- Email: [email protected]
Prognostic impact and therapeutical options for breast cancer patients with micrometastatic disease
54
THURSDAY,
SEPTEMBER
FC4.17.07 A STUDY OF APOmOSIS IN NORMAL BREAST TISSUE IN TAMOXIFEN-TREATED PREMENOPAUSAL WOMEN WITH FIBROADENOMA L.H. Gebrim, M.T. Seixas, E.C. Baracat, G.R. Lima, Dept. of
FC4.17.09 PROGNOSTIC IMPACT AND THERAPEUTICAL OmIONS FOR BREAST CANCER PATIENTS WITH MICROMETASTATIC DISEASE. I.J. E.F. Solomayer, Ch. Gollan, F. Schiitz, G. Bastert. Dept.
Gynecology,
OBIGYN, University Hospital, Voss-Str. 9, 69115 Heidelberg, Germany.
University Federal of SBo Paula, SBo Paula, Brazil.
Objectives: Tamoxifen (TAMi20mgiday) has been used in breast cancer chemoprevention and exerts a long-term suppressive effect on human breast cancer cell proliferation. The aim of the study is to determine the apoptosis rate in the normal human breast epithelium adjacent to fibroadenoma during TAM treatment (10 and 20 mgiday). Study Methods: We evaluated a group of 40 premenopause fibroadenoma patients during 22 days of therapy. By using a doubleblind randomized study patients were divided according to the following groups: Group I - 14 patients used as a control group. Group II - 13 patients receiving 10 mg of TAM/day and Group III - 13 patients receiving 20 mg TAM/day. Treatment was started on the first day of menstrual cycle and biopsies were performed on the 22”d day of therapy. Serum levels of estradiol, progesterone, prolactin, FSH, LH and SHBG were measured on the 22”d of the last cycle as well as on the biopsy day. Mammary samples were immediately fixed on 10% buffered formalin and included in paraffin. H.E. staining was performed in order to quantitate the number of apoptotic cells in 10 different fields at 400x magnification. Results: The mean number of apoptotic cells in group I was 34.8 cells. Group II (10 mgiday) 12.9 and group III (20 mgiday) 12.0 apoptotic cells. Statistical analysis showed significant reductions in groups II and III when compared to the control group. On the other hand, there were no differences between groups II and III (Fisher’s test p
FC4.17.08 TUMOR CELLS IN BONE MARROW OF BREAST CANCER PATIENTS WITH LOCOREGIONAL RECURRENCE OR DISTANT METASTASES W. S. Gastroph, F. Hepp, Ch. Kentenich, D. Rjosk, Ch.
Schindlbeck, H. Sommer, S. Braun. I. Universitaetsfrauenklinik, Klinikum Innenstadt, Munich, Germany Objectives: Using cytokeratin (CK) as histogenetic marker to identify tumor cells in bone marrow (BM) of breast cancer (BC) patients, a subgroup of patients with poor clinical outcome can be identified. This study was designed to evaluate the frequency and prognostic relevance of such cells in BC-patients at the time of either locoregional or distant relapse. Study Methods: BM-aspirates from 65 consecutive patients with either locoregional recurrence (n=32) or distant metastases (n=33) were analysed immunocytochemically for the presence of cytokeratin(CK)positive cells. We used a quantitative immunoassay with monoclonal antibody A45B/B3 and evaluated 2 x106 bone marrow cells per patient. For prognostic evaluation a cut off level of the number of detected tumor cells was calculated in analogy to classification and regression tree (CART) analysis. Patients were monitored prospectively for a median of 32 months. Results: BM-micrometastases were present in 5 of 32 patients (15.6 %) with locoregional recurrence, while significantly more patients (24 of 33 patients [72.7 %]) had positive BM-results. No prognostic relevance was found in patients with locoregional recurrence. Significant separation of prognosis for patients with distant disease was found at a cut off level of 5 CK-positive cells per 2 x 106 screened BM-cells. The mean overall survival in patients with higher micrometastatic tumor load was 5.6 months (2.0.9.1,95 % CI) compared to 16.8 months (11.6.22.0,95 % CI) in patients with 5 or less cells (P= .0004). Multivariate analysis allowing for hormone receptor status, disease free interval prior to recurrence, manifestation site of metastases, age and micrometastases in BM, revealed BM-status to be an independent risk factor for reduced overall survival (RR 4.66 [1.63-13.32,95 % CI]). Conclusions: Number of micrometastases present in BM of patients with metastatic BC represents an independent prognostic indicator with potential influence on therapeutic strategies.
