Enrichment with anti-cytokeratin alone or combined with anti-EpCAM antibodies significantly increases the sensitivity for circulating tumor cell detection in metastatic breast cancer patients

Abstract

Introduction Circulating tumor cells (CTCs) are detectable in most cancer patients and they can meet an existing medical need to monitor cancer patients during a course of treatment and to help determine recurrent disease. CTCs are rarely found in the blood of cancer patients and enrichment is necessary for sensitive CTC detection. Most CTC enrichment technologies are anti-EpCAM antibody based even though CTC identification criteria are cytokeratin positive (CK + ), CD45 negative (CD45 - ) and 4'6-diamidino-2-phenylindole (nuclear stain) positive (DAPI + ). However, some tumor cells express low or no EpCAM. Here we present a highly sensitive and reproducible enrichment method that is based on binding to anti-CK alone or a combination of anti-CK and anti-EpCAM antibodies. Methods Blood samples from 49 patients with metastatic breast cancer were processed using the CellSearch™ system (Veridex, LLC, Raritan, NJ, USA), in parallel with our CTC assay method. We used anti-CK alone or in combination with anti-EpCAM antibodies for CTC enrichment. Brightfield and fluorescence labeled anti-CK, anti-CD45 and DAPI (nuclear stain) images were used for CTC identification. The Ariol ® system (Genetix USA Inc, San Jose, CA, USA) was used for automated cell image capture and analysis of CTCs on glass slides. Results Our method has the capability to enrich three types of CTCs including CK + &EpCAM + , CK + &EpCAM -/low , and CK -/low &EpCAM + cells. In the blind method comparison, our anti-CK antibody enrichment method showed a significantly higher CTC positive rate (49% vs. 29%) and a larger dynamic CTC detected range (1 to 571 vs. 1 to 270) than that of the CellSearch™ system in the total of 49 breast cancer patients. Our method detected 15 to 111% more CTCs than the CellSearch™ method in patients with higher CTC counts (>20 CTCs per 7.5 ml of blood). The three fluorescent and brightfield images from the Ariol ® system reduced the number of false-positive CTC events according to the established CTC criteria. Conclusion Our data indicate that the tumor-specific intracellular CK marker could be used for efficient CTC enrichment. Enrichment with anti-CK alone or combined with anti-EpCAM antibodies significantly enhances assay sensitivity. The three fluorescent and brightfield superior images with the Ariol ® system reduced false-positive CTC events.

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT–PCR in patients with metastatic breast cancer

“…Several authors reported the heterogeneous expression of EpCAM in mammary carcinomas (Thurm et al, 2003) and downregulation of EpCAM has been reported for disseminated tumour cells in bone marrow and CTCs in peripheral blood (Thurm et al, 2003;Rao et al, 2005). Recently, Deng et al (2008) showed that CTC enrichment with anti-cytokeratin antibodies, in combination with anti-EpCAM antibodies, significantly enhances assay sensitivity.…”

Minimal Residual Disease and Circulating Tumor Cells in Breast Cancer

“…Several slide-based automated microscopic scanning devices, such as the Ikoniscope® [17] and the Ariol® system [18,19], have been applied for standardized micrometastatic cell detection and characteri zation. Another approach has been developed that uses fi ber-optic array scanning technology (FAST) for rare cell detection [20].…”

Real-time RT-PCR systems for CTC detection from blood samples of breast cancer and gynaecological tumour patients (Review)

“…Another system for CTC detection is the so-called Ariol system, which is based on image capturing. Three fluorescent images of cell surface antigens like CK or EpCAM and one brightfield image of one cell are taken together for detection of tumour cells (72). Flow cytometry can also be used for CTC detection, with the advantage, that multiple cell parameters, such as DNA-content, cell size and cellular markers can be analysed simultaneously and has thereby advanced to be a specific method to monitor breast cancer patients receiving chemotherapy (73).…”