Abstract
The vast majority of breast cancer patients present with tumors that are clinically localised to the breast and axillary lymph nodes, due to the wide spread of screening tests. Despite highly efficient locoregional therapy a significant number of patients later experience a systemic recurrence. Α crucial step in the metastasis cascade is the spreading of cancer cells from the primary site to distant parts of the body. This happens via two distinct routes: via lymphatics and then into the blood circulation or directly via the blood circulation. Additionally, it seems that distinct biologic mechanisms are required for each route. The development of metastases is the final step in the natural history of the disease and the definitive confirmation of tumor aggressiveness. Minimal residual disease, in form of disseminated (DTCs) and circulating tumor cells (CTCs), lies between primary tumor evolution and metastasis development. In general, CTCs in peripheral blood preserve basic genotype ch ...
The vast majority of breast cancer patients present with tumors that are clinically localised to the breast and axillary lymph nodes, due to the wide spread of screening tests. Despite highly efficient locoregional therapy a significant number of patients later experience a systemic recurrence. Α crucial step in the metastasis cascade is the spreading of cancer cells from the primary site to distant parts of the body. This happens via two distinct routes: via lymphatics and then into the blood circulation or directly via the blood circulation. Additionally, it seems that distinct biologic mechanisms are required for each route. The development of metastases is the final step in the natural history of the disease and the definitive confirmation of tumor aggressiveness. Minimal residual disease, in form of disseminated (DTCs) and circulating tumor cells (CTCs), lies between primary tumor evolution and metastasis development. In general, CTCs in peripheral blood preserve basic genotype characteristics of the primary tumor. Nevertheless, these cells acquire certain genetic abnormalities that permit them to detach from the primary tumour, migrate through the basal membrane and the extracellular matrix, penetrate the surrounding vessels and translocate into a new site. The clinical significance of detection of CTCs in peripheral blood of the early breast cancer patient is still uncertain in contrast to bone marrow DTCs. The purpose of this study was to investigate the prognostic and predictive value of the detection of CTCs, as well as the influence of adjuvant chemotherapy and hormonotherapy on these cells. For the detection of CTCs we used a quantitative real-time PCR approach because of its sensitivity. Cytokeratin-19 mRNA (CK-19mRNA) was chosen as molecular marker for identification of CTCs, because of its higher sensitivity and specificity compared to other markers for the recognition of epithelial tumor cells in mesenchymal tissues such as blood. From February ’97 until December ’04, 450 patients with early breast cancer were enrolled in study. Peripheral blood before and after systemic chemotherapy was obtained from all 450 patients, as well as during follow-up. All blood samples were examined for CK-19mRNA(+) cells with real-time PCR. Before any systemic treatment CK-19mRNA(+) cells were detected in 183 (40.7%). Among the examined clinicopathological factors, menopausal status was the only variable that was associated, but not statistical significant, with the detection of CTCs before adjuvant therapy. Primary tumors in premenopausal patients reveal CTCs in circulation, more frequently than tumors in postmenopausal patients, while there was a reverse relation between menopausal status and lymph node status. This inverse correlation of detection of CTCs and lymph node involvement with menopausal status is in accordance with the hypothesis that distinct, independent pathways are used for hematogenic and lymphatic malignant cell spread. After completion of chemotherapy, CK-19mRNA(+) cells were detected in 147 (32.7%) patients’ blood samples. Detection of CK-19mRNA(+) cells was significantly associated only with the number of positive lymph nodes (Ν>3, p=0.007) but not with other patients’ or tumors’ clinicopathologic characteristics. CK-19mRNA(+) cells were detected in 42% of patients with more than 3 positive lymph nodes (Ν>3), while in patients without or with 3 or less positive lymph nodes (Ν0-3), only 29% had detectable cells after chemotherapy. Chemotherapy (T/EC, FEC or CMF) achieved elimination of circulating in 51,4% of patients. Different treatment regimens didn’t show any significant difference in their efficiency to eradicate positive cells from blood. The T/EC regimen was slightly superior than FEC or CMF (55%, 46% and 57%, respectively). In respect of measured CK-19mRNA(+) cells before and after chemotherapy, T/EC statistically significant reduced more efficiently the number of measured cells, compared to FEC or CMF. Chemoresistance of CTCs, which was more pronounced in patients with disease Ν>3 and T2,3 or PR(+) tumors, may be due to the fact that numerous of these cells are in a quiescent, non-cycling stage and overexpress genes that prevent apoptosis. Adjuvant hormonotherapy (anti-estrogens, aromatase inhibitors or sequential administration) eradicated CK-19mRNA(+) cells in 65% of post-chemotherapy positive blood samples. Moreover, aromatase inhibitors succeeded in the elimination of cells in 65% of samples that had remained persistently positive during administration of anti-estrogens. Hormonotherapy was more efficacious in patients with ER(+)/PR(-) tumors than in those with ER(+)/PR(+) or ER(-)/PR(+) tumors and overexpression of HER2/neu reduced the effectiveness of anti-estrogens. Patients with detectable CK-19mRNA(+) cells before chemotherapy relapsed and died because of disease progression more frequently than patients without detectable cells (31% vs 16%, p=0,0001 and 15% vs 6%, p=0,001, respectively) and they also displayed reduced disease free interval and overall survival (78 vs 92 months, p<0,00001 and 94 vs 101 months, p=0,0001). Furthermore, the median number of CK-19 mRNA-positive CTCs in patients who recurred was significant higher than in patients who did not (p=0,02). The influence of positive cell detection on recurrence probability was not significant only in patients with Τ1, or ER+ tumors. In the multivariate analysis, the presence of CK-19mRNA(+) CTCs before chemotherapy, T2,3 tumor size, involvement of more than 3 axillary lymph nodes, radiotherapy omitting and anti-estrogens administration were all found to be independent adverse prognostic factors for early relapse. Detection of CK-19mRNA(+), involvement of more than 3 axillary lymph nodes and anti-estrogens administration were found to be independent prognostic factors for reduced overall survival. In a separate analysis for node-negative patients, we found that patients with detectable CK-19mRNA(+) cells relapsed and died from disease more frequently than patients without positive cells. In the multivariate analysis, the presence of CK-19mRNA(+) cells before chemotherapy, ER negativity, histology grade III, postmenopausal status and CMF administration were independent prognostic factors for early relapse, while only the detection of circulating cells was independent prognostic factor for reduced overall survival. The adverse influence of detectable CK-19mRNA(+) cells in disease recurrence persisted after completion of chemotherapy also. Risk for relapse was higher for patients with positive cells before and after chemotherapy (38%) and lower for those who were always negative for detectable cells (14%). The same association was found between risk of death from disease progression and detection of CK-19mRNA(+) cells (19% and 4%). Disease free interval and overall survival were considerable reduced in patients with detectable cells before and after chemotherapy than in patients without such cells (72 vs 94 months and 90 vs 102 months, respectively). Independent prognostic factors for early relapse were CK-19mRNA(+) cells detection after chemotherapy, T2,3 tumor size, involvement of more than 3 axillary lymph nodes, radiotherapy omitting and anti-estrogens administration. Only the presence of CK-19mRNA(+) cells after completion of chemotherapy and more than 3 involved axillary lymph nodes were found to be independent prognostic factors for reduced overall survival. Presence of CK-19mRNA(+) cells during hormonotherapy correlated with increased risk of relapse and death from disease progression, as well as with reduced disease free interval and overall survival. Additionally, disease free interval was significant higher in premenopausal patients who received anti-estrogens and in postmenopausal patients who received aromatase inhibitors. In conclusion, detection of CK-19mRNA(+) cells in peripheral blood with real-time PCR is a sensitive and reliable method for identification of circulating tumor cells in patients with early breast cancer. Adjuvant chemotherapy and hormonotherapy failed to eliminate soundly cells from circulation. Moreover, the presence of CK-19mRNA(+) cells before and after chemotherapy and during hormonotherapy is an independent adverse prognostic factor for reduced disease free interval and overall survival. The ability to detect circulating tumor cells opens interesting clinical perspectives. Potential applications include more precise estimation of prognosis and necessity of adjuvant systemic therapy, earlier assessment of the efficacy of chemotherapy and disease progression.
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