Prognostic relevance of induced and spontaneous apoptosis of disseminated tumor cells in primary breast cancer patients
- Natalia Krawczyk†1,
- Andreas Hartkopf†2,
- Malgorzata Banys1,
- Franziska Meier-Stiegen1,
- Annette Staebler3,
- Markus Wallwiener4,
- Carmen Röhm2,
- Juergen Hoffmann1,
- Markus Hahn2 and
- Tanja Fehm1Email author
© Krawczyk et al.; licensee BioMed Central Ltd. 2014
Received: 28 December 2013
Accepted: 20 May 2014
Published: 3 June 2014
An imbalance between cell proliferation and programmed cell death can result in tumor growth. Although most systemic cytotoxic agents induce apoptosis in tumor cells, a high apoptotic rate in primary breast cancer correlates with poor prognosis. The aim of this study was to investigate the incidence and the prognostic significance of apoptotic disseminated tumor cells (DTC) in the bone marrow (BM) of breast cancer patients who either underwent primary surgery or primary systemic chemotherapy (PST).
A total of 383 primary breast cancer patients with viable DTC in the BM were included into this study. Eighty-five patients were initially treated with primary systemic chemotherapy whereas 298 patients underwent surgery first. Detection of apoptotic DTC were performed by immunocytochemistry using the M30 antibody which detects a neo-epitope expressed after caspase cleavage of cytokeratin 18 during early apoptosis. The median follow up was 44 months (range 10–88 months).
Eighty-two of 298 (27%) primary operated patients and 41 of 85 (48%) patients treated with primary systemic systemic therapy had additional apoptotic DTC (M30 positive). In the neoadjuvant group M30-positive patients were less likely to suffer relapse than those without apoptotic DTC (7% vs. 23% of the events, p = 0.049). In contrast, the detection of apoptotic DTC in patients treated by primary surgery was significantly associated with poor overall survival (5% vs. 12% of the events, p = 0.008).
Apoptotic DTC can be detected in breast cancer patients before and after systemic treatment. The presence of apoptotic DTC in patients with PST may be induced by the cytotoxic agents. Thus, both spontaneous and chemotherapy-induced apoptosis may have different prognostic significance.
KeywordsApoptosis M30 Breast cancer Survival Disseminated tumor cell
30-40% of primary breast cancer patients present with disseminated tumor cells (DTC) at the time of diagnosis and the detection of these cells in the bone marrow has been shown to be a strong independent prognostic factor for disease free and overall survival . Furthermore, DTC are able to survive systemic treatment and their persistence is associated with a poor outcome . However, not all of these patients develop distant metastatic disease during follow-up suggesting that the majority of detected DTC has a short half-life and not the capability to induce tumor growth at secondary sites ("metastatic inefficiency"). Beyond mere detection of DTC, it is therefore important to further characterize these cells with respect to their phenotype and apoptotic status .
Apoptosis is a strongly regulated process that occurs in biological organisms leading to destruction of individual cells [4, 5]. The role of this programmed cell death in oncogenesis has been intensively investigated in the past two decades. Since the survival of genetically altered cells results in carcinogenesis, an inadequate ratio of apoptosis leads to uncontrolled cell proliferation and thereby tumor growth. This is considered to be a result of mutations in oncogenes which are responsible for the regulation of apoptosis, including BCL-2, C-MYC and P53 [6–8]. Paradoxically, several studies have shown that a high ratio of apoptotic cells in untreated primary breast cancer generally correlates with increased cell proliferation, negative hormonal status, high grading and thus with a poor clinical outcome [8–10]. Whether this phenomenon is restricted to primary tumor or if it takes place in DTC remains to date unclear.
In contrast, chemotherapeutic agents can induce apoptosis of tumor cells leading to disease regression. This process can be explored in vivo in patients treated with PST . We have previously reported that PST may induce apoptosis not only in primary tumor, but in DTC as well .
The purpose of this study was to investigate the incidence and prognostic significance of apoptotic DTC in two different subgroups of primary breast cancer patients: 1) patients who underwent surgery first and 2) patients treated with PST.
Clinical data for patients who underwent primary surgery
M30 positive DTC
N = 298
Clinical data for patients who underwent neoadjuvant therapy
M30 positive DTC
N = 85
Collection and analysis of BM
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.
The chi-squared test was used to evaluate the association between apoptotic DTC and clinicopathological factors. Statistical analysis was performed by SPSS (version 19). Values of p < 0.05 were considered statistically significant. Survival intervals were measured from the time of BM biopsy until death or the first diagnosis of relapse. Relapse was defined as either local recurrence or distant metastasis. Survival was calculated using Kaplan-Meier method and compared by the log-rank test.
Apoptotic DTC and clinical response to neoadjuvant chemotherapy
M30 positive DTC
Presence of apoptotic DTC in patients treated with primary surgery
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.
Presence of apoptotic DTC in patients treated with PST
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).
Survival analysis of patients depending on M30 status of DTC
Patients with primary surgery
Patients with NST
(M30 positive vs M30 negative)
Relapse free survival*
(M30 positive vs M30 negative)
Survival analysis of neoadjuvant patients
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.
Survival analysis of patients treated with primary surgery
The presence of DTC in BM of patients with primary breast cancer is an independent prognostic factor associated with poor clinical outcome . Although this phenomenon can be seen in 30-40% of breast cancer patients, only a minority of DTC positive patients will develop distant metastasis in course of disease ("metastatic inefficiency").
The aim of the present study was to evaluate the incidence and prognostic relevance of apoptotic DTC in breast cancer patients. We focused on two subsets of patients: 1) untreated patients whose bone marrow status was assessed at the time of primary surgery, and 2) pre-treated patients after completion of neoadjuvant cytotoxic therapy.
Clinical relevance of apoptotic DTC in untreated patients at time of 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 . 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 . 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.
Clinical relevance of therapy-induced apoptosis in DTC
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 . Beyond this indication NST offers the additional possibility to test in vivo the chemosensitivity of the primary tumor . 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 . Since apoptosis is the main mechanism of chemotherapy-induced disease regression , 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 . In addition, patients converted to node negative disease after neoadjuvant treatment have high survival rates, despite residual tumor in the breast . These data indicate that in these patients apoptotic tumor cells are associated with therapy response.
Although the observations made in primary tumors cannot be directly extrapolated to DTC in secondary sites, our data suggest that high level of spontaneous apoptosis in minimal residual disease (MRD) is an indicator of poor prognosis. Hypothetically, if the presence of apoptotic DTC reflects an active status of MRD; accordingly, dormant (inactive) tumor cells would appear as non-apoptotic. In contrast, therapy-induced apoptosis of DTC was correlated to pathological response of the tumor and may be regarded as a favorable event. Our data demonstrate for the first time that the biological significance of apoptotic status of DTC is contingent on whether the apoptosis occurs spontaneously or was induced by treatment.
Automated cellular imaging system
Alkaline phosphatase-antialkaline phosphatase
Circulating tumor cell
Disseminated tumor cell
Minimal residual disease
Neoadjuvant systemic therapy
We thank Dr. Mustafa Anjari from Department of Neurosurgery of The National Hospital for Neurology and Neurosurgery, London, UK for revising the manuscript.
- Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC, Schlimok G, Diel IJ, Gerber B, Gebauer G, Pierga JY, Marth C, Oruzio D, Wiedswang G, Solomayer EF, Kundt G, Strobl B, Fehm T, Wong GY, Bliss J, Vincent-Salomon A, Pantel K: A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med. 2005, 353: 793-802. 10.1056/NEJMoa050434.View ArticlePubMedGoogle Scholar
- Janni W, Rack B, Schindlbeck C, Strobl B, Rjosk D, Braun S, Sommer H, Pantel K, Gerber B, Friese K: The persistence of isolated tumor cells in bone marrow from patients with breast carcinoma predicts an increased risk for recurrence. Cancer. 2005, 103: 884-891. 10.1002/cncr.20834.View ArticlePubMedGoogle Scholar
- Fehm T, Becker S, Becker-Pergola G, Sotlar K, Gebauer G, Durr-Storzer S, Neubauer H, Wallwiener D, Solomayer EF: Presence of apoptotic and nonapoptotic disseminated tumor cells reflects the response to neoadjuvant systemic therapy in breast cancer. Breast Cancer Res. 2006, 8: R60-10.1186/bcr1611.View ArticlePubMedPubMed CentralGoogle Scholar
- Kerr JF, Winterford CM, Harmon BV: Apoptosis. Its significance in cancer and cancer therapy. Cancer. 1994, 73: 2013-2026. 10.1002/1097-0142(19940415)73:8<2013::AID-CNCR2820730802>3.0.CO;2-J.View ArticlePubMedGoogle Scholar
- Kerr JF, Wyllie AH, Currie AR: Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972, 26: 239-257. 10.1038/bjc.1972.33.View ArticlePubMedPubMed CentralGoogle Scholar
- Koskinen PJ, Alitalo K: Role of myc amplification and overexpression in cell growth, differentiation and death. Semin Cancer Biol. 1993, 4: 3-12.PubMedGoogle Scholar
- Hockenbery DM: The bcl-2 oncogene and apoptosis. Semin Immunol. 1992, 4: 413-420.PubMedGoogle Scholar
- van Slooten HJ, van de Vijver MJ, van de Velde CJ, van Dierendonck JH: Loss of Bcl-2 in invasive breast cancer is associated with high rates of cell death, but also with increased proliferative activity. Br J Cancer. 1998, 77: 789-796. 10.1038/bjc.1998.128.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang GJ, Kimijima I, Abe R, Watanabe T, Kanno M, Hara K, Tsuchiya A: Apoptotic index correlates to bcl-2 and p53 protein expression, histological grade and prognosis in invasive breast cancers. Anticancer Res. 1998, 18: 1989-1998.PubMedGoogle Scholar
- Lipponen P, Aaltomaa S, Kosma VM, Syrjanen K: Apoptosis in breast cancer as related to histopathological characteristics and prognosis. Eur J Cancer. 1994, 30A: 2068-2073.View ArticlePubMedGoogle Scholar
- Li D, Yao Q, Li L, Wang L, Chen J: Correlation between hybrid 18F-FDG PET/CT and apoptosis induced by neoadjuvant chemotherapy in breast cancer. Cancer Biol Ther. 2007, 6: 1442-1448. 10.4161/cbt.6.9.4621.PubMedGoogle Scholar
- Miller AB, Hoogstraten B, Staquet M, Winkler A: Reporting results of cancer treatment. Cancer. 1981, 47: 207-214. 10.1002/1097-0142(19810101)47:1<207::AID-CNCR2820470134>3.0.CO;2-6.View ArticlePubMedGoogle Scholar
- Fehm T, Braun S, Muller V, Janni W, Gebauer G, Marth C, Schindlbeck C, Wallwiener D, Borgen E, Naume B, Pantel K, Solomayer E: A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation. Cancer. 2006, 107: 885-892. 10.1002/cncr.22076.View ArticlePubMedGoogle Scholar
- Borgen E, Naume B, Nesland JM, Kvalheim G, Beiske K, Fodstad O, Diel I, Solomayer EF, Theocharous P, Coombes RC, Smith BM, Wunder E, Marolleau JP, Garcia J, Pantel K: Standardization of the immunocytochemical detection of cancer cells in BM and blood. I. Establishment of objective criteria for the evaluation of immunostained cells. Cytotherapy. 1999, 5: 377-388.View ArticleGoogle Scholar
- Caulin C, Salvesen GS, Oshima RG: Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol. 1997, 138: 1379-1394. 10.1083/jcb.138.6.1379.View ArticlePubMedPubMed CentralGoogle Scholar
- Leers MP, Kolgen W, Bjorklund V, Bergman T, Tribbick G, Persson B, Bjorklund P, Ramaekers FC, Bjorklund B, Nap M, Jörnvall H, Schutte B: Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol. 1999, 187: 567-572. 10.1002/(SICI)1096-9896(199904)187:5<567::AID-PATH288>3.0.CO;2-J.View ArticlePubMedGoogle Scholar
- Fehm T, Solomayer EF, Meng S, Tucker T, Lane N, Wang J, Gebauer G: Methods for isolating circulating epithelial cells and criteria for their classification as carcinoma cells. Cytotherapy. 2005, 7: 171-185. 10.1080/14653240510027082.View ArticlePubMedGoogle Scholar
- Larson CJ, Moreno JG, Pienta KJ, Gross S, Repollet M, O’Hara SM, Russell T, Terstappen LW: Apoptosis of circulating tumor cells in prostate cancer patients. Cytometry A. 2004, 62: 46-53.View ArticlePubMedGoogle Scholar
- Mehes G, Witt A, Kubista E, Ambros PF: Circulating breast cancer cells are frequently apoptotic. Am J Pathol. 2001, 159: 17-20. 10.1016/S0002-9440(10)61667-7.View ArticlePubMedPubMed CentralGoogle Scholar
- Pantel K, Brakenhoff RH: Dissecting the metastatic cascade. Nat Rev Cancer. 2004, 4: 448-456. 10.1038/nrc1370.View ArticlePubMedGoogle Scholar
- Weiss L: Metastatic inefficiency. Adv Cancer Res. 1990, 54: 159-211.View ArticlePubMedGoogle Scholar
- Frankfurt OS, Robb JA, Sugarbaker EV, Villa L: Apoptosis in breast carcinomas detected with monoclonal antibody to single-stranded DNA: relation to bcl-2 expression, hormone receptors, and lymph node metastases. Clin Cancer Res. 1997, 3: 465-471.PubMedGoogle Scholar
- Vakkala M, Lahteenmaki K, Raunio H, Paakko P, Soini Y: Apoptosis during breast carcinoma progression. Clin Cancer Res. 1999, 5: 319-324.PubMedGoogle Scholar
- Liu S, Edgerton SM, Moore DH, Thor AD: Measures of cell turnover (proliferation and apoptosis) and their association with survival in breast cancer. Clin Cancer Res. 2001, 7: 1716-1723.PubMedGoogle Scholar
- Aihara M, Scardino PT, Truong LD, Wheeler TM, Goad JR, Yang G, Thompson TC: The frequency of apoptosis correlates with the prognosis of Gleason Grade 3 adenocarcinoma of the prostate. Cancer. 1995, 75: 522-529. 10.1002/1097-0142(19950115)75:2<522::AID-CNCR2820750215>3.0.CO;2-W.View ArticlePubMedGoogle Scholar
- Lipponen PK, Aaltomaa S: Apoptosis in bladder cancer as related to standard prognostic factors and prognosis. J Pathol. 1994, 173: 333-339. 10.1002/path.1711730408.View ArticlePubMedGoogle Scholar
- Kaufmann M, Hortobagyi GN, Goldhirsch A, Scholl S, Makris A, Valagussa P, Blohmer JU, Eiermann W, Jackesz R, Jonat W, Lebeau A, Loibl S, Miller W, Seeber S, Semiglazov V, Smith R, Souchon R, Stearns V, Untch M, von Minckwitz G: Recommendations from an international expert panel on the use of neoadjuvant (primary) systemic treatment of operable breast cancer: an update. J Clin Oncol. 2006, 24: 1940-1949. 10.1200/JCO.2005.02.6187.View ArticlePubMedGoogle Scholar
- Kuerer HM, Newman LA, Smith TL, Ames FC, Hunt KK, Dhingra K, Theriault RL, Singh G, Binkley SM, Sneige N, Buchholz TA, Ross MI, McNeese MD, Buzdar AU, Hortobagyi GN, Singletary SE: Clinical course of breast cancer patients with complete pathologic primary tumor and axillary lymph node response to doxorubicin-based neoadjuvant chemotherapy. J Clin Oncol. 1999, 17: 460-469.PubMedGoogle Scholar
- Hickman JA: Apoptosis induced by anticancer drugs. Cancer Metastasis Rev. 1992, 11: 121-139. 10.1007/BF00048059.View ArticlePubMedGoogle Scholar
- Untch M, Fasching PA, Konecny GE, Hasmuller S, Lebeau A, Kreienberg R, Camara O, Muller V, du Bois A, Kuhn T, Stickeler E, Harbeck N, Höss C, Kahlert S, Beck T, Fett W, Mehta KM, von Minckwitz G, Loibl S: Pathologic complete response after neoadjuvant chemotherapy plus trastuzumab predicts favorable survival in human epidermal growth factor receptor 2-overexpressing breast cancer: results from the TECHNO trial of the AGO and GBG study groups. J Clin Oncol. 2011, 29: 3351-3357. 10.1200/JCO.2010.31.4930.View ArticlePubMedGoogle Scholar
- Hennessy BT, Hortobagyi GN, Rouzier R, Kuerer H, Sneige N, Buzdar AU, Kau SW, Fornage B, Sahin A, Broglio K, Singletary SE, Valero V: Outcome after pathologic complete eradication of cytologically proven breast cancer axillary node metastases following primary chemotherapy. J Clin Oncol. 2005, 23: 9304-9311. 10.1200/JCO.2005.02.5023.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/394/prepub
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