Monitoring of disseminated tumor cells in bone marrow in high-risk breast cancer patients treated with high-dose chemotherapy Vilde Drageset 1 , Jahn M. Nesland 2 , Bjorn Erikstein 1 , Eva Skovlund 1 , Hilde Sommer 3 , Gun Anker 4 , Erik Wist 3 , Steinar Lundgren 5 , Jonas Bergh 6 and Gunnar Kvalheim 1 * 1 Laboratory for Cellular Therapy, Department of Medical Oncology and Radiotherapy, The Norwegian Radium Hospital, University of Oslo, Oslo, Norway 2 Department of Pathology The Norwegian Radium Hospital, Oslo, Norway 3 Department of Medical Oncology and Radiotherapy, Ullev ˚ al University Hospital, Oslo; Norway 4 Department of Medical Oncology and Radiotherapy, Haukeland Hospital, Bergen, Norway 5 Department of Oncology, St Olavs Hospital, Trondheim University Hospital, Trondheim, Norway 6 Radiumhemmet, Karolinska Institutet, Stockholm, Sweden The present study aimed to investigate the clinical relevance of disseminated tumor cells (DTC) in breast cancer patients before and after high-dose adjuvant chemotherapy with or without pro- genitor stem-cell support. One hundred and eighteen high-risk stage II breast cancer patients entering the Scandinavian Study Group multicenter trial were randomized to 9 cycles of tailored and dose-escalated FEC (5-flurouracil, epirubicin, cyclophospha- mide) or 3 cycles of standard FEC followed by high-dose chemo- therapy. Bone marrow (BM) samples at diagnosis and 6 months after completion of chemotherapy were assessed for the presence of cytokeratin positive (CK1) cells. Before treatment, 29% of the patients were CK1 (21% in the dose-escalated group and 36% in the high-dose-group). Six months after treatment, 17% of the patients were CK1 (17 and 16% respectively). Of the 95 patients who were evaluated 6 months after treatment, 60% were consis- tently CK2. CK1 cells in BM was evaluated as a prognostic and predictive marker and compared to other defined prognostic fac- tors of the primary tumor. Monitoring BM changes at the time of diagnosis and 6 months posttreatment is an independent predic- tive factor for breast-cancer-specific survival (BCS) (p 5 0.001). Those who have consistent CK negative (2) BM findings consti- tute a group of patients with good prognosis. Our results suggest that changes in CK1 cells in BM before and after chemotherapy can be used clinically as a surrogate maker to predict outcome in breast cancer patients. ' 2005 Wiley-Liss, Inc. Key words: disseminated tumor cells; high-dose chemotherapy; prediction High-dose chemotherapy with stem-cell support has been studied in breast cancer patients with both metastatic and early- stage, high-risk disease. Phase II studies 1,2 suggested that the out- come of patients treated with high-dose therapy was superior to standard-dose chemotherapy. To confirm these results, several large randomized studies have been conducted. Even if a longer follow-up is required until final conclusions can be drawn, the results reported show that progenitor stem-cell-supported high- dose therapy does not appear to be sufficient as tumor reductive therapy. 3–5 Several breast cancer studies 6,7 have shown a significant associ- ation between the detection of tumor cells in the BM, both at diag- nosis and in follow-up, and increased risk of systemic relapse. Both Braun et al. 8 and Wiedswang et al. 9 reported that presence of cytokeratin (CK) staining cells in the BM after adjuvant con- ventional chemotherapy of breast cancer patients was associated with distant metastases and cancer-related death. The Scandinavian Breast Cancer Group (9401 protocol) pub- lished their first results of a randomized adjuvant study in high- risk breast cancer in year 2000. 4 Patients treated with 9 cycles of tailored and dose-escalated high-dose FEC with granulocyte col- ony-stimulating factor (G-CSF) support had significantly fewer breast cancer relapses compared with those treated with 3 cycles of FEC plus cyclophosphamide, thiotepa, carboplatin (CTCb) with autologous stem-cell support (p 5 0.04). No significant difference in overall survival (OS) was demonstrated, nor in overall health related quality of life. 10 From this study, all the Norwegian patients (n 5 128) were asked to participate for further analysis. The aim was to analyze the association between DTC, standard prognostic factors and clinical outcome. Of the 128 Norwegian patients, 11 patients were excluded because of lack of consent for this substudy. The remaining 118 patients were examined for the presence of DTC in the BM before and after therapy, using stand- ardized immunocytochemistry (ICC) and immunomagnetic-based enrichment procedures. 7,11,12 Standard prognostic factors (age, number of positive lymph nodes, hormone receptor status, histo- logic grading) have been registered. Associations between these findings and DTC in the BM are reported. Material and methods Subjects 525 Scandinavian women with high-risk breast cancer were randomized after surgery to receive one of two regimens of high- dose chemotherapy with or without hematopoietic progenitor cell support. 4 The study was initiated in March 1994 and closed in March 1998. Of the 128 women entering the study from Norway, 118 were assessed for the presence of DTC in BM before therapy, and of them, 95 were reexamined 6 months posttreatment. The dif- ference between numbers of subjects at the 2 points in time is due to dropouts and not deaths. The median follow-up at the time of analysis was 68 months. The study included patients with an expected 5-year relapse- free survival (RFS) of about 30% or less and a life expectancy exceeding 3 months. The clinical inclusion criterion was primary breast cancer with either 8 or more nodes positive or 5 or more metastatic axillary nodes combined with receptor negativity and high nuclear grade. A bone scan was mandatory for inclusion to- gether with bilateral Jamshidi biopsies. 4 The subject characteristics are given in Table I. No differences between the study groups with regard to age, number of involved axillary lymph nodes, histology grade or receptor status were observed. *Correspondence to: Laboratory for Cellular Therapy, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway. Fax: 147 22934596. E-mail: gunnar.kvalheim@klinmed.uio.no Received 25 July 2005; Accepted after revision 4 October 2005 DOI 10.1002/ijc.21709 Published online 27 December 2005 in Wiley InterScience (www. interscience.wiley.com). Abbreviations: BCS: breast cancer specific survival; BM: bone marrow, CK1: cytokeratin positive; CK2: cytokeratin negative; CTCb: cyclophos- phamide, thiotepa, carboplatin; DTC: disseminated tumor cells; FEC: 5- flurouracil, epirubicin, cyclophosphamide; G-CSF: granulocyte colony- stimulating factor; ICC: immunocytochemistry; OS: overall survival; PBPC: peripheral blood progenitor cells; RFS: relapse-free survival. Int. J. Cancer: 118, 2877–2881 (2006) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer