P50 CA92629 Pilot Project 7 and 14), the European Union 6th Framework contract LSHC-CT-2004-503011 (P-Mark), the Sidney Kimmel Center for Prostate and Urologic Cancers, the Prostate Cancer Foundation, William H. Goodwin and Alice Goodwin, and the Commonwealth Foundation for Cancer Research, and the Experimental Therapeutics Cancer of Memorial Sloan-Kettering Cancer Center. R.M. Väänänen and K. Pettersson were supported by the Academy of Finland. We thank Janet Novak of Helix Editing for substantive editing of the manuscript; this work was paid for by MSKCC.

Abbreviations

CPE
concordance probability estimate
CRPC
castration-refractory prostate cancer
CTC
circulating tumor cell
MSKCC
Memorial Sloan-Kettering Cancer Center
PSA
prostate-specific antigen
RT-PCR
reverse transcription PCR
UKE
University Hospital of Hamburg

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