The purpose of this study was to evaluate the presence of micrometastatic cells in the apheresis products from patients with breast cancer, and also to determine if repeated infusion of contaminated products had any clinical impact. A total of 94 patients with high-risk breast cancer were enrolled in a prospective single center study to evaluate the use of dose-intensified chemotherapy (doxorubicine 75 mg/m2 and cyclophosphamide 3000 or 6000 mg/m2 for four cycles) with repeated (× 2) stem cell reinfusion. All women were monitored for the presence of metastatic cells in aphereses, collected after first course of intensive chemotherapy, and following additional mobilization with rhG-CSF. Epithelial cells were screened with monoclonal antibodies directed to cytokeratin. Eight of the 94 patients had detectable tumor cells in one or several aphereses collected after intensive chemotherapy; this was unrelated to other tumor characteristics, including size, histology, Scarff Bloom and Richardson (SBR) grading (presence or absence of hormone receptors). Hemato-poietic reconstitution was similar in the cells from these eight patients, and in the total patient population. Three of these eight patients relapsed. This study has confirmed that contamination of apheresis products remains a rare event, which does not seem to affect clinical evolution, even when reinfused into the patient.
Breast cancer is the most frequent malignancy in developed countries, affecting approximately one in seven women. Adjuvant chemotherapy has been shown to produce consistent improvements in long-term disease-free survival (DFS) and overall survival (OS) in patients with primary breast cancer. However, certain subgroups of patients retain a dismal prognosis. Axillary lymph node involvement and tumor size are two strong predictors of survival upon which are based most of the decisions regarding adjuvant chemotherapy. Despite the impact of adjuvant treatment, 30% of patients relapse and finally die of metastatic breast cancer. Considering this relatively high risk of relapse, alternative strategies including new drugs such as taxanes or high-dose chemotherapy with stem cell support have been tried and are still under evaluation. Based on preliminary pilot studies showing activity, at least in terms of response for high-dose alkylating agent with stem cell support in metastatic breast cancer,1,2 we developed, as have others,3 studies aimed at evaluating multiple high-dose alkylating agents combined with anthracyclines and stem cell support in the adjuvant setting.
Undetected micrometastases can contribute to failure of primary treatment. Epithelial cells are identified as cytokeratin-positive cells, using either immunocytochemistry,4,5 flow cytometry,6 or molecular techniques, including PCR.7,8 The presence of tumor cells in the bone marrow raises several questions, including the need for disease re-staging,9,10 and the prognostic value of such findings. The prognostic importance of bone marrow micrometastases in patients with localized breast cancer has been confirmed in various prospective clinical studies.4,9,11 Several of these studies have confirmed this as an independent prognostic factor even in women without axillary lymph node involvement.4 In all of these studies, poor prognosis was assessed in patients treated with conventional therapy. Other studies evaluated the incidence of tumor cell contamination in aphereses obtained from breast cancer patients with metastatic disease, who were candidates for intensive chemotherapy with stem cell rescue. In this setting, detection of breast cancer cells in hematopoietic grafts5,12 also raises questions about the potential contribution of these cells to relapse after high-dose chemotherapy supported with the reinfusion of autologous cells and progenitors. Gene marking studies have provided limited arguments for the contribution of contaminating tumor cells to relapse in patients with acute myeloid leukemia, neuroblastoma or chronic myeloid leukemia;13,14 these reports remain unconfirmed in the case of breast cancer15,16 and the value of purging products for transplantation is unclear, both in breast cancer,17,18,19 and in other malignancies, such as multiple myeloma.20 Observation of tumor cell contamination of apheresis products has mostly been carried out in the setting of autologous stem cell transplantation for metastatic breast cancer, where the impact on outcome may be diluted by several important prognostic factors, such as tumor sites or chemoresistance. In the setting of autologous stem cell transplant as adjuvant treatment for poor risk breast cancer with significant axillary involvement, few studies21 have reported the incidence of this phenomenon or examined its impact.
The goal of the present study was to assess the presence of micrometastatic cells in aphereses collected from patients with more than four involved axillary homolateral lymph nodes, and to determine whether the reinfusion of these contaminated products had any clinical impact. The study was conducted by analyzing tumor cell contamination in aphereses collected after the first cycle of intensive chemotherapy, from 94 patients with stage II/III breast cancer receiving four cycles of sequential high-dose chemotherapy with repeated stem cell reinfusion.
Patients and methods
From June 1994 to November 1998, 94 patients were analyzed for the presence of micro-metastatic cells in apheresis products, while undergoing intensified chemo-therapy for node positive, nonmetastatic breast cancer. Patients were between the ages of 18 and 60 years and had newly diagnosed and untreated noninflammatory stage II–III breast cancer, with at least four positive axillary lymph nodes. Patients were required to have undergone tumorectomy or mastectomy, to have tumor-free margins, and to have had chemotherapy initiated within 35 days of surgery. Patients with clinical, radiographic or pathologic evidence of metastatic breast cancer were excluded. Other patient eligibility requirements included WHO performance status less than grade 3, and hematological, renal and hepatic functions within normal limits. In addition, normal cardiac function was required, as demonstrated by echocardiogram or by nuclear-gated heart analysis. All patients had to give written informed consent; the protocol was approved by the Marseille Ethics Committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale, CCPPRB 2).
Chemotherapy was a sequential dose intense regimen, combining two cytotoxic agents: three successive cohorts of patients received four cycles of chemotherapy with cyclophosphamide (3 or 6 g/m2) and doxorubicin (75 mg/m2). The first cohort (36 patients – cohort A) was treated with four cycles of 3 g/m2 of cyclophosphamide and 75 mg/m2 of doxorubicin per cycle, every 21 days. The second cohort (30 patients – cohort B) received the same drug combination and doses, but cycles were spaced at 15 days. In the third cohort (28 patients – cohort C), cycles were given every 21 days at a dose of 6 g/m2 of cyclophosphamide and 75 mg/m2 of doxorubicin per cycle. Supportive care included uromitexan for bladder protection, and systematic administration of rhG-CSF (Neupogen®, Amgen, Thousand Oaks, CA, USA); rhG-CSF was given at a daily dosage of 5 μg/kg (maximum 300 μg/kg/day) by subcutaneous injection, starting on day 5 after each cycle, until the ANC reached 0.5 × 109/l on three consecutive days, or aphereses were completed.
At 1 month after completing all chemotherapy, patients received standard radiation therapy; in addition, post-menopausal patients with estrogen receptor and/or progesterone receptor positive tumors began a planned 5-year course of oral tamoxifen treatment at 20 mg daily.
Progenitor cell collection
Aphereses were performed after the first cycle of chemo-therapy using an automated continuous-flow blood cell separator (Cobe Spectra, Lakewood, CO, USA). Approximately, two blood volumes were processed during an average 3 h procedure, as previously described.22 The procedure was started when the absolute number of CD34+ cells in the peripheral blood rose above 20/μl. A minimum of 5.5 × 106 CD34+ cells/kg was required. Cells were divided into at least two bags, to allow reinfusion of a minimum of 2 × 106 CD34+ cells/kg after cycles 3 and 4, and were stored in the vapor phase of liquid nitrogen. No attempt was made to purge hematopoietic stem cells of possible tumor cells, when immunocytochemistery was positive (see below), apheresis products were entirely reinfused to patients. All hematopoietic stem cells were reinfused on day 5 of cycles 3 and 4.
Immunocytochemical analysis for occult tumor cells
Epithelial cells were detected using an immunocytochemical staining technique that detects as few as one tumor cell in 106 hematopoietic cells. The technique has been slightly modified since our previous report.23 Samples were obtained from each apheresis, and four cytospin slides were prepared using the Hettich cytocentrifuge, according to the manufacturer's recommendations. In all, 500 000 cells were centrifuged onto individual slides. After fixation in formal-acetone, different slides from each individual were incubated with one of the following antibodies: AE1/AE3mAb (Dako, Trappes, France), CK19mAb (Dako) or CK18mAb (Dako). A fourth slide was incubated with an irrelevant isotype-matched control antibody. The SW480 cell line was stained with the AE1/AE3mAb, and used as positive control. Intracellular binding of the mAb to their respective antigens was revealed with an Alkaline Phosphatase Anti-Alkaline Phosphatase (APAAP) technique, using the Evision kit (Dako). Apheresis was considered positive when at least one of the three slides revealed the presence of cytokeratin-positive cells. The frequency of tumor cells was calculated by summation of positive events on the three slides; results were expressed as numbers of cytokeratin-positive cells per 1.5 × 106 analysed cells.
Descriptive statistics are reported as frequencies or medians. OS was calculated from the date of diagnosis, death being scored as an event, with censoring of other patients at the time of last follow-up. DFS was also calculated from the date of diagnosis, first recurrence, local or distant, being scored as an event, with censoring of other patients at the time of presentation, follow-up or death. OS and DFS curves were drawn using Kaplan and Meier estimates. Follow-up was truncated at 60 months. Survival rates are presented with their 95% confidence intervals (CI). Analyses were performed using SPSS Version 10.0.5.
The 94 patients enrolled into this study had histologically proven breast cancer with more than four involved axillary hipsilateral lymph nodes. Table 1 lists patient characteristics by age, tumor size, number of axillary lymph nodes involved, grade and receptor status. The median age was 46 years (range, 27–60). In total, 88% (84 patients) of patients were premenopausal status at diagnosis. Of these, 67% (71 patients) of patients presented a ductal histological subtype.
Micrometastatic cells in aphereses
A total of 137 aphereses were collected from these 94 patients. A median of 1 (range, 1–4) apheresis yielded 9.7 × 106 CD34+ cells/kg (range, 5.5–42.8): nine out of 137 aphereses (6.5%) were positive for epithelial cells. Finally, eight (8.5%) out of 94 patients had detectable tumor cells in at least one apheresis: for seven out of these eight patients, tumor cells were detected on only one occasion in the different aphereses for the same patient. When detected, a median of one tumor cell (range, 1–8) was identified. Characteristics of these eight patients are detailed in Table 2. Overall, these patients with circulating tumor cells were indistinguishable from other patients in terms of age, TNM stage, histological subtype, number of involved axillary lymph nodes and hormone receptor status.
Impact of occult tumor cell contamination on hematological toxicity and recovery
Patients with epithelial cells in their aphereses had overall similar patterns of hematological toxicity following sequential intensive chemotherapy: Table 3 summarizes the hematological toxicity of 32 cycles in the eight patients with tumor cell contamination, in comparison to 344 cycles in the other 86 patients: after the first two cycles, during which no stem cells were infused, patients with or without tumor cell contamination experienced similar hematological toxicities with no difference in severity or frequency. After the last two cycles, following which comparable numbers of peripheral blood stem cells were infused, hematological recovery was observed with similar delays.
Table 4 shows the frequency of local and distant relapses, as well as the number of deaths in each group, with a minimum follow-up of 33 months (median, 48 months; range, 33–59). Of the eight patients displaying micrometastases in apheresis products, three relapsed (two had local relapses and the third developed liver metastases). No significant differences were found between the three cohorts in terms of tumor cell contamination, OS and DFS. Figures 1 and 2 show OS and DFS for all patients. The actuarial probabilities of OS and DFS at 5 years were 81 and 71%, respectively, with no differences between the two groups.
