Prognostic relevance of induced and spontaneous apoptosis of disseminated tumor cells in primary breast cancer patients

Between 10 and 20 ml of BM were aspirated from the anterior iliac crest and processed within 24 hours. All specimens were obtained after written informed consent from patients. BM samples were separated by density centrifugation over Ficoll (Biochrom, Germany) with a density of 1,077 g/ml. 10⁶ mononuclear cells were spun onto a glass slide using a cytocentrifuge (Hettich, Tuttlingen, Germany). Slides were than fixed in a 4% neutral buffered formalin solution for 10 minutes and were rinsed in phosphate-buffered saline. Automatic immunostaining was performed on the DAKO autostainer using the monoclonal mouse A45–B/B3 antibody (Micromet, Munich, Germany), and the DAKO-APAAP detection kit (DakoCytomation, Glostrup, Denmark) according to the manufacturers’ instructions. For each patient, 2 × 10⁶ cells were analyzed. Slides were automatically scanned using the ACIS™ imaging system (ChromaVision, Medical Systems Inc., San Juan, Capistrano, CA, USA) and evaluated based on the recommendations for standardized tumor cell detection and the criteria of the European ISHAGE Working group [13, 14]. The MCF-7 cell line served as a positive control. Leukocytes from healthy volunteers were used as a negative control. All BM specimens were evaluated qualitatively, as positive and negative for DTC.

383 primary breast cancer patients with A45-B/B3 positive DTC in BM were included into this study. In order to evaluate the apoptotic status of DTC in this group, additional BM slides were stained using the M30 antibody (Roche Applied Science, Mannheim, Germany) and analyzed by use of the APAAP kit detection method as described above. The M30 antibody reacts with a neo-epitope expressed only after caspase cleavage of cytokeratin 18 during early apoptosis [15]. M30 antibody does not bind intact, full-length cytokeratin 18 in viable or necrotic cells and can, therefore, be used specifically to recognize apoptotic cells [16]. Identification of apoptotic DTC was based on positive M30 staining and cytomorphological criteria as described elsewhere [17‐19]. MCF-7 cells treated with sodium azide served as a positive control; untreated MCF-7 cell line and leukocytes from healthy volunteers were used as a negative control. Figures 1 and 2 show M30 and pan-cytokeratin staining.

A total of 383 DTC positive breast cancer patients were included in this study. Two-hundred ninety-eight patients underwent breast surgery first. 204 out of 298 patients (68%) had T1 tumors and 214 (71%) were node negative. The most common histological tumor type was invasive ductal carcinoma. Estrogen and progesterone receptor status were positive in 85% and 82% of these patients, respectively. Fifty-two of 284 (18%) patients had HER2 positive tumors. Clinical data of this group are summarized in Table 1. Eighty-five patients were treated with PST. The majority of these patients was premenopausal (48 of 85 cases). 22% achieved pathological complete response after chemotherapy while 53% responded partially. Stable disease was observed in 19% of patients whereas 6% of patients (5 cases) developed progressive disease (Table 3). Clinical data of patients treated with PST are summarized in Table 2.

In eighty-two of 298 (27%) patients with pan-cytokeratin positive DTC in BM who underwent primary surgery additional apoptotic DTC could be detected. No correlation could be found between positive M30 status and any established prognostic factors, including tumor size, lymph node status, hormone receptor status or grading.

Forty-one of 85 (48%) patients had additional M30 positive DTC after completion of PST. Patients with apoptotic DTC were less likely to have nodal metastasis (35% vs. 64%; p = 0.007). No significant correlation could be observed between the positive M30 status of DTC and other clinicopathological factors. The presence of apoptotic DTC was associated with response to PST. M30 positive cells were found in 63% of patients with complete remission, 53% with partial remission and 31% with stable disease, respectively. None of the patients with progressive disease had M30 positive DTC (p = 0.034; Table 3).

The median follow-up was 44 months (range: 10–88 months). 32 of 383 patients were diagnosed with relapse (either local recurrence or distant metastasis) and 28 died during follow-up. Clinical outcome data are summarized in Table 4.

Thirteen of 85 neoadjuvant patients (15%) presented with relapse during follow-up. Patients with additional M30 positive DTC were less likely to suffer from relapse than patients with only non-apoptotic DTC (7% vs. 23%; p = 0.049). However, the association between disease-free interval and M30 status of DTC assessed by Kaplan-Meier analysis did not reach statistical significance (p = 0.128; 70 months, 95% CI: 63–78 months vs. 81 months, 95% CI: 74–87 months). Seven of 85 neoadjuvant patients (8%) died during follow-up. No correlation could be found between M30 status of DTC and overall survival in this group of patients.

Twenty-one out of 298 (7%) patients in this group died during follow-up. The overall survival was significantly shorter among patients with M30 positive DTC as compared to M30 negative patients (75 months, 95% CI: 68–81 months vs. 84 months, 95% CI: 81–86 months; p = 0.008). However, there was no association between disease free survival and M30 status (83 months in M30 positive patients, 95% CI: 80–85 months vs. 78 months in M30 negative patients, 95% CI: 73–84 months; p > 0.05). Figure 3 shows the Kaplan-Meier analysis of overall survival in patients who underwent primary surgery.

M30 antigen is an early apoptotic marker of epithelial cells, detectable after caspase cleavage of cytokeratin 18. This antibody was used in our study to assess the apoptotic status of DTC in 298 patients who received no treatment prior to surgery. Patients with apoptotic DTC had a significantly shorter overall survival compared to patients with only non-apoptotic DTC (75 months vs. 84 months, p < 0.008). This seems not conclusive, since apoptotic DTC are generally assumed to result from a phenomenon described as "metastatic inefficiency". According to this conceptual model, only a small percentage of tumor cells are able to survive and persist at secondary homing sites. Large numbers of cancer cells are shed from the primary site into the systemic circulation; only a small subset will give rise to overt metastases. To successfully reach and colonize a secondary site, a tumor cell must complete a series of steps (metastatic cascade): migration from the primary tumor, intravasation into the blood stream, survival of the vigorous passage in blood, extravasation, and development of micrometastases in distant organs [20]. Failure in any one of these steps leads to elimination of tumor cells; 99,9% of shed cells are thought to perish during the process, while only a minor subpopulation attains metastatic capacity [21]. The key regulatory points that contribute to metastatic inefficiency remain unclear; initiation of apoptosis has been assumed to be a major component of this mechanism. Yet, in our study apoptotic status of DTC in bone marrow in untreated patients resulted in significantly worse survival.

In the past decades, major research efforts have been conducted to study the role of apoptosis in the primary tumor [10, 22]. The association between apoptosis rates and cell proliferation is well established; increased apoptosis reflects a high cell turnover in the tumor. In breast cancer, high levels of apoptosis correlate with enhanced cellular proliferation and biological markers of increased malignancy, such as negative hormone receptor status, high histological grade, HER2 overexpression, positive lymph nodes, tumor aneuploidy and a decreased expression of bcl-2 protein [10, 23, 24]. Furthermore, high apoptotic counts are associated with shortened disease-free and overall survival [9, 10, 24]. Similar observations were made in other solid tumors, including prostate and bladder cancer [25, 26]. These data seem to disprove the concept that the elimination of apoptosis in tumor cells is a necessary condition for autonomous uncontrolled cancer growth. On the contrary, an enhanced rate of spontaneous apoptosis in the primary tumor is an indicator of high proliferation and negative prognostic markers.

Over the last two decades, neoadjuvant systemic therapy (NST) has become the standard treatment strategy for locally advanced breast cancer, conducted primarily to enhance the possibility of breast-conserving surgery [27]. Beyond this indication NST offers the additional possibility to test in vivo the chemosensitivity of the primary tumor [28]. The pathological response to NST is associated with favorable clinical outcome and is considered by some as a surrogate marker for complete eradication of micrometastatic disease. However, up to 25% of patients who achieve complete pathological remission will suffer relapse within five years of diagnosis, suggesting subclinical persistence of isolated tumor cells beyond systemic treatment.

We reported previously a high incidence of persistent DTC after NST [3]. Since apoptosis is the main mechanism of chemotherapy-induced disease regression [29], we aimed to assess the apoptotic status of DTC in patients who received neoadjuvant therapy. In 41 out of 85 (48%) DTC positive patients additional apoptotic tumor cells were detected after therapy. Compared to patients with only viable DTC, patients with apoptotic DTC responded better to therapy, reaching complete or partial remission in 88% (vs. 64% in the M30-negative group, p = 0.034). Moreover, additional apoptotic cells in BM were significantly associated with negative nodal status after completion of neoadjuvant treatment but not with any other clinicopathological factor. Pathological response of the primary tumor and lymph nodes metastases was therefore reflected by changes in tumor cells in secondary sites, such as bone marrow. Further, patients with positive M30-status were less likely to suffer from a relapse (p = 0.049). This is in accordance with clinical studies: patients who achieve complete pathological response to neoadjuvant treatment regime perform favorably with regard to overall and disease-free survival [30]. In addition, patients converted to node negative disease after neoadjuvant treatment have high survival rates, despite residual tumor in the breast [31]. These data indicate that in these patients apoptotic tumor cells are associated with therapy response.