Colonic Stent and Tumor Cell Dissemination

• Changes of circulating tumor cell level before and after colonic stenting for patients with obstructive colorectal cancer [ Time Frame: Before and first 3 days after procedure ] [ Designated as safety issue: No ]
  The patients' peripheral blood will be collected before and on first 3 days after the procedure.
  
  

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).