We prospectively studied the incidence of micrometastatic cells in autologous aphereses collected from 94 women with nonmetastatic breast cancer at high risk of relapse, treated with a high dose of cyclophosphamide and doxorubicin with reinfused autologous hematpoietic stem cells. Micrometastatic cells were detected as cytokeratin-positive cells, using murine monoclonal antibodies to human cytokeratin and a standard immunocytochemistry technique. The monoclonal antibodies employed (AE1/AE3, anti-cyto-keratin 18 and 19) have been widely used to detect bone marrow micrometastases.4,24,25,26 These antibodies are able to detect as few as 1–2 epithelial cells in 1 × 106 bone marrow mononuclear cells with a very high affinity.27,28,29,30. This type of anticytokeratin antibody was initially used to demonstrate that the existence of bone marrow micrometastases is a poor prognosis factor for DFS and OS in patients treated with standard therapy.4,25,26 The technique used in the present report has a similar sensitivity.
The existence of micrometastases in the circulating blood appears to be a rare event compared to the development of bone marrow micrometastases, especially among patients with advanced as well as metastatic breast cancer. The physiologic implication of this observation is still unknown. Our results, with a small percentage of patients with circulating micrometastatic cells (less than 10% of patients), confirm previously published studies on circulating blood cells31 as well as studies on apheresis products.6,21,32 Nevertheless, our study is the first performed on such a large number of patients with advanced, nonmetastatic breast cancer. It is worth noting that patients were studied after a single course of intensive chemotherapy, and thus after minimal in vivo purging, while most reported studies were performed in the context of conventional high-dose chemotherapy, followed by autologous peripheral blood stem cell transplantation, when patients had already been treated with three or four cycles of standard drug combinations.33,34 Finally, unlike findings of other published studies,12,35 it does not appear that chemotherapy used for bone marrow cell mobilization caused an increase in the contamination of apheresis products.
In this series, with a median follow-up of 4 years, the existence of micrometastases in aphereses had no influence on clinical evolution. A number of hypotheses can be put forward. Unlike previously published clinical studies on bone marrow micrometastases, all patients treated in the present study were initially given an unfavourable prognosis. Furthermore, all patients were given intensive primary chemotherapy which may have contributed to in vivo purging and a decrease in the amount of tumor contamination in aphereses. This may also be related to the phenotype of these micrometastatic cells as well as their pathogenicity, which may differ from the primitive tumour and as well as the medullar micrometastatic cells. Phenotyping was mainly performed on bone marrow cells. Interestingly, the presence of elevated angiogenic activity within the primitive tumour was associated with the presence of medullar micrometastasis,36 erbB2 overexpression,37 a deficit in major histocompatibility (MHC) class I molecule expression or the expression38,39 of proliferation markers, such as KI-67 or p120.40 In all these studies, it has been hypothesized that micrometastatic cells were in a quiescent state (G0), and therefore more likely to remain refractory to chemotherapy.34 These observations may not translate to peripheral blood micrometastatic cells, and this may therefore explain the different prognosis value. Another key element of this study is the fact that contaminated cellular products have been reinfused twice, and despite this, patients displayed the same progression profile as those who had not been reinfused with contaminated products. All patients recovered blood counts after each of the four cycles, with reasonable delays, although numbers of patients are small, neutrophil and platelet recoveries did not differ in patients with occult metastatic cells.
In conclusion, this study based on 94 nonmetastatic breast cancer patients with an unfavorable prognosis confirms that contamination of aphereses products remains a rare event. The absence of impact on clinical evolution needs to be confirmed in a larger number of patients. Finally, it may be interesting to investigate a possible correlation between this event and tumor phenotype.
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This work was supported by special grants from the ‘Association pour la Recherche sur le cancer’.
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Viret, F., Chabannon, C., Sainty, D. et al. Occult tumor cell contamination in patients with stage II/III breast cancer receiving sequential high-dose chemotherapy. Bone Marrow Transplant 32, 1059–1064 (2003). https://doi.org/10.1038/sj.bmt.1704283
- tumor cell contamination
- apheresis products
- breast cancer
- sequential high-dose chemotherapy
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