Early Detection of Prostate Cancer. CETCs/CTCs combined with PSMA Scan
A pilot study with significant correlation
Presentation at Early Detection Conference 2021
Eng Peter, Pachmann Katharina, Lux Daniel, Fluhrer Joachim
Abstract
Prostate cancer is one of the leading causes of mortality in men. However, prostate cancer screening using PSA levels is still a matter of debate and the effect on long term mortality is unclear. Instead, screening may be associated with increased harms such as over-diagnosis and complications of treatment for indolent disease.
2-5 Difficulty of shared, informed decision-making between patients and primary care providers about PSA screening may also contribute to practice variations.
In the present report the enumeration of tumor cells in blood was used to obtain information about the risk of developing prostate cancer and correlated to the detection of prostate cancer in PSMA-PET. CETC/CTCs were prospectively enumerated in 64 men aged between 50 and 85 years using an immunofluorimetric assay and semiautomated detection of fluorescent events.
PSMA-PET was significantly more frequently positive in patients with higher CETC/CTC with the best discriminatory power above a value of 450 tumor cells/ml.
In men at risk of prostate cancer CETC/CTC numbers above a cut off limit or increasing CETC/CTC numbers additional PSMA-PET examinations may help to early detect patients with prostate cancer in whom further watchful waiting is warranted.
Background
Prostate cancer is one of the most frequent malignant diseases in men and after the age of 80 is detected in almost all men during autopsy.
Monitoring the PSA level over time has been the most frequently used tool in prostate cancer surveillance but the usefulness of this test is still debated [1] and two trials with discrepant results, rather than solving the problem, contributed to further polarize the discussion [2,3].
Men with PSA levels below a threshold of 4ng/ml can have PCA [4] and an increase in PSA is not prostate cancer specific [5]. With prostate tissue still present PSA rise can also be due to other reasons [6,7] such as genetic background [8] or disintegration of PSA-positive cells [9].
The 10-year and 15-year relative survival rates for prostate cancer are 98% and 95%, respectively [10] and not every tumor needs to be treated invasively. It is often possible to just watch and wait [11] and to change life style habits.
In patients with elevated PSA biopsy is recommended. Overdiagnosis [12] as well as upgrading of the tumor may occur during active surveillance [13]. However, biopsy may also lead to dissemination of tumor cells [14]. Due to these in part severe adverse side effects men often are reluctant to turn to prostate cancer screening in the present form [15] as exemplified by the suspension of the PREFER-study [16] and less invasive procedures are eagerly looked for.
Such a procedure may become the PSMA-PET [17] which gains rapid acknowledgment [18]. It is, however, still expensive and cannot be repeated often, therefore it is important to determine the optimal time for screening with the PSMA-PET. It is known that growing tumors can seed cells into the circulation already at early times during development.
Tracking of such cells which are believed to be the origin of metastatic disease due to having left the primary tumor and capable to settle in distant organs [19] with a blood test that analyzes circulating tumor cells may hold promise [20].
We have therefore monitored circulating epithelial cells in men who requested this analysis and compared the numbers of cells to PSMA-PET positivity.
Materials and Methods
Blood samples were obtained from 64 patients aged between 50 and 85 years without a diagnosis of prostate cancer from 2014 until 2018.
CETC/CTCs were enumerated repeatedly, using an approach avoiding loss of cells [25]. Briefly, 1 ml blood was subjected to red blood cell lysis using 15 ml of erythrocyte lysis solution (Qiagen, Hilden, Germany) for 15 min in the cold, spun down at 700 g and re-diluted in 500 μl of PBS-EDTA.
Subsequently, 20µl of cell suspension were incubated with 20µl of a mastermix (120 µl EpCAM-FITC (fluoroisothiocyanate (FITC) -conjugated anti-EpCAM antibody (CD-326, Miltenyi, Bergisch Gladbach, Germany)), 100 µl 10% BSA, 4µl 7-AAD (7-Aminoactinomycin D Sigma-Aldrich Co., Taufkirchen, Germany) and 776 µl PBS-EDTA) for 1hr in the cold.
The corresponding isotypic control for EpCAM (Mouse IgG1K FITC, Miltenyi Biotec GmbH) was used at the same final concentration. The samples were subsequently diluted with 440 μl PBS-EDTA.
A defined volume of the cell suspension was transferred to flat bottom wells of ELISA plates (Greiner Bio-One, Monroe, NC, USA).
The analysis of red and green fluorescence of the cells was performed using a Fluorescence Scanning Microscope, ScanR, (Olympus, Tokyo, Japan), enabling the detection and relocation of cells for the visual examination of EpCAM‑positive cells.
For data analysis, we used the ScanR Analysis software (Olympus). Live CETC/CTCs were defined as EpCAM‑positive cells, lacking nuclear 7-AAD staining and with intact morphology, and only these cells were counted.
