Sensitive detection of micrometastases in bone marrow from patients with breast cancer using immunomagnetic isolation of tumor cells in combination with reverse transcriptase/polymerase chain reaction for cytokeratin-19

Abstract

We report a highly sensitive method to detect rare human breast cancer cells, which combines an immunomagnetic separation (IMS) using antibody BM2 against MUC-1 with cytokeratin-19 (CK19) and the reverse transcriptase/polymerase chain reaction (RT-PCR). The IMS-RT-PCR technique allows the detection of 1 tumor cell/10(7)-10(8) mononuclear cells. This is at least ten times more sensitive than CK19 RT-PCR alone, or immunocytochemistry. All 117 peripheral blood and 8 bone marrow samples obtained from healthy donors as negative controls were positive for beta2-microglobulin by RT-PCR but negative for CK19 by IMS-RT-PCR or RT-PCR alone. Out of 26 bone marrow samples from breast cancer patients, 18 had CK19 transcripts detectable by IMS-RT-PCR. In contrast, only 14 and 13 samples from the 26 were found to be positive by RT-PCR alone or by routine immunocytochemical staining. In conclusion, IMS-RT-PCR for CK19 is a highly sensitive and specific method for detecting very low numbers of micrometastatic breast cancer cells in bone marrow amidst an excess of nonmalignant cells. For the early diagnosis of disseminating disease, this assay is more efficient than RT-PCR alone and routine immunocytochemistry.

The detection of circulating breast cancer cells in blood

“…45 Immunomagnetic separation, used before RT-PCR for tumour cell enrichment from BM, resulted in the detection of one CK19 expressing tumour cell in 10 7 -10 8 mononuclear cells and was therefore at least 10 times more sensitive than using RT-PCR alone in this study. 51 The detection of tumour cells by this method was also approximately 100 times more sensitive than the detection of CK19 by IHC. 27 28 51 However, the use of such a technique to enrich the circulating tumour cell population from blood samples would ultimately increase both the cost and time taken to prepare and analyse blood samples from patients with breast cancer.…”

Review: Biological relevance of disseminated tumor cells in cancer patients

“…[40][41][42] Therefore, measurements of epithelium-specific or more organspecific mRNA species such as CK19 and 20 or mammaglobin or PSA mRNA by RT (reverse transcriptase)-PCR have been proven as more promising to detect DTC in BM samples. [43][44][45][46][47][48][49][50] The main advantage of very sensitive RT-based techniques for the detection of DTC at the single-cell level is the nearly ''unlimited'' availability of primers for almost every gene of interest. However, one of the major problems of RT-PCR approaches is low RNA stability.…”

Enrichment methods to detect bone marrow micrometastases in breast carcinoma patients: clinical relevance

“…The methods currently used to evaluate the spreading of micrometastases are immunocytochemistry (IC) [4,5], RT-PCR [6,7], flow cytometry [8,9], fluorescence in situ hybridization [10,11], and immunomagnetic (IM) bead enrichment [12-15]. Most of these methods rely on the expression of epithelial markers on the membrane or the cytoskeleton of carcinoma cells collected from blood or BM after density gradient centrifugation (Ficoll) of the mononuclear cell (MNC) fraction.…”