A Comparison of Immunohistochemistry, Two-Color Immunofluorescence, and Flow Cytometry with Cell Sorting for the Detection of Micrometastatic Breast Cancer in the Bone Marrow

Abstract

A significant percentage of women with primary breast cancer have micrometastatic disease in the bone marrow. Bone marrow involvement may be an adverse prognostic factor, and more aggressive therapy may be indicated for these patients. There are a number of different techniques and antibodies used to detect tumor cells in the bone marrow. We used the same panels of four antibreast cancer antibodies and compared three immunodetection techniques: two-color immunofluorescence, immunohistochemical staining, and fluorescence-activated cell sorting with cytologic examination of the sorted cells. The two-color immunofluorescence technique was superior and consistently detected one tumor cell contaminating one million normal bone marrow cells and had no reactivity with normal bone marrow.

Detection of circulating tumour cells in patients with breast or ovarian cancer by molecular cytogenetics

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The detection and elimination of minimal disseminated disease in patients with solid tumours is one of the main current topics in clinical oncology (Pantel, 1996).A variety of assays of widely varying sensitivity have been utilized for the detection of circulating tumour cells such as light microscopy, cytogenetic analyses, fluorescence in situ hybridization (FISH), Southern blot analysis, immunocytochemistry, polymerase chain reaction (PCR) and flow cytometry (Kvalheim, 1996;Pantel, 1996;Vrendenburgh et al, 1996;Schoenfeld et al, 1997;Traystman et al, 1997).Because of the fact that breast and ovarian cancers do not appear to have tumour-specific chromosomal aberrations, tumour cell detection by molecular methods is based on the amplification of tissue-specific transcripts Bostick et al, 1998). In immunocytochemical assays, epithelial specific antibodies have been used to detect isolated tumour cells in bone marrow (BM) and blood (Cote et al, 1991(Cote et al, , 1995(Cote et al, , 1996Diel et al, 1996;Franklin et al, 1996).

In an effort to obtain greater sensitivity, several investigators have developed techniques for the enrichment of tumour cells before their identification by immunocytochemistry, PCR or FISH (Hardingham et al, 1993(Hardingham et al, , 1995Berois et al, 1997;Eaton et al, 1997;Hildebrandt et al, 1997;Naume et al, 1997;Martin et al, 1998).

Therefore, the aim of our study was to analyse the presence and frequency of circulating tumour cells in the peripheral blood of patients with breast or ovarian cancer by using a combination of magnetic activated cell sorting (MACS) and interphase FISH.

MATERIALS AND METHODS

Patients

In this study we included 48 adult patients with a median age of 60.6 ± 13.3 years (range 30-86).

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“…A variety of assays of widely varying sensitivity have been utilized for the detection of circulating tumour cells such as light microscopy, cytogenetic analyses, fluorescence in situ hybridization (FISH), Southern blot analysis, immunocytochemistry, polymerase chain reaction (PCR) and flow cytometry (Kvalheim, 1996;Pantel, 1996;Vrendenburgh et al, 1996;Schoenfeld et al, 1997;Traystman et al, 1997).…”

Ex vivo expansion of bone marrow from breast cancer patients: reduction in tumor cell content through passive purging

“…This technique can detect one tumor cell in 10 6 normal BM cells. 27 Ex vivo expansion of tumor cell positive C/T BM MNC obtained from stage IV breast cancer patients resulted in passive purging in all samples examined. Tumor cell frequency decreased over the course of ex vivo expansion in all cases.…”

“…25,26 Immunocytochemical staining Quantitation of the tumor cell population present in the C/T MNC samples pre-and post-expansion was determined by ICC staining, as previously described. 27 Ten coded slide preparations containing 5.0 × 10 5 nucleated cells per slide were prepared for each sample. The slides were air dried and stored for up to 1 month prior to staining.…”

Cancer-Immune Equilibrium: Questions Unanswered

“…Flowcytometry using monoclonal antibodies has also been tried with limited success [43]. Different types of enrichment methods have been used to overcome this problem such as use of immunomagnetic beads for separation of tumor cells from peripheral blood, bone marrow, ascetic fluid or pleural effusions.…”