Abstract
Transrectal ultrasound-guided prostate biopsy (TRUS) is a standard procedure for prostate cancer diagnosis. Because prostate cancer is a multifocal disease in many patients, multiple sampling (n ≥ 10) is required, which may bear the risk of systemic spread of cancer cells.
Using the standardized CellSearch® system that allows for the detection of single epithelial cell adhesion molecule-positive circulating tumor cells (CTCs) in blood, we investigated whether prostate biopsy is associated with release of prostatic tumor cells into the circulation. Peripheral blood was obtained before and within 30 min after performing prostate biopsy from 115 men with increased serum prostate-specific antigen.
The number of CTCs significantly increased after biopsy in men with histologically confirmed prostate cancer (odds ratio, 7.8; 95% CI, 4.8–12.8), whereas no biopsy-related changes could be detected in men without confirmed prostate cancer. Multivariable analysis showed that biopsy-related increase of CTCs was significantly correlated with a worse progression-free survival (hazard ratio, 12.4; 95% CI, 3.2–48.6) within the median follow-up of 41 months.
Prostate biopsies may lead to a tumor-associated release of CTCs into the blood circulation. Larger confirmatory trials with longer follow-up periods are required before any change in clinical practice can be recommended.
Prostate cancer (PCa)5 is the most common cancer in men, with an incidence rate of 110.8 cases per 100000 men in the European Union (1). The main tools to diagnose PCa include digital rectal examination, determination of serum concentration of prostate-specific antigen (PSA), and transrectal ultrasound (TRUS)-guided core biopsy. PSA is a glycoprotein enzyme secreted by the epithelial cells of the prostate gland. Although it is the most valuable tumor marker of PCa currently available in clinical practice, PSA is also often increased in men with benign prostatic hyperplasia, prostatitis, and other nonmalignant disorders (2). Therefore, TRUS biopsy has become the standard way to obtain material for histopathological examination to diagnose or exclude PCa. For PCa diagnosis, a series of core needle biopsies (18 gauge and 1.25-mm diameter) are taken according to a routine scheme, usually 5 to 8 biopsies from each side under ultrasound guidance. This extensive sampling is necessary because PCa is a multifocal disease in many patients, and the current imaging technologies do not allow identifying the suspicious lesions with unequivocal precision. The biopsy needle penetrates the prostate tissue with high velocity, thus causing substantial risk for local trauma releasing prostate cells into the surrounding tissue or blood vessels (3). Previous studies performed >10 years ago suggested that TRUS biopsy may cause hematogenous dissemination of tumor cells, but the existing assays for detecting these circulating tumor cells (CTCs) in blood were not reliable enough at that time to have an impact on medical practice (4, 5). More recently, no evidence was found that transurethral resection of bladder tumors influences the release of tumor cells into circulation (6). In contrast, core needle biopsies of tumors in a breast cancer mouse model have been shown to lead to an increase of lung metastasis compared with mice not biopsied (7), which could be confirmed in breast cancer patients as well (8). Therefore, it is of high importance to further investigate the role TRUS biopsy has on the spread of cancer cells and the formation of metastasis.
Assessment of CTCs using the semiautomated CellSearch® system has been reported as the most accurate and independent predictor of overall survival in castration-resistant PCa, leading to its clearance by the Food and Drug Administration for this disease (9–11). CTCs detected using the CellSearch have also been reported as a surrogate marker for predicting therapy response in castration-resistant PCa (12, 13). Furthermore, CTCs were also found in the bloodstream of early-stage PCa patients, albeit with lower frequency (14–16).
The aim of this study was to investigate whether prostate biopsy was associated with the release of malignant prostatic cells into the peripheral blood and whether this release led to an increase in relapse. Although it cannot be proven that all epithelial cell adhesion molecule (EpCAM)+/K+/CD45− cells detected by the CellSearch system are actually tumor cells, in this study we refer to these cells as CTCs because it is the internationally accepted definition supported by >100 publications comprising findings on 10000s of blood analyses using the CellSearch system (17).
