Detection of cancer cells in peripheral blood stem cells of women with breast cancer by RT-PCR and cell culture.

ABSTRACT

The benefit of high-dose therapy and blood stem cell reinfusion for women with high-risk breast cancer is currently under investigation. Contaminations of autologous blood stem cells with cancer cells have been described. Cancer micrometastases may be detected by immunocytochemistry, culture techniques and cytokeratin-19 mRNA reverse transcriptase PCR. Women with breast cancer received adjuvant HD-CTM with peripheral blood stem cell (PBSC) support after surgical therapy and 4 cycles conventional chemotherapy. Peripheral blood stem cells were mobilised by G-CsF and harvested after the third or fourth cycle of standard therapy. Aliquots of PBSC-collections (10(7)-2*10(7) cells) were subjected to CK19-mRNA reverse transcriptase PCR. RNA was extracted by standard methods and reverse transcription was performed with MMV-RT. Integrity of RNA was checked by coamplification of housekeeping sequences. Aliquots of the RT-mix were subjected to PCR-amplification with outer and inner primer pairs, subsequently. A second aliquot of 2*10(7) cells was cultured over 42 days in liquid culture. Cytospins were prepared weekly from cultured cells and evaluated by light microscopy with or without prior immunocytochemistry. Ten leukaphereses from 6 women were available for PCR-analysis and cell culture. Six leukaphereses were negative for CK19-mRNA and for detection of cancer cells by culture technique, two samples were positive for CK19-mRNA and culturally enriched cells and two samples were positive for CK19-mRNA and negative for cultured cancer cells. No sample was positive for cultured cells and negative for CK19-mRNA. Overall, the results corresponded in 80%. Two sensitive techniques for the detection of cancer micrometastases were applied to aliquots from 10 leukaphereses of six breast cancer patients with corresponding results in 80%. PCR-mediated detection of cancer cells was confirmed by culture technique and light microscopy, however, further comparison of CK19-PCR with standard techniques like cell culture and immunocytochemistry is still necessary.