Objectives: The detection of disseminated tumor cells in the bone marrow of patients with breast cancer is associated with a poorer prognosis. These cells are also the focus of new treatment modalities. Because detection methods are not only diverse but have also usually been tested in only small groups, our aim was to investigate a detection method in a large number of patients under standardized conditions. Study Methods: Intraoperative, bilateral iliac crest biopsies were performed in 1338 patients with primary breast cancer. The aspirated bone marrow was subjected to differential centrifugation, smeared onto slides and stained with a monoclonal antibody that recognizes the MUC1 gene and targets the tumor-associated glycoprotein TAG 12. Patients underwent follow-up examinations at regular intervals. The results were statistically evaluated. Results: After a median follow-up period of 56 months, distant metastasis was observed in 368 patients (28%). 225 (61%) were tumorcell positive at the time of surgery. Of the 238 patients who died 161 (68%) were positive. In a multivariate Cox regressions analysis tumor cell detection was by far the best prognostic factor in patients with small breast tumors (Tl). In women with tumors larger than 2 cm nodal status and tumor cell detection had the same prognostic value. Conclusions: Our investigations show that the dissemination marker micrometastasis has a greater important for the prognosis of the disease in women with small breast tumors than the classic prognostic factors. In this group of patients it might be better to dispense with axillary lymphadenectomy in favor of tumor cell detection. The goal must be to destroy these individual cells in the bone marrow, which are not accessible to standard cytotoxic treatments, by means of a new therapeutic modality (immunotargeting, gene therapy, bisphosphonates).
FC4.17.10 ANTI-HER-2/NEU ANTIBODY INDUCES APOPTOSIS IN HER-YNEU OVEREXPRESSING BREAST CANCER CELLS INDEPENDENT FROM INTACT P53 T. Brodowicz’, s. Tomek’, d. Kandioler-eckersberger3, m. Rudas“, w.j.
Kestler’, c. Ludwig, a. Budinsky’, m. Hejna’, m. Krainer’, c. Wiltschke I, C.C. Zielinski’,z,5 ‘Clinical Division of Oncology and ‘Chair of Medical Experimental Oncology, Department of Medicine I, 3Department of Surgery and “Department of Clinical Pathology, University Hospital, and ‘Ludwig Boltzmann Institute for Clinical Experimental Oncology, Vienna, Austria. Objectives: Anti-HER-2/neu antibody is known to induce apoptosis in HER-2/neu overexpressing breast cancer cells. However, exact regulatory mechanisms mediating and controlling this phenomenon are still unknown. Methods: In the present study, we have investigated the effect of antiHER-2/neu antibody on apoptosis of HER-2/neu overexpressing human breast cancer cell lines SK-BR-3, HTB-24, HTB-25, HTB-27, HTB-128, HTB-130 and HTB-131 in relation to ~53 genotype and bcl-2 status. HER-2/neu density and bcl-2 expression were determined by flow cytometry, ~53 status by sequence analysis and apoptosis was measured flow cytometrically using a DNA fragmentation assay. Results: SK-BR-3, HTB-24, HTB-128 and HTB-130 cells exhibited mutant ~53, whereas wild type ~53 was found in HTB-25, HTB-27 and HTB-131 cells. All seven native HER-2/neu positive cell lines (SK-BR3, HTB-24, HTB-25, HTB-27, HTB-128, HTB-130, HTB-131) weakly expressed bcl-2 protein (10.20%). Anti-HER-2/neu antibody, irrespective of ~53 and bcl-2 status, induced apoptosis in all 7 cell lines dose-and time-dependently. Incubation of the cell lines with anti-HER2/neu antibody did not alter ~53 or bcl-2 status. Furthermore, anti-HER2/neu antibody did not induce apoptosis in HER-2/neu negative HBL100 and HTB-132 cell lines. Conclusion: Our results indicate that within the panel of tested breast cancer cell lines, anti-HER-2/neu antibody induced apoptosis seems to be independent of the presence of intact ~53.