Materials and Methods
PATIENT RECRUITMENT AND MATERIAL COLLECTION
A total of 115 men with increased serum PSA concentrations (>4 ng/mL) or abnormal findings on digital rectal examination suspicious for PCa were recruited for this study after written informed consent was obtained. The study was approved by the local ethical review board under number PV3779. TRUS-guided diagnostic core biopsies were performed between August 2012 and January 2016. Histologic diagnosis of 8 to 12 tissue cores was carried out according to the Gleason score by an experienced pathologist. Two CellSave Preservative tubes were each filled with 7.5 mL of peripheral blood, 1 before and 1 within 30 min after performing prostate biopsy. The tubes contained Na2EDTA for clotting prevention and a cell preservative to maintain the morphology and cell-surface antigen expression of epithelial cells for phenotyping. The samples were directly processed to detect CTCs using the CellSearch system. Researchers analyzing the samples were blinded to all patient-related data, including time point of blood sampling. Characteristics of the patient cohort are presented in Table 1. Patients with histological diagnosis of PCa were treated with radical prostatectomy, radiation therapy, or active surveillance.
Characteristics of the total cohort of participants.a
| PCa− (n = 40) | PCa+ (n = 75) | Total (n = 115) | P value | |
|---|---|---|---|---|
| Age at diagnosis, years (mean) | 40–82 (61.4) | 46–79 (66.1) | 40–82 (64.5) | 0.0086 |
| Total PSA, ng/mL (median) | 1.9–13.4 (7.1) | 2.5–304.6 (7.7) | 1.9–304.6 (7.7) | 0.0586 |
| Free PSA, ng/mL (median) | 0.2–2.8 (1.1) | 0.2–3.2 (0.9) | 0.2–3.2 (0.9) | 0.2438 |
| Free PSA/total PSA ratio, % (median) | 7.8–25.9 (14.9) | 2.2–35.6 (10.8) | 2.2–35.6 (12.9) | <0.001 |
| Gleason score | ||||
| 3 + 3 | n = 24 | |||
| 3 + 4 | n = 26 | |||
| 4 + 3 | n = 5 | |||
| ≥4 + 4 | n = 20 | |||
| T stage | ||||
| T1 | n = 10 | |||
| T2 | n = 34 | |||
| T3 | n = 20 | |||
| Treatment | ||||
| Prostatectomy | n = 46 | |||
| Prostatectomy + other | n = 7 | |||
| Radiotherapy | n = 13 | |||
| Other/none | n = 9 |
| PCa− (n = 40) | PCa+ (n = 75) | Total (n = 115) | P value | |
|---|---|---|---|---|
| Age at diagnosis, years (mean) | 40–82 (61.4) | 46–79 (66.1) | 40–82 (64.5) | 0.0086 |
| Total PSA, ng/mL (median) | 1.9–13.4 (7.1) | 2.5–304.6 (7.7) | 1.9–304.6 (7.7) | 0.0586 |
| Free PSA, ng/mL (median) | 0.2–2.8 (1.1) | 0.2–3.2 (0.9) | 0.2–3.2 (0.9) | 0.2438 |
| Free PSA/total PSA ratio, % (median) | 7.8–25.9 (14.9) | 2.2–35.6 (10.8) | 2.2–35.6 (12.9) | <0.001 |
| Gleason score | ||||
| 3 + 3 | n = 24 | |||
| 3 + 4 | n = 26 | |||
| 4 + 3 | n = 5 | |||
| ≥4 + 4 | n = 20 | |||
| T stage | ||||
| T1 | n = 10 | |||
| T2 | n = 34 | |||
| T3 | n = 20 | |||
| Treatment | ||||
| Prostatectomy | n = 46 | |||
| Prostatectomy + other | n = 7 | |||
| Radiotherapy | n = 13 | |||
| Other/none | n = 9 |
Significant test for mean: Welch 2-sample t-test; significant test for median: Wilcoxon rank-sum test with continuity correction.
