Cellsearch

Digitally stored CellSearch® (Menarini Silicon Biosystems, Huntingdon Valley, PA, USA) image files from 98 metastatic breast cancer patients from a previously reported study (CLCC-IC-2006-04 study, ClinicalTrials.gov Identifier: NCT00898014) were re-analyzed [3 (link), 9 (link)]. The original study included 267 patients with either HER2+ or HER2− primary tumor, whose characteristics at the baseline have been previously described [9 (link)]. Of these samples, 114 were further screened for HER2 expression on CTCs. These samples had been processed with the CellSearch system using the CTC kit, as previously described [7 ] including an additional fluorescein (FITC)-conjugated monoclonal antibody recognizing HER2 (clone Her81) in the staining mixture [11 (link)]. The specific clone recognizes an epitope in the extracellular domain of HER2, and it does not cross block binding of the FDA-cleared trastuzumab used in the clinics [11 (link)]. Ninety-eight out of 114 image datasets (where the anti-HER2 had been included in the staining mix) were available for the present analysis.

The enriched samples were cyto-centrifuged onto glass slides and CTCs identified by immunocytochemistry (ICC) using the CellSearch^® antibody cocktail (pan-Cytokeratin/CD45/DAPI; Menari-Silicon Biosystems, PA, USA). Briefly, the slides were air dried overnight and incubated with the combination of CellSearch^® (Menarini Silicon Biosystems Inc, PA, USA) reagents (20 µL staining reagent, 20 µL permeabilization buffer, 20 µL fixation buffer, 10 µL DAPI and 130 µL 1× PBS). The slide was incubated for 1.5 h at room temperature, washed three times in 1× PBS. Putative CTCs were further characterized for EGFR exon 19 deletion (1:100 dilution) (EGFR E746-A750, Cell Signaling, Beverly, MA, USA) and PD-L1 (1:200 dilution) (anti-PD-L1 (28-8)). The slides were mounted with Prolong Gold mounting medium (Molecular Probes, Eugene, OR, USA) to prevent photo-bleaching and preserve the fluorescent labelled molecules for long term storage, coverslipped and imaged using a Nikon Epifluorescence microscope (Nikon, Tokyo, Japan). CTCs were identified as intact, pan-cytokeratin⁺, DAPI⁺, CD45⁻ cells larger than 4 µm. The mean fluorescence intensity (MFI) was measured per CTC by determining the fluorescence intensity per CTC and subtracting this from the local background intensity. This was compared to the MFI of known NSCLC cancer cell lines and controls.

Circulating tumor cells were detected through two methods, one EpCAM-dependent (CellSearch) and the other size based (ScreenCell). Peripheral blood samples were drawn after informed consent into two tubes, after discarding the first milliliters to avoid contamination with cutaneous epithelial cells taken by the needle during the sampling. 7.5 mL of blood was collected in CellSave preservative tube (Menarini Silicon Biosystems, Castel Maggiore, Bo, Italy) and processed by CellSearch (Menarini Silicon Biosystems), employing CellSearch CTC kit, according to manufacturer's instructions. Six milliliters of blood was collected in tube containing K₂EDTA and processed by ScreenCell Cyto kit (ScreenCell, Sarcelles, France) to isolate fixed cells for cytological studies. Briefly, in order to fix the cells and lyse red blood cells, 3 mL of blood was diluted in 4 mL of FC2 filtration buffer (ScreenCell). After 8 min of incubation at room temperature, 7 mL of diluted sample was added into device tank and filtered under a pressure gradient using a vacutainer tube. After washing with PBS to remove red blood cells debris, the filter was left on absorbing paper to dry at room temperature and then stored at −20°C. The filtration was carried out in duplicate and completed within 3 min.