Establishment of immunocytochemical and molecular methods for characterization of circulating tumor cells in breast cancer patients

Abstract

The presence of CTCs in peripheral blood has been proven to be an independent prognostic and predictive factor for patients with metastatic breast cancer. The major aims of the study were to detect and characterize CTCs in peripheral blood from breast cancer patients with the help of the CellSearchTM system, as well as to establish and optimize a set of methods to further phenotypically and molecularly characterize CTCs. CTCs were detected in 65 breast cancer patients with the CellSearchTM system and additionally characterized for HER2 expression in 51 cases through processing blood samples. HER2-positive CTCs were detected in 15 of 51 patients (29.4%), and heterogeneity of HER2 expression in CTCs among individual patients was observed. Furthermore, previously described discrepancies between the HER2 status in primary tumors and CTCs in blood were confirmed by the present study. In this study, for the further characterization of CTCs a set of methods including double immunofluorescence, FISH, combined FISH and IF, as well as real time PCR was established. The application of a new antigen retrieval technique prior to FISH analysis was the most important prerequisite for the establishment of a combined method for FISH and immunocytochemistry on CTCs. Thus far, there are no hints that processing the blood with the CellSearchTM system significantly interferes with the downstream molecular analysis of CTCs. Using these established methods, we confirmed the expression of CK, HER2 and hormone receptor as well as the HER2 gene status in seven breast cancer cell lines, presented various Ki-67 staining patterns, and revealed a serial change in CK and M30 expression as well as morphological changes during apoptosis from early stage to late stage. The newly developed method of combined FISH and IF was utilized for various kinds of specimens, and appeared to be a robust tool for phenotyping and genotyping individual cells simultaneously in pleural effusion, CTCs and other cytological specimens. Our real time PCR protocol using an internal control located on chromosome 17 appeared to be comparable to the current gold-standard FISH for the evaluation of HER2 gene status in tissue sections of breast cancer patients. For real time PCR with DNA obtained by whole genome amplification, the TaqMan assay was superior to the SYBR Green assay because of the higher specificity of the former one. Although the number of clinical samples analyzed in the study is small, the feasibility of the established methods has been evident. All these methods might be employed in the oncoming large scale analyses of clinical samples. However, further investigations of primary tumors are needed to identify tumor-specific markers that can be used both for the detection of CTCs as well as for their application in therapeutic monitoring.