2.2.1
Patient and control population
Studies were carried out under protocol # 15569E approved by the University
of Western Ontario’s Health Sciences Research Ethics Board (Appendix 1). Twenty-six
PCa patients with a rising PSA following RP who consented to salvage RT were enrolled
in this study following informed consent. To be included in this study, patients were
required to have a PSA value > 0.1 ng/mL and a minimum of three PSA values taken one
month apart, in order to calculate doubling time. Pre-RT bone scan and CT scan were
performed at the treating physicians’ discretion. Blood was drawn before radiation and
baseline PSA and CTC levels were determined using standard clinical immunoassay for
PSA and the CSS for CTC analysis as described below. Baseline characteristics of each
patient were noted, including pre-radiation PSA, Gleason score, pathologic T (pT) stage,
presence of extracapsular extension (ECE), presence of seminal vesicle invasion (SVI),
margin status, lymph node status, months free from relapse, and PSADT. All patients
were treated with radiation to the prostate bed as per Radiation Therapy Oncology Group
(RTOG) guidelines with 6600 cGy in 33 fractions using a 5 field intensity modulated
radiation therapy (IMRT) technique. Three months following the completion of RT, a
second set of blood samples were drawn and follow-up PSA and CTC levels were
determined.
Blood from 7 healthy individuals was also collected following informed
consent and analyzed for CTCs as a negative control. In addition, blood from 4
biochemically controlled patients with undetectable PSA for a minimum of 3 years was
consisted of two post-RP patients and two post-salvage RT patients. Blood was collected
at enrolment and at a 3 month follow-up visit for PSA and CTC analysis.
2.2.2 PSA determination
Blood samples were collected into a 6mL red topped Vacutainer® venous blood collection tube and analyzed for total PSA concentration by the London Health Sciences
Centre Endocrinology Laboratory on the AutoDelfia using a time resolved
fluorimmunoassay from Perkin Elmer.
2.2.3 Circulating tumor cell enumeration
Blood samples were collected into CellSave Preservative tubes (Janssen
Diagnostics) and CTCs were enumerated using the CSS as per the manufacturer’s
directions within 96 hours of sample collection13. The CSS consists of two components, (1) the CellTracks™ AutoPrep system, which automates the blood sample preparation,
and (2) the CellTracks™ Analyzer II, which scans the prepared samples. The AutoPrep
system uses an antibody mediated, ferrofluid-based magnetic separation technique and
differential staining with fluorescent particles to distinguish CTCs from contaminating
leukocytes in blood samples (Figure 2.1). Initially, an EpCAM (epithelial cell adhesion
molecule) selection is performed using anti-EpCAM antibodies conjugated to iron
nanoparticles incubated in a magnet. The only EpCAM+ cells in the blood should be the tumor cells. The remainder of the fluid is then aspirated from the sample, selected tumor
cells are resuspended, and differential staining antibodies are added to the samples.
Samples are then incubated in a magnetic cartridge called a MagNest™ and scanned
using the CellTracks™ Analyzer II. Samples are scanned using three different filters,
Figure 2.1. Schematic overview of the step-by-step processing of CTC blood
samples using the CellSearch®system (Source: Immunicon [adapted]).Following
sample collection into a CellSave tube, 7.5ml of blood is mixed with dilution buffer and
centrifuged (800g x 10 min) to collect blood and tumour cells. The centrifuged sample is
then loaded onto the CellSearch®AutoPrep, the plasma is aspirated, and anti-EpCAM
ferrofluid is added. Following a magnetic incubation, unlabelled cells are aspirated and the
remaining sample is permeabilized and incubated with the appropriate staining reagents
(CK/CD45/DAPI). The completed sample is then transferred to a MagNest®and
incubated for a minimum of 20 min (up to 24 hours). The MagNest device is then loaded
identified as events bound by anti-EpCAM and stained with anti-pan-cytokeratin (CK)-
phycoerythrin (PE) (CK 8, 18 and 19 are characteristic of epithelial cells), and the DNA
stain 4’, 6-diamidino-2-phenylindole (DAPI). Leukocytes are identified as events bound
by anti-CD45-allophycocyanin (APC) and DAPI. After the scan is complete, a gallery of
computer-defined, potential tumor cells is presented. These galleries were reviewed by 3
independent and blinded observers, and CTCs were confirmed via qualitative analysis
based on the differential staining criteria discussed above. If any discrepancies in the
number of selected events were noted between observers, these events were discussed
until a consensus was reached.
2.2.4 Statistical analysis
Mean and standard deviations were calculated for the following variables: age,
PSA, PSADT and Gleason score. The mean and standard deviations were compared with
CTC count (0 vs >0 and <2 vs ≥2) using the Independent-Samples t-test procedure.
Fisher’s Exact test was used to compare CTC count (0 vs >0 and <2 vs ≥2) with pT stage,
presence of ECE, SVI and margin status. In addition, the Pearson Correlation Coefficient
and the Spearman Correlation Coefficient were used to look for associations between
age, PSADT, Gleason score, change in PSA (pre-RT vs post-RT) and change in CTC
number (pre-RT vs post-RT). CTC cut-off values were chosen based on results
suggesting that CTC levels in early-stage PCa patients appear to be lower than those