7
THURSDAY,
SEPTEMBER
FC4.17.07 A STUDY OF APOmOSIS IN NORMAL BREAST TISSUE IN TAMOXIFEN-TREATED PREMENOPAUSAL WOMEN WITH FIBROADENOMA L.H. Gebrim, M.T. Seixas, E.C. Baracat, G.R. Lima, Dept. of
FC4.17.09 PROGNOSTIC IMPACT AND THERAPEUTICAL OmIONS FOR BREAST CANCER PATIENTS WITH MICROMETASTATIC DISEASE. I.J. E.F. Solomayer, Ch. Gollan, F. Schiitz, G. Bastert. Dept.
Gynecology,
OBIGYN, University Hospital, Voss-Str. 9, 69115 Heidelberg, Germany.
University Federal of SBo Paula, SBo Paula, Brazil.
Objectives: Tamoxifen (TAMi20mgiday) has been used in breast cancer chemoprevention and exerts a long-term suppressive effect on human breast cancer cell proliferation. The aim of the study is to determine the apoptosis rate in the normal human breast epithelium adjacent to fibroadenoma during TAM treatment (10 and 20 mgiday). Study Methods: We evaluated a group of 40 premenopause fibroadenoma patients during 22 days of therapy. By using a doubleblind randomized study patients were divided according to the following groups: Group I - 14 patients used as a control group. Group II - 13 patients receiving 10 mg of TAM/day and Group III - 13 patients receiving 20 mg TAM/day. Treatment was started on the first day of menstrual cycle and biopsies were performed on the 22”d day of therapy. Serum levels of estradiol, progesterone, prolactin, FSH, LH and SHBG were measured on the 22”d of the last cycle as well as on the biopsy day. Mammary samples were immediately fixed on 10% buffered formalin and included in paraffin. H.E. staining was performed in order to quantitate the number of apoptotic cells in 10 different fields at 400x magnification. Results: The mean number of apoptotic cells in group I was 34.8 cells. Group II (10 mgiday) 12.9 and group III (20 mgiday) 12.0 apoptotic cells. Statistical analysis showed significant reductions in groups II and III when compared to the control group. On the other hand, there were no differences between groups II and III (Fisher’s test p
FC4.17.08 TUMOR CELLS IN BONE MARROW OF BREAST CANCER PATIENTS WITH LOCOREGIONAL RECURRENCE OR DISTANT METASTASES W. S. Gastroph, F. Hepp, Ch. Kentenich, D. Rjosk, Ch.
Schindlbeck, H. Sommer, S. Braun. I. Universitaetsfrauenklinik, Klinikum Innenstadt, Munich, Germany Objectives: Using cytokeratin (CK) as histogenetic marker to identify tumor cells in bone marrow (BM) of breast cancer (BC) patients, a subgroup of patients with poor clinical outcome can be identified. This study was designed to evaluate the frequency and prognostic relevance of such cells in BC-patients at the time of either locoregional or distant relapse. Study Methods: BM-aspirates from 65 consecutive patients with either locoregional recurrence (n=32) or distant metastases (n=33) were analysed immunocytochemically for the presence of cytokeratin(CK)positive cells. We used a quantitative immunoassay with monoclonal antibody A45B/B3 and evaluated 2 x106 bone marrow cells per patient. For prognostic evaluation a cut off level of the number of detected tumor cells was calculated in analogy to classification and regression tree (CART) analysis. Patients were monitored prospectively for a median of 32 months. Results: BM-micrometastases were present in 5 of 32 patients (15.6 %) with locoregional recurrence, while significantly more patients (24 of 33 patients [72.7 %]) had positive BM-results. No prognostic relevance was found in patients with locoregional recurrence. Significant separation of prognosis for patients with distant disease was found at a cut off level of 5 CK-positive cells per 2 x 106 screened BM-cells. The mean overall survival in patients with higher micrometastatic tumor load was 5.6 months (2.0.9.1,95 % CI) compared to 16.8 months (11.6.22.0,95 % CI) in patients with 5 or less cells (P= .0004). Multivariate analysis allowing for hormone receptor status, disease free interval prior to recurrence, manifestation site of metastases, age and micrometastases in BM, revealed BM-status to be an independent risk factor for reduced overall survival (RR 4.66 [1.63-13.32,95 % CI]). Conclusions: Number of micrometastases present in BM of patients with metastatic BC represents an independent prognostic indicator with potential influence on therapeutic strategies.