Characteristics of the total cohort of participants.a
| PCa− (n = 40) | PCa+ (n = 75) | Total (n = 115) | P value | |
|---|---|---|---|---|
| Age at diagnosis, years (mean) | 40–82 (61.4) | 46–79 (66.1) | 40–82 (64.5) | 0.0086 |
| Total PSA, ng/mL (median) | 1.9–13.4 (7.1) | 2.5–304.6 (7.7) | 1.9–304.6 (7.7) | 0.0586 |
| Free PSA, ng/mL (median) | 0.2–2.8 (1.1) | 0.2–3.2 (0.9) | 0.2–3.2 (0.9) | 0.2438 |
| Free PSA/total PSA ratio, % (median) | 7.8–25.9 (14.9) | 2.2–35.6 (10.8) | 2.2–35.6 (12.9) | <0.001 |
| Gleason score | ||||
| 3 + 3 | n = 24 | |||
| 3 + 4 | n = 26 | |||
| 4 + 3 | n = 5 | |||
| ≥4 + 4 | n = 20 | |||
| T stage | ||||
| T1 | n = 10 | |||
| T2 | n = 34 | |||
| T3 | n = 20 | |||
| Treatment | ||||
| Prostatectomy | n = 46 | |||
| Prostatectomy + other | n = 7 | |||
| Radiotherapy | n = 13 | |||
| Other/none | n = 9 |
| PCa− (n = 40) | PCa+ (n = 75) | Total (n = 115) | P value | |
|---|---|---|---|---|
| Age at diagnosis, years (mean) | 40–82 (61.4) | 46–79 (66.1) | 40–82 (64.5) | 0.0086 |
| Total PSA, ng/mL (median) | 1.9–13.4 (7.1) | 2.5–304.6 (7.7) | 1.9–304.6 (7.7) | 0.0586 |
| Free PSA, ng/mL (median) | 0.2–2.8 (1.1) | 0.2–3.2 (0.9) | 0.2–3.2 (0.9) | 0.2438 |
| Free PSA/total PSA ratio, % (median) | 7.8–25.9 (14.9) | 2.2–35.6 (10.8) | 2.2–35.6 (12.9) | <0.001 |
| Gleason score | ||||
| 3 + 3 | n = 24 | |||
| 3 + 4 | n = 26 | |||
| 4 + 3 | n = 5 | |||
| ≥4 + 4 | n = 20 | |||
| T stage | ||||
| T1 | n = 10 | |||
| T2 | n = 34 | |||
| T3 | n = 20 | |||
| Treatment | ||||
| Prostatectomy | n = 46 | |||
| Prostatectomy + other | n = 7 | |||
| Radiotherapy | n = 13 | |||
| Other/none | n = 9 |
Significant test for mean: Welch 2-sample t-test; significant test for median: Wilcoxon rank-sum test with continuity correction.
CTC ANALYSIS
Blood analysis with the CellSearch system was performed within 96 h as previously described (18) using the CTC Kit according to the manufacturer's recommendations. Briefly, epithelial cells among the cells captured by anti–epithelial cell adhesion molecule (EpCAM) antibodies were detected by binding of antibodies C11 and A.53B/A2, directed against keratins 8, 18, and 19, and potentially also recognizing keratins 4 through 6, 10, and 13 (19, 20). An anti-CD45 antibody was used to define and exclude leukocytes. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). After enrichment and immunocytochemical staining, immunomagnetically labeled cells were kept in a strong magnetic field and scanned using the CellSpotter Analyzer (Menarini-Silicon Biosystems). Experienced researchers interpreted results of these analyses. A blood sample of 7.5 mL was considered positive with the identification of at least 1 keratin-positive, DAPI-positive, CD45-negative cell showing no morphological signs of apoptosis. This cutoff was adapted from our studies on early-stage breast cancer because so far no other prognostic cutoff has been determined by previous publications for early-stage PCa.
STATISTICAL ANALYSIS
Statistical analyses were performed with Matlab R2016a (The Mathworks), R version 3.3.3 (R Foundation for Statistical Computing), and In-Silico Online version 2.0 (21). Two-sample t-tests and Wilcoxon rank-sum tests were used to calculate the statistical difference between means and medians, respectively. To calculate the odds ratios (ORs) in CTC change, the CTC counts were normalized to the amount of blood volume and correlated to the following independent variables using a Poisson distributed generalized linear mixed-effect model, corrected for age, with patients nested in time point: (a) time point of blood collection (before/after biopsy), (b) presence of PCa cells in the biopsies (yes/no), and (c) interaction effects. Progression-free survival from date of biopsy was modeled using the Kaplan–Meier function, and univariable and multivariable hazard ratios (HRs) were calculated using Cox proportional hazards analysis. Patients undergoing active treatment (radical prostatectomy or radiation therapy without concurrent androgen deprivation therapy) were evaluated with respect to the effect of CTC increase after therapy (increase of at least 1 CTC) on biochemical recurrence for progression-free survival. Biochemical recurrence after radical prostatectomy was defined as a PSA increase >0.2 ng/mL and PSA nadir + 2 after radiation therapy. Covariates consisted of age, PSA, number of positive cores, biopsy Gleason score, and treatment (radical prostatectomy vs radiation therapy). For calling statistical significance, the α level of 0.005 that was recently proposed by Benjamin et al. to increase credibility was applied (22).
Results
PATIENT COHORT
All tissue biopsy samples underwent histopathological investigation, and PCa was diagnosed in 75 patients with 41% having a Gleason score of 7. The biopsies from the remaining 40 patients were tumor-free (Table 1). The median ratio of free PSA/total PSA was measured before study admission and turned out to be significantly less in the PCa-positive (PCa+) group compared with the PCa-negative (PCa−) group (10.8% vs 14.9%; P < 0.001; Wilcoxon rank-sum test; Table 1). The PCa+ group was a mean 4.7 years older than the men in whom no PCa was detected (P = 0.0086; Welch 2-sample t-test; Table 1). Although P > 0.005, we decided to correct further analyses for age to prevent potential covariance. All patients underwent an internationally accepted diagnostic workup, and none had metastatic disease at primary diagnosis.
CTC COUNTS BEFORE AND AFTER BIOPSY
In 21 of 105 participants (20%), tumor cells were detected in the blood before biopsy (median CTC count, 1; range, 1–34/7.5 mL of blood), while 34 of 101 (34%) individuals were positive for tumor cells after biopsy (median CTC count, 1.5; range, 1–39/7.5 mL of blood). The CTC status of 100 cases with measurement before and after biopsy changed as follows: 54 cases remained CTC negative, 25 became positive, 12 became negative, and 9 remained positive over the course of taking the biopsy. To investigate whether the increase of CTCs was associated with TRUS biopsy and PCa status, paired analyses were performed on the number of detected CTCs before and after biopsy. In univariable and multivariable analyses, taking a biopsy had a significant effect on the increase of CTCs (P < 0.0001; Table 2). The main effect of having a biopsy was a highly significant increase of CTCs in the blood (OR, 5.06; 95% CI, 3.27–7.48) in the total cohort. In univariable and multivariable analyses, PCa status was not found significantly associated with CTCs as expected from early-stage PCa (P = 0.036; Table 2). However, the analysis showed a significant interaction between PCa status and the time point of blood collection on the number of CTCs (P = 0.0023; Table 2). Dividing the cohort based on PCa status, the negative participants were not affected by an increase of CTCs after biopsy (OR, 0.429; 95% CI, 0.180–1.023), whereas the effect in PCa+ participants was highly significant (OR, 7.882; 95% CI, 4.844–12.825).
ORs of CTC increase for biopsy and PCa status.a
| Covariate | Univariable analysis | Multivariable analysis | ||||||
|---|---|---|---|---|---|---|---|---|
| Coefficient (bi) | OR [exp(bi)] | 95% CI | P value | Coefficient (bi) | OR [exp(bi)] | 95% CI | P value | |
| Biopsy | 0.8532 | 2.3470 | 1.6610–3.3165 | <0.0001 | 1.6217 | 5.0617 | 3.2674–7.8413 | <0.0001 |
| PCa status | 0.2433 | 1.2754 | 0.5828–2.7910 | 0.5409 | 1.0962 | 2.9928 | 1.0762–8.3220 | 0.0358 |
| Biopsyb * PCa | 1.3405 | 3.8210 | 1.6213–9.0009 | 0.0023 | ||||
| Biopsy: PCa− | −0.8467 | 0.4288 | 0.1797–1.0235 | 0.1260c | ||||
| Biopsy: PCa+ | 2.0646 | 7.8821 | 4.8443–12.8250 | <0.0001c | ||||
| Covariate | Univariable analysis | Multivariable analysis | ||||||
|---|---|---|---|---|---|---|---|---|
| Coefficient (bi) | OR [exp(bi)] | 95% CI | P value | Coefficient (bi) | OR [exp(bi)] | 95% CI | P value | |
| Biopsy | 0.8532 | 2.3470 | 1.6610–3.3165 | <0.0001 | 1.6217 | 5.0617 | 3.2674–7.8413 | <0.0001 |
| PCa status | 0.2433 | 1.2754 | 0.5828–2.7910 | 0.5409 | 1.0962 | 2.9928 | 1.0762–8.3220 | 0.0358 |
| Biopsyb * PCa | 1.3405 | 3.8210 | 1.6213–9.0009 | 0.0023 | ||||
| Biopsy: PCa− | −0.8467 | 0.4288 | 0.1797–1.0235 | 0.1260c | ||||
| Biopsy: PCa+ | 2.0646 | 7.8821 | 4.8443–12.8250 | <0.0001c | ||||
Estimated coefficient of CTC increase with corresponding OR, 95% CI, and P value in univariable and multivariable analyses, corrected for age. Depicted are the effects of biopsy and PCa status on CTC count, as well as their interaction effect (Biopsy * PCa) and the effect of biopsy in PCa negative (Biopsy: PCa−) and PCa positive (Biopsy: PCa+) separately.
Biopsy = time point of blood collection (before biopsy/after biopsy).
Bonferroni adjusted P value.
ORs of CTC increase for biopsy and PCa status.a
| Covariate | Univariable analysis | Multivariable analysis | ||||||
|---|---|---|---|---|---|---|---|---|
| Coefficient (bi) | OR [exp(bi)] | 95% CI | P value | Coefficient (bi) | OR [exp(bi)] | 95% CI | P value | |
| Biopsy | 0.8532 | 2.3470 | 1.6610–3.3165 | <0.0001 | 1.6217 | 5.0617 | 3.2674–7.8413 | <0.0001 |
| PCa status | 0.2433 | 1.2754 | 0.5828–2.7910 | 0.5409 | 1.0962 | 2.9928 | 1.0762–8.3220 | 0.0358 |
| Biopsyb * PCa | 1.3405 | 3.8210 | 1.6213–9.0009 | 0.0023 | ||||
| Biopsy: PCa− | −0.8467 | 0.4288 | 0.1797–1.0235 | 0.1260c | ||||
| Biopsy: PCa+ | 2.0646 | 7.8821 | 4.8443–12.8250 | <0.0001c | ||||
| Covariate | Univariable analysis | Multivariable analysis | ||||||
|---|---|---|---|---|---|---|---|---|
| Coefficient (bi) | OR [exp(bi)] | 95% CI | P value | Coefficient (bi) | OR [exp(bi)] | 95% CI | P value | |
| Biopsy | 0.8532 | 2.3470 | 1.6610–3.3165 | <0.0001 | 1.6217 | 5.0617 | 3.2674–7.8413 | <0.0001 |
| PCa status | 0.2433 | 1.2754 | 0.5828–2.7910 | 0.5409 | 1.0962 | 2.9928 | 1.0762–8.3220 | 0.0358 |
| Biopsyb * PCa | 1.3405 | 3.8210 | 1.6213–9.0009 | 0.0023 | ||||
| Biopsy: PCa− | −0.8467 | 0.4288 | 0.1797–1.0235 | 0.1260c | ||||
| Biopsy: PCa+ | 2.0646 | 7.8821 | 4.8443–12.8250 | <0.0001c | ||||
Estimated coefficient of CTC increase with corresponding OR, 95% CI, and P value in univariable and multivariable analyses, corrected for age. Depicted are the effects of biopsy and PCa status on CTC count, as well as their interaction effect (Biopsy * PCa) and the effect of biopsy in PCa negative (Biopsy: PCa−) and PCa positive (Biopsy: PCa+) separately.
Biopsy = time point of blood collection (before biopsy/after biopsy).
Bonferroni adjusted P value.
CLINICAL FOLLOW-UP OF PCa-POSITIVE CASES
Twenty-two of the PCa+ cases had an increase in the number of CTCs after biopsy compared with baseline (Fig. 1). To investigate whether the release of tumor cells was associated with progression of disease, we performed a prospective follow-up study on the PCa+ participants and recorded the time until biochemical relapse or diagnosis of distant metastasis as event after surgery. During the study's follow-up (median observation time, 41 months), data could be collected on 74 PCa+ individuals; 1 participant was lost to follow-up. Kaplan–Meier estimate and log-rank test showed a significantly poorer progression-free survival in participants with an increase of at least 1 CTC per 7.5 mL of blood following biopsy (P = 0.0021; Fig. 2). Progression of disease was seen in 10 of 22 participants with CTC increase (median time to progression, 51.6 months) and in 8 of 52 cases without CTC increase after biopsy. Univariable and multivariable analyses showed that an increase of CTCs is significantly correlated with progression of disease (HR, 12.43; 95% CI, 3.18–48.60), whereas none of the other clinical variables was associated with survival (Table 3). Because the number of CTCs before biopsy alone was not correlated with progression of disease (HR, 1.25; 95% CI, 0.91–1.71; P = 0.153), these data supported that the additional mechanical release of prostate cells into the circulation of PCa+ participants influenced prognosis.
CTC change.
Waterfall plot showing the change of CTCs after biopsy compared with baseline for the PCa+ cases.
Progression-free survival probability.
Kaplan–Meier function for biochemical and metastatic relapse in months (median, 41.1) correlated to increase or stable count of tumor cells after biopsy (P = 0.0021, log-rank test).
Cox proportional HRs.a
| Covariate | Univariable analysis | Multivariable analysis | ||||||
|---|---|---|---|---|---|---|---|---|
| Coefficient (bi) | HR [exp(bi)] | HR 95% CI | P value | Coefficient (bi) | HR [exp(bi)] | HR 95% CI | P value | |
| CTC increase | 1.384 | 3.989 | 1.57–10.14 | 0.0036 | 2.520 | 12.429 | 3.178–48.604 | 0.0003 |
| Age | 0.011 | 1.011 | 0.953–1.073 | 0.7190 | −0.003 | 0.997 | 0.875–1.136 | 0.9660 |
| PCa-positive cores | 1.0375 | 2.822 | 0.50–16.04 | 0.2420 | 2.536 | 12.636 | 0.66–241.62 | 0.0920 |
| PSA | 0.006 | 1.006 | 0.994–1.014 | 0.1190 | −0.077 | 0.925 | 0.850–1.007 | 0.0707 |
| Gleason score (3 + 3) | ||||||||
| 3 + 4 | −0.744 | 0.475 | 0.119–1.901 | 0.2930 | −1.825 | 0.161 | 0.027–0.976 | 0.0470 |
| 4 + 3 | 0.449 | 1.567 | 0.316–7.769 | 0.5830 | 1.090 | 2.976 | 0.194–45.549 | 0.4334 |
| ≥4 + 4 | 0.419 | 1.520 | 0.448–4.734 | 0.4700 | −0.176 | 0.839 | 0.148–4.763 | 0.8429 |
| Treatment | 0.604 | 1.828 | 0.625–5.3 | 0.271 | 1.486 | 4.420 | 0.863–22.630 | 0.0745 |
| Covariate | Univariable analysis | Multivariable analysis | ||||||
|---|---|---|---|---|---|---|---|---|
| Coefficient (bi) | HR [exp(bi)] | HR 95% CI | P value | Coefficient (bi) | HR [exp(bi)] | HR 95% CI | P value | |
| CTC increase | 1.384 | 3.989 | 1.57–10.14 | 0.0036 | 2.520 | 12.429 | 3.178–48.604 | 0.0003 |
| Age | 0.011 | 1.011 | 0.953–1.073 | 0.7190 | −0.003 | 0.997 | 0.875–1.136 | 0.9660 |
| PCa-positive cores | 1.0375 | 2.822 | 0.50–16.04 | 0.2420 | 2.536 | 12.636 | 0.66–241.62 | 0.0920 |
| PSA | 0.006 | 1.006 | 0.994–1.014 | 0.1190 | −0.077 | 0.925 | 0.850–1.007 | 0.0707 |
| Gleason score (3 + 3) | ||||||||
| 3 + 4 | −0.744 | 0.475 | 0.119–1.901 | 0.2930 | −1.825 | 0.161 | 0.027–0.976 | 0.0470 |
| 4 + 3 | 0.449 | 1.567 | 0.316–7.769 | 0.5830 | 1.090 | 2.976 | 0.194–45.549 | 0.4334 |
| ≥4 + 4 | 0.419 | 1.520 | 0.448–4.734 | 0.4700 | −0.176 | 0.839 | 0.148–4.763 | 0.8429 |
| Treatment | 0.604 | 1.828 | 0.625–5.3 | 0.271 | 1.486 | 4.420 | 0.863–22.630 | 0.0745 |
Estimated coefficients of progress-free survival on PCa+ individuals. Calculated are the corresponding HR, 95% CI of the HR, and P value in univariable and multivariable Cox proportional hazards analysis for CTC number increase after biopsy, age, the fraction of PCa+ cores, PSA at time of diagnosis, Gleason scores with 3 + 3 as reference, and prostatectomy vs radiotherapy as treatment covariate.
