Although only less than 10% of women with primary breast cancer have clinicopathologic signs of overt metastases, metastatic relapse occurs in about half of the cases with apparently localized tumors within five years after surgery. In 23% of the patients, bone marrow metastases are detectable at first relapse and this rate even increases in patients with metastatic breast cancer. However, hematogeneous or lymphatic spread of occult tumor cells can arise before diagnosis at an early stage of primary tumor growth and is regularly underestimated by currently available clinical and pathologic staging procedures. We studied cytokeratin-positive (CK+) cells in the bone marrow (BM) and tumor markers in the blood of 128 patients with primary breast cancer in order to obtain an early diagnosis of residual disease. In a second study, we monitored cytokeratin (CK)/17-1A positive cells in the BM and peripheral blood stem cells (PBSC) to evaluate whether dose intensive or high-dose (HD)-chemotherapy can eliminate micrometastases in high-risk breast cancer patients. The overall CK+ rate was 34% (44/128 patients), 29% (15/51) for patients with T1 tumors, 33% (28/84) for N0 patients and 31% (26/82) for patients with G1-2 breast carcinoma. Interestingly, 67% of CK+ patients were only positive in one of the two BM aspirates studied. At least one tumor marker including carcinoembryonic antigen, carbohydrate antigen 15-3 and tissue polypeptide antigen, was increased in 58/128 (45%) patients [21/58 (36%) were CK+ in the BM]. Surprisingly, levels for the extracellular domain of Her-2/neu in serum samples were within the normal range in every patient studied. After a 2-year follow-up, 7/128 patients relapsed (3/7 CK+/TM-; 2/7 CK-/TM+; 2/7 CK-/TM-). We concluded that studying two BM aspirates for CK+ cells by immunocytochemistry in combination with tumor marker determination is useful for identifying patients with a higher risk for relapse. A tumor cell enrichment technique, applied in 70 patients prior to immunocytochemistry using dynabeads directly coupled to an antibody (BerEp4) targeting the 17-1A antigen, did not enhance the detection rate of disseminated tumor cells in this patient group. We monitored CK+/17-1A+ cells in the BM and PBSC and studied Her-2/neu serum levels of patients with locally advanced (n=13, group 1) and metastatic breast cancer (n=30, group 2). CK+ cells were found in the BM of 3/13 (23%) group 1 patients before but not after chemotherapy resulting in an overall survival (OS) of 92% after a median follow-up of 33 months. Contamination of PBSC in 2/9 (22%) patients was not associated with decreased survival. In group 2 patients, the CK+ rate was 60% (18/30 patients) before and 40% (4/10 patients) after therapy with an OS rate of 43% after 29 months. PBSC samples were positive in 7/24 (29%) patients. CK+ BM and PBSC led to a rapid progress and short OS whereas tumor cell free BM and PBSC resulted in a mean OS of 30 months. The antigen 17-1A was detected on most CK+ cells in both patient groups before therapy, on all CK+ PBSC and on CK+ cells in group 2 patients after therapy. Increased Her-2/neu levels were found in group 2 patients before chemotherapy. In conclusion, micrometastatic cells are present in blood and PBSC grafts of high-risk breast cancer patients and can survive even HD-chemotherapy. Immunotherapeutic target antigens on the cell surface of these cells support the idea that a combined chemoimmunotherapy might be successful in eliminating minimal residual disease.

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