We used fluorospheres (Flow-Check 770, Beckman Coulter) for daily verification of optical components and detectors of the microscope, which are required to ensure the consistent analysis of samples. This resulted in a trajectory of cell numbers during the observation time.
Serum PSA levels were determined at the time of performance of the PSMA-PET in 51 men.
PET scanning was performed from vertex to upper thighs on a hybrid PET/CT scanner, following intravenous administration of 69.6 MBq of Gallium-68 prostate specific membrane antigen (Ga-68 PSMA).
A contemporaneous low dose non-contrast CT scan was performed for attenuation correction and anatomical correlation. Numbers of cells determined at the time of PSMA-PET were correlated to PSMA-PET positive or negative results. Follow up was up to 4 years.
Fig. 1
Live CETC/CTCs were defined as EpCAM-positive cells, lacking PI-staining and with intact morphology, and only these cells were counted (Fig.1).
Circulating epithelial-antigen positive cells were found repeatedly during the time of observation (Fig 2). This resulted in a trajectory of cell numbers during the observation time and numbers of cells were typically increasing before or at the time the PSMA-PET became positive. PSMA-PET Scan positivity and negativity was defined by the examiner performing the PET-scan.
In subjects in whom PSA had simultaneously been determined at the time of PSMA-PET who had shown a positive PSMA-PET (n=27) PSA levels were not different from that in subjects with a negative PSMA-PET (n=24) (Fig. 3a).
In contrast, in subjects in whom CETC/CTCs had been determined (n=25) those with a positive PSMA-PET had cell numbers significantly higher than in subjects (n=24) with a negative PSMA-PET (Fig. 3b).
Numbers of EpCAM-positive cells were, however, significantly higher in the group of male subjects between the age of 50 and 85 years than in randomly screened subjects (n=35) (Fig. 3c).
The cut-off distinguishing best between patients with a high probability to be positively screened for prostate cancer was at 450 cells/ml with a specificity of 0.7 and a sensitivity of 0.6 as shown from the ROC curve (Fig 4).
A decline in cell numbers was seen to a different extent during comprehensive cancer care and a re-increase often after terminating therapy (see Fig 2).
Discussion
Early detection of prostate cancer aiming at reducing mortality from prostate cancer is controversially discussed with respect to PSA screening [26]. The American Urological Association (AUA) Guideline from 2013 recommends “for men ages 55 to 69 years” ..”the decision to undergo PSA screening involves weighing the benefits of preventing prostate cancer mortality in 1 man for every 1,000 men screened over a decade against the known potential harms associated with screening and treatment”[21]. As a downstream consequence of prostate-specific antigen (PSA) –based screening Active surveillance (AS) has become an accepted strategy for reducing overtreatment of favorable-risk prostate cancer [22] but which still may be underused [23]. However, also repeated biopsy as recommended in active surveillance is laden with severe side effects [24] and even after therapy with curative intent treatment failure is a frequent event. On the other hand prostate-cancer–specific mortality is low with no significant difference among treatments [25].
To explain the presence of treatment failure in men with pathologically organ confined prostate cancer dissemination of tumor cells must be an early event, prior to treatment [31]. Detection of these circulating tumor cells has methodological pitfalls [32,33] and even in metastatic disease circulating tumor cells so far are not used for treatment decision [34].
Using an approach of epithelial cell enumeration with minimal cell loss, circulating cells are already detectable before detection of an asymptomatic prostate cancer. Often the number of these cells already present at an elevated level in men at the age when they become at risk for prostate cancer are increasing obviously during growth of the tumor warranting at that time implementation of a PSMA-PET. In contrast to PSA levels which were not different in patients with negative or positive PSMA-PET it could be shown that numbers of circulating cells of potential tumor origin were significantly higher in patients with a subsequent positive PSMA-PET than in patients with a negative PSMA-PET. Most of these tumors presumably are of low Gleason score. It has been shown that overall survival in patients without invasive treatment is not different from patients undergoing prostatectomy, but in patients choosing watchful waiting metastatic disease may be a burden during the subsequent course of disease [35]. Detection of elevated levels of CETC/CTCs in patients with positive PSMA-PET subsequently allows serial determination of the numbers of these cells in order to early monitor increased tumor activity. We have, however, shown that lifestyle changes and the respective integrative cancer care [36] can even in this situation reduce the number of epithelial cells of potential tumor origin thus possibly reducing the risk of metastatic disease.
Conclusion
Repeated counting of CTC/CETCs during integrative therapy or change in life-style reveals that subjects at risk for prostate cancer due to advanced age can improve their chance to avoid aggressive treatment for detection and therapy of prostate cancer.
References
Available upon request
Acknowledgement and Contact
The authors like to thank the participating patient and clinics for supplying for supplying the data and information for this pilot study.
The authors also thank the Laboratory Pachmann for anlaysing the sample and Simfo for data collection and statistical evaluation.
For further details and queries, contact Dr. Joachim Fluhrer here.