Cox proportional HRs.a
| Covariate | Univariable analysis | Multivariable analysis | ||||||
|---|---|---|---|---|---|---|---|---|
| Coefficient (bi) | HR [exp(bi)] | HR 95% CI | P value | Coefficient (bi) | HR [exp(bi)] | HR 95% CI | P value | |
| CTC increase | 1.384 | 3.989 | 1.57–10.14 | 0.0036 | 2.520 | 12.429 | 3.178–48.604 | 0.0003 |
| Age | 0.011 | 1.011 | 0.953–1.073 | 0.7190 | −0.003 | 0.997 | 0.875–1.136 | 0.9660 |
| PCa-positive cores | 1.0375 | 2.822 | 0.50–16.04 | 0.2420 | 2.536 | 12.636 | 0.66–241.62 | 0.0920 |
| PSA | 0.006 | 1.006 | 0.994–1.014 | 0.1190 | −0.077 | 0.925 | 0.850–1.007 | 0.0707 |
| Gleason score (3 + 3) | ||||||||
| 3 + 4 | −0.744 | 0.475 | 0.119–1.901 | 0.2930 | −1.825 | 0.161 | 0.027–0.976 | 0.0470 |
| 4 + 3 | 0.449 | 1.567 | 0.316–7.769 | 0.5830 | 1.090 | 2.976 | 0.194–45.549 | 0.4334 |
| ≥4 + 4 | 0.419 | 1.520 | 0.448–4.734 | 0.4700 | −0.176 | 0.839 | 0.148–4.763 | 0.8429 |
| Treatment | 0.604 | 1.828 | 0.625–5.3 | 0.271 | 1.486 | 4.420 | 0.863–22.630 | 0.0745 |
| Covariate | Univariable analysis | Multivariable analysis | ||||||
|---|---|---|---|---|---|---|---|---|
| Coefficient (bi) | HR [exp(bi)] | HR 95% CI | P value | Coefficient (bi) | HR [exp(bi)] | HR 95% CI | P value | |
| CTC increase | 1.384 | 3.989 | 1.57–10.14 | 0.0036 | 2.520 | 12.429 | 3.178–48.604 | 0.0003 |
| Age | 0.011 | 1.011 | 0.953–1.073 | 0.7190 | −0.003 | 0.997 | 0.875–1.136 | 0.9660 |
| PCa-positive cores | 1.0375 | 2.822 | 0.50–16.04 | 0.2420 | 2.536 | 12.636 | 0.66–241.62 | 0.0920 |
| PSA | 0.006 | 1.006 | 0.994–1.014 | 0.1190 | −0.077 | 0.925 | 0.850–1.007 | 0.0707 |
| Gleason score (3 + 3) | ||||||||
| 3 + 4 | −0.744 | 0.475 | 0.119–1.901 | 0.2930 | −1.825 | 0.161 | 0.027–0.976 | 0.0470 |
| 4 + 3 | 0.449 | 1.567 | 0.316–7.769 | 0.5830 | 1.090 | 2.976 | 0.194–45.549 | 0.4334 |
| ≥4 + 4 | 0.419 | 1.520 | 0.448–4.734 | 0.4700 | −0.176 | 0.839 | 0.148–4.763 | 0.8429 |
| Treatment | 0.604 | 1.828 | 0.625–5.3 | 0.271 | 1.486 | 4.420 | 0.863–22.630 | 0.0745 |
Estimated coefficients of progress-free survival on PCa+ individuals. Calculated are the corresponding HR, 95% CI of the HR, and P value in univariable and multivariable Cox proportional hazards analysis for CTC number increase after biopsy, age, the fraction of PCa+ cores, PSA at time of diagnosis, Gleason scores with 3 + 3 as reference, and prostatectomy vs radiotherapy as treatment covariate.
Discussion
The present results show that malignant epithelial cells can be released into the blood vessels during prostate biopsy in cancer patients, which might be associated with an increased risk of disease progression as indicated by follow-up analysis.
There are a few previous reports suggesting that TRUS prostate biopsy can be associated with the release of prostatic cells (4, 5) or cellular epithelial material (3) into the blood circulation. However, the assays applied were less validated than the CellSearch system, and no follow-up data were presented. The method applied in our study has been clinically validated in several studies including PCa patients at early disease stages (23–28), suggesting that the assay is able to detect even very low numbers of CTCs. Moreover, the clinical specificity of the CellSearch system has been demonstrated in several previous studies on larger control cohorts as prerequisite of the Food and Drug Administration clearance (18, 29).
A significant increase in tumor cell counts after biopsy was observed in only patients diagnosed with PCa, whereas such difference was not observed in patients without confirmed PCa (“control group”). Furthermore, we observed a worse progression-free survival of cancer patients who were diagnosed with an increase in tumor cells after biopsy. It remains unclear whether these cells really contribute to cancer progression or whether they are surrogates of a more aggressive biology of the tumor (e.g., less tight cell adhesions owing to epithelial-mesenchymal transition) (30) or of progression-inducing changes in the primary tumor (e.g., CTC release might indicate more tissue damage in the biopsied prostate tissue, which could lead to an increased rate of local infections and/or wound healing, both processes that can induce tumor progression) (31).
TRUS biopsy is a standard method to obtain histological material to make a clinical diagnosis in PCa. Moreover, current literature shows that systematic ultrasound-guided prostate biopsies are the preferred diagnostic method for PCa (32–34). Thus, we do not advocate to abandon this important diagnostic tool. However, our present study shows that TRUS-guided biopsy can lead to an increase in CTCs in some patients, and we cannot exclude that this process might contribute to disease progression. To our best knowledge, this is the first report that supports the hypothesis that PCa cells released by needle biopsies might be relevant for relapse. Previous studies in pancreas and breast cancer had mixed results showing such a link between needle aspiration-induced tumor cell dissemination and clinical outcome (8, 35, 36). Besides technical considerations, 1 possible explanation for this discrepancy could be the more extensive sampling required to diagnose PCa (at least 12 needle biopsies).
The current study has obvious limitations. The observation time of our follow-up analysis is still short (41 months) for early-stage PCa, which might explain the finding that the Gleason score in our analysis was not a significant prognosticator of relapse. Further research should include the genomic characterization of these disseminated cells to investigate their malignancy and metastatic potential as has recently been made possible by single-cell genomic sequencing (37). Higher capture rates are required for further molecular analysis of CTCs in patients with early PCa. Other CTC assays produce higher “CTC” counts than the CellSearch system (38), but the clinical specificity of these assays is questionable because of a lack of validation by independent expert groups (39). For the analysis of higher blood volumes, diagnostic leukapheresis has been recently highlighted as an approach for this purpose (40, 41). Future studies motivated by our present results might incorporate diagnostic leukapheresis in patients with early-stage PCa.
We conclude that this study generates an interesting hypothesis with potential clinical implications, but larger confirmatory trials with longer follow-up periods are obviously needed before any change in clinical practice can be recommended. We do not advocate abandoning diagnosis by tissue biopsy; however, further investigation on the effect of released CTCs on prognosis is required. Our current results might induce the design of future multicenter clinical trials to improve current diagnostic strategies: The ongoing development of blood- or urine-based PCa biomarkers (“liquid biopsy”) and the improvement of imaging procedures might help to reduce the number of prostate biopsies and/or limit the biopsies to the suspicious regions of the prostate (42); furthermore, drugs that prevent CTCs from extravasation might be a future outlook for short-term intervention around the time of biopsy (31), similar to the concept of using antibiotics perioperatively to avoid the spread of germs.
Footnotes
(see editorial on page )
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.
S.A. Joosse, statistical analysis; P. Tennstedt, statistical analysis; T. Schlomm, provision of study material or patients; K. Pantel, financial support, administrative support.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership: K. Pantel, guest editor, Clinical Chemistry, AACC.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: K. Pantel, Veridex, Janssen, Menarini.
Research Funding: C. Alix-Panabières, DGOS and INCA grants, FEDER support; K. Pantel, ERC Advanced Investigator Grant INJURMET, grant support from Veridex, Janssen, Menarini.
Expert Testimony: None declared.
Patents: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, preparation of manuscript, or final approval of manuscript.
Acknowledgments
The authors thank Professor Markus Graefen for critically reading the manuscript, as well as Cornelia Coith, Antje Andreas, Malgorzata Stoupiec, Susanne Hoppe, and Olivier Mauermann for their technical assistance.
References
Author notes
B. Beyer, C. Gasch, P. Nastały, and A. Kuske contributed equally.
