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a Department of Internal Medicine V;
b Department of Gynecology and Obstetrics;
c Department of Pathology, University of Heidelberg, Heidelberg, Germany;
d Department of Hematology and Oncology, University of Düsseldorf, Düsseldorf, Germany
Key Words. Metastatic breast cancer • High-dose chemotherapy • Prognostic factors • CD34+ cell dose • Blood stem cell transplantation
Anthony D. Ho, M.D., Department of Medicine V, Hospitalstrasse 3, D-69115 Heidelberg, Germany. Telephone: 49-6221-568001; Fax: 49-6221-565813; e-mail: Anthony_Ho{at}med.uni-heidelberg.de; www.poliklinik-hd.de
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ABSTRACT |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionConclusionReferences |
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With a median follow-up period of 40 months (range, 7-89 months), no more than two metastatic sites (relative risk [RR] = 3.84 [95% confidence interval (CI) 1.49-10]; p =.005) and hyperploidy (RR = 2.58 [95% CI 1.26-5.26]; p = .009) were independent predictors of longer PFS according to multivariate analysis. Independent prognostic factors of longer OS included one or two metastatic sites (RR = 4.16 [95% CI 1.96-4.16]; p < .001), a positive combined hormone receptor status (RR = 2.45 [95% CI 1.45-4.14]; p = .001) and a high number of infused stem cells (>7.8 x 106 CD34+ cells per kg body weight) (RR = 2.0 [95% CI 1.17-3.42]; p = .01).
In conclusion, positive hormone receptors, 
2 metastatic sites, high DNA-index and high CD34+ cell dose (>7.8 x 106 CD34+ cells per kg) are predictors for a favorable outcome after autotransplantation for MBC. Our observation might indicate a favorable effect of HDCT in MBC patients with overexpression of Her2/neu who might have a worse prognosis when treated with conventional chemotherapy.
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INTRODUCTION |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionConclusionReferences |
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The Her2/neu receptor is a member of the epidermal growth factor receptor family of receptor tyrosine kinases [8]. It is overexpressed in 25%-30% of patients with MBC, and overexpression correlates with shorter survival [9]. On the other hand, overexpression of Her2/neu might predict an improved sensitivity to a doxorubicin-based regimen [10,11].
p53 is a tumor-suppressor gene, and mutations are detected in 18%-45% of breast tumors. So far no conclusive findings regarding the prognostic value of p53 mutation have been reported [12,13]. However, p53 accumulation might be a negative predictor of response to conventional treatment with doxorubicin [14] and cyclophosphamide, methotrexate, and fluorouracil [15,16]. Increased DNA content (hyperploidy), increased tumor proliferation as measured by the S-phase fraction, and an increased level of Ki67+ tumor cells have been shown to be associated with earlier relapse and death [17,18]. The bcl-2 proto-oncogene, which encodes for a protein that inhibits programmed cell death (apoptosis), is expressed in 80% of breast cancers derived from women with primary tumors [19]. It has been shown to predict the efficacy of adjuvant hormonal treatments [20]. Expression of bcl-2 is commonly associated with a favorable prognosis in breast cancer [21-23].
Even though most of the patients in our clinical trials did not previously receive intensive chemotherapy, the CD34+ cell dose that could be harvested varied greatly among patients The significance of the stem cell dose for long-term outcome in patients with acute leukemia after HDCT and autologous stem cell transplantation (ASCT) has been demonstrated [24]. So far, no data have been published on the significance of the CD34+ cell dose for patients with solid tumors. Therefore, we analyzed this parameter in addition to the other prognostic factors as reported above.
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PATIENTS AND METHODS |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionConclusionReferences |
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in detail.
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Inclusion criteria for the HDCT trials were: MBC with no prior palliative chemotherapy (except eight patients with one and two patients with two and five cycles of prior palliative chemotherapy), age between 18 and 65 years, Karnofsky performance score 
90%, normal hematological, cardiac, renal and hepatic function, and no relevant concomitant disease. Patients with only bone metastases or in whom the central nervous system was involved were excluded from the trials.
Chemotherapy and Stem Cell Harvesting
In 31 patients the D-HDCT regimen consisted of conