Detection of circulating tumour cells in peripheral blood with an automated scanning fluorescence microscope

Abstract

We have developed an automated, highly sensitive and specific method for identifying and enumerating circulating tumour cells (CTCs) in the blood. Blood samples from 10 prostate, 25 colorectal and 4 ovarian cancer patients were analysed. Eleven healthy donors and seven men with elevated serum prostate-specific antigen (PSA) levels but no evidence of malignancy served as controls. Spiking experiments with cancer cell lines were performed to estimate recovery yield. Isolation was performed either by density gradient centrifugation or by filtration, and the CTCs were labelled with monoclonal antibodies against cytokeratins 7/8 and either AUA1 (against EpCam) or anti-PSA. The slides were analysed with the Ikoniscope robotic fluorescence microscope imaging system. Spiking experiments showed that less than one epithelial cell per millilitre of blood could be detected, and that fluorescence in situ hybridisation (FISH) could identify chromosomal abnormalities in these cells. No positive cells were detected in the 11 healthy control samples. Circulating tumour cells were detected in 23 out of 25 colorectal, 10 out of 10 prostate and 4 out of 4 ovarian cancer patients. Five samples (three colorectal and two ovarian) were analysed by FISH for chromosomes 7 and 8 combined and all had significantly more than four dots per cell. We have demonstrated an Ikoniscope based relatively simple and rapid procedure for the clear-cut identification of CTCs. The method has considerable promise for screening, early detection of recurrence and evaluation of treatment response for a wide variety of carcinomas.

Figure 1

Screen captures of the Viewer software that displays the data produced by the Ikoniscope® scanning system (C). C32 cells were spiked in normal blood, isolated by Lymphoprep^TM and immunostained with AUA1 and Cam5.2 followed by FISH with CEP probes for chromosomes 17 and 18. (A): High magnification ( × 100) target gallery screen showing composite images of C32 cells for AUA1 (Cy5), Cam5.2 (green) and nuclei (DAPI/blue) staining. (B): Screenshot displaying a target C32 cell at high magnification. Pseudo colored images in the DAPI (nucleus), green (Cam5.2), Cy5 (AUA1), orange (CEP 17) and aqua (CEP 18) channels are shown on the left side of the screen shot (clockwise from top left corner). A composite image of all five channels is shown at the bottom right of the screen shot. After automatic identification of FISH signals, a pseudo colored composite image of the nucleus, in which chromosomes 17 appear in yellow and chromosomes 18 appear in aqua, is shown at the top right of the screenshot.

Figure 2

Circulating tumour cells isolated by Lymphoprep from the peripheral blood of colorectal and prostate cancer patients. Composite pseudo colored images of cells at high magnification ( × 100) in the DAPI, green and Cy5 channels, are shown. (A) CTCs were isolated from colorectal cancer patients Co 1, 2 and 9 and were immunostained with AUA1 (green) and Cam5.2 (Cy5). (B) CTCs were isolated from prostate cancer patients 1 and 2 and were immunostained with Cam5.2 (Cy5) and AUA1 or PSA (green), as indicated.

Figure 3

Circulating tumour cells isolated by filtration from the peripheral blood of colorectal and ovarian cancer patients. Composite pseudo colored images of cells at high magnification ( × 100) in the DAPI, green, Cy5 and aqua channels are shown on the left of each panel, followed by cell images in the aqua channel in the middle of each panel. After automatic identification of FISH signals (dot count), a pseudo colored composite image of the nucleus, in which chromosomes 7 and 8 appear in aqua, is shown on the right of each panel. (A) CTCs were isolated from colorectal cancer patient Co 11 and were immunostained with AUA1 (green) and Cam5.2 (Cy5), followed by FISH with CEP 7/aqua and CEP 8/aqua. A total of 7 aqua dots are present in each nucleus, indicating polysomy for at least one of the chromosomes 7 and 8. (B) CTCs were isolated from ovarian cancer patient Ov 4 and were immunostained with AUA1 (green) and Cam5.2 (Cy5), followed by FISH with CEP 7/aqua and CEP 8/aqua. A total of 8 aqua dots are present in each nucleus, indicating polysomy for at least one of the chromosomes 7 and 8.