Objectives: The detection of disseminated tumor cells in the bone marrow of patients with breast cancer is associated with a poorer prognosis. These cells are also the focus of new treatment modalities. Because detection methods are not only diverse but have also usually been tested in only small groups, our aim was to investigate a detection method in a large number of patients under standardized conditions. Study Methods: Intraoperative, bilateral iliac crest biopsies were performed in 1338 patients with primary breast cancer. The aspirated bone marrow was subjected to differential centrifugation, smeared onto slides and stained with a monoclonal antibody that recognizes the MUC1 gene and targets the tumor-associated glycoprotein TAG 12. Patients underwent follow-up examinations at regular intervals. The results were statistically evaluated. Results: After a median follow-up period of 56 months, distant metastasis was observed in 368 patients (28%). 225 (61%) were tumorcell positive at the time of surgery. Of the 238 patients who died 161 (68%) were positive. In a multivariate Cox regressions analysis tumor cell detection was by far the best prognostic factor in patients with small breast tumors (Tl). In women with tumors larger than 2 cm nodal status and tumor cell detection had the same prognostic value. Conclusions: Our investigations show that the dissemination marker micrometastasis has a greater important for the prognosis of the disease in women with small breast tumors than the classic prognostic factors. In this group of patients it might be better to dispense with axillary lymphadenectomy in favor of tumor cell detection. The goal must be to destroy these individual cells in the bone marrow, which are not accessible to standard cytotoxic treatments, by means of a new therapeutic modality (immunotargeting, gene therapy, bisphosphonates).
FC4.17.10 ANTI-HER-2/NEU ANTIBODY INDUCES APOPTOSIS IN HER-YNEU OVEREXPRESSING BREAST CANCER CELLS INDEPENDENT FROM INTACT P53 T. Brodowicz’, s. Tomek’, d. Kandioler-eckersberger3, m. Rudas“, w.j.
Kestler’, c. Ludwig, a. Budinsky’, m. Hejna’, m. Krainer’, c. Wiltschke I, C.C. Zielinski’,z,5 ‘Clinical Division of Oncology and ‘Chair of Medical Experimental Oncology, Department of Medicine I, 3Department of Surgery and “Department of Clinical Pathology, University Hospital, and ‘Ludwig Boltzmann Institute for Clinical Experimental Oncology, Vienna, Austria. Objectives: Anti-HER-2/neu antibody is known to induce apoptosis in HER-2/neu overexpressing breast cancer cells. However, exact regulatory mechanisms mediating and controlling this phenomenon are still unknown. Methods: In the present study, we have investigated the effect of antiHER-2/neu antibody on apoptosis of HER-2/neu overexpressing human breast cancer cell lines SK-BR-3, HTB-24, HTB-25, HTB-27, HTB-128, HTB-130 and HTB-131 in relation to ~53 genotype and bcl-2 status. HER-2/neu density and bcl-2 expression were determined by flow cytometry, ~53 status by sequence analysis and apoptosis was measured flow cytometrically using a DNA fragmentation assay. Results: SK-BR-3, HTB-24, HTB-128 and HTB-130 cells exhibited mutant ~53, whereas wild type ~53 was found in HTB-25, HTB-27 and HTB-131 cells. All seven native HER-2/neu positive cell lines (SK-BR3, HTB-24, HTB-25, HTB-27, HTB-128, HTB-130, HTB-131) weakly expressed bcl-2 protein (10.20%). Anti-HER-2/neu antibody, irrespective of ~53 and bcl-2 status, induced apoptosis in all 7 cell lines dose-and time-dependently. Incubation of the cell lines with anti-HER2/neu antibody did not alter ~53 or bcl-2 status. Furthermore, anti-HER2/neu antibody did not induce apoptosis in HER-2/neu negative HBL100 and HTB-132 cell lines. Conclusion: Our results indicate that within the panel of tested breast cancer cell lines, anti-HER-2/neu antibody induced apoptosis seems to be independent of the presence of intact ~53.
7