Trial Information

Impact of Metallic Stent Insertion for Obstructing Colorectal Cancer on Circulating Tumor Cell Dissemination

1. Patient recruitment:

Patients will be recruited from the Division of Colorectal Surgery, Department of
Surgery, the University of Hong Kong, over a 12 - 18 months period. Our institution is
a tertiary referral center for colorectal cancer patients in Hong Kong. Informed
consent will be obtained from the patients after the inclusion criteria are met.

The study plans to recruit 25 patients in the colonic stenting group to compare with 15
patients in the control group.

Inclusion criteria:

Colonic stenting group

1. Patients with obstructive colorectal cancer that requires colonic stenting as
treatment

2. Age > 18 years and is able to give informed consent

3. Informed consent for blood collection obtained

Control group

1. Patients with colorectal cancer who have colonoscopy without stenting

2. Age > 18 years and is able to give informed consent

3. Informed consent for blood collection obtained

2. Blood collection and examination for circulating tumor cell

About 10ml of EDTA blood will be collected for examination before the procedure
(colonoscopic stenting or colonoscopy in the control group) and blood collection daily will
be repeated for 3 days after procedure. Blood will be sent to the laboratory for processing
within 2 hours of withdrawal. Circulating tumour cells, which are characterized by CD45- (to
exclude hematopoietic cells), CD326+ (to mark epithelial cells), CK18+ and CK19+ (to mark
intestinal cells), i.e. cells expressing CD45-CD326+CK18/19+, will be detected by
fluorescence activated cell sorting (FACS).

For each ml of blood, 1 ml of FCM Lysing solution will be added and allowed to incubate for
5 minutes to lyse the red blood cells, followed by centrifugation at 1000 RPM for 5
minutes. Supernatant will then be aspirated, and the pellet resuspend in approximately 1 ml
cold 1X PBS. 10 µl of the appropriate antibody will be added into the tubes, vortexed and
incubated for 15 in dark and room temperature. After incubation, cells will be washed with
PBS and then incubated with the second antibody. The process is repeated until all 4
antibodies have been labelled to the cells. Cells will then be analysed on a FACS machine
(MoFlo, Beckman Coulter).