Cytology-based Circulating Tumour Cell (CTC) Testing in cancer patients and for early detection screening: The ISET-CTC study

A doctor's referral is required to undertake the CTC test as part of this study. Local participants: Participants will attend the National Institute of Integrative Medicine to have their blood samples taken at two separate visits. Non-local participants: A test kit will be posted to the participant at 2 time points. CTC blood samples will be collected by the participant's referring doctor at their usual clinic and will then be sent to the National Institute of Integrative Medicine for analysis. CTC reports will be sent to the referring doctor to discuss with the patient. The CTC test will allow: a) Monitoring of treatment effectiveness in cancer patients. b) Monitoring of cancer patients to facilitate detection of relapse, tumour progression. c) Screening test: Early Detection of CTC and other rare cells in patients with a family history of cancer or patients with chronic illness, advanced age, or as part of a health check. For all groups a-c, follow-up testing time frame is dependent on CTC count, e.g. - if no CTC are detected or the CTC count is low (<3 CTC/ml), repeat testing is suggested on a yearly bases. - if CTC count is 3-10 CTC/ml, repeat testing is recommended at 6-9 months. - if CTC count is >10 CTC/ml, repeat testing is recommended at 3-6 months. Method: CTC are isolated for blood (10ml in ACD) by ISET (Rarecells, France) using filtration technology before treatment (baseline) and 3-4 weeks after treatment (follow-up). CTC analysis by microscopy using cytological criteria. Participants will be followed up for 5 years. Add-on secondary testing available (Marker testing) on same participants' blood sample as initial CTC test. No additional visits required. Immuno-cyto-chemistry (ICC) staining with cancer markers (= specific antibodies) or viral markers ICC staining to be conducted as per protocol * Markers include: PSA = Prostate specific marker Mammaglobin (MG) = breast specific marker Other cancer markers, including CD45, prostein, etc EBV = Epstein-Barr-Virus specific marker (to ascertain viral reactivation often associated with cancer) HSV = Herpes Simplex Virus specific marker CMV = Cyto-Megalo-Virus specific marker HPV = Human Papilloma Virus specific marker *ICC Protocol (published in Ried et al 2020 Front Oncol 10:582): “ICC staining was conducted with the Dako EnVision Flex Mini Kit, high pH, and antibodies a) PSA (Dako monoclonal mouse anti-human prostate-specific antigen Clone ER-PR8, concentrate), and b) Prostein/P501S (Dako Flex Monoclonal mouse anti-human Prostein Clone 10E3, Ready-to-Use (RTU)). For antigen retrieval, ISET® filters were placed into a 50 ml tube and heated in a water bath at 99 ºC for 40 min in 50 mL of 2% v/v target retrieval solution (high pH (50x): 3x30 mL, 50x concentrate Tris/EDTA buffer, pH9; Dako Agilent). Next, the ISET® filters were washed 15x in 0.2M PBS and incubated with permeabilisation buffer (0.1% Triton) for 2 min. After washing the ISET® filter as before, the ISET® filter was incubated for 30 min with peroxidase-blocking reagent (80 µL of H2O2, 15 mmol/L Na3N and detergent), and washed with distilled water 15 times. Each ISET®-filter spot was incubated overnight at 4 ºC in 600 µL solution of the primary antibody (a) 1:200 dilution of concentrated PSA antibody or b) 1:4 dilution of Prostein antibody RTU in a buffer of 1xPBS / 5%BSA in a 1.5 ml Eppendorf tube. After incubation with the primary antibody, ISET®-filter spots were washed in PBS as before, placed on a clean slide, for incubation with 80 µL of the secondary antibody (Dako Envision Flex Kit goat secondary antibody) for 45 min at RT. The ISET®-filter spots were washed again, before incubation with 80 µL of chromogen (Dako Liquid DAB + Substrate Chromogen (3,3 diaminobenzidine tetrahydrochloride) on a glass slide in the dark for 10 min. The excess chromogen was removed by washing in 0.2 M PBS. The filter spots were allowed to air-dry, then counterstained with hematoxylin for 2 min. After washing in PBS as before, each air-dried immuno-stained spot was mounted on a clean glass slide for microscopic analysis.”