Detection of micrometastatic clonogenic growth in bone marrow of early stage breast cancer patients presenting with or without lymph node involvement

Micrometastases found in the bone marrow (BM) of early stage patients has been associated with a poor clinical outcome for breast cancer (BrCa). We have previously demonstrated that the presence of clonogenic breast cancer cells in the BM or peripheral blood stem cell(PBSC)products of stage IV patients is also strongly correlated with an extremely poor prognosis. In this study, we have utilized the clonogenic assay to add accuracy to staging BrCa at diagnosis. Methods: We have evaluated the presence of clonogenic breast cancer cells in bone marrow aspirates from 98 BrCa patients with I, II, III and IV stage of disease at diagnosis. The clonogenic assay utilized mononuclear cells plated in Iscove's medium containing 0.3% agarose, 10% fetal bovine serum and growth factors; the plates were incubated at 370C for 10 days. Verification that colonies are BrCa in origin was achieved by immunocytochemical (ICC) staining. Results: Using the standard ICC analysis, tumor cells were detected in 27/98 (27.5%) of BM specimens; 13/71 (18%) negative specimens turned positive when the specimen was enriched for tumor cells using an ultra-sensitive tumor enrichment (EICC) assay system. Clonogenic tumor cell growth was found in 24/98 (24.5%) of BM specimens. There were 44 patients where the clinical stage was known: One stage I, 28 stage II, 16 stage III, and 5 stage IV patients. Seven of twenty eight (7/28). 4/16 and 1/5 patient's BM showed clonogenic cell growth for stages II, III and IV, respectively. Interestingly, in the 27 patients with no lymph node involvement, 5/13 (38%) stages IIA and 3/14 (21%) stages IIIA had clonogenic tumor cell growth. Moreover, clonogenic growth was detected in 9/71 patients with undetectable tumor cells by standard ICC analysis. When EICC was performed on 2 of these negative specimens, micrometastatic tumor cells were detected in both. Conclusion: We conclude that the clonogenic assay can be utilized to allow early stage cancer to be more accurately staged at diagnosis and have better assessment of residual disease after adjuvant therapy.

Duke Scholars

Author Stephen L. George Biostatistics & Bioinformatics, Division of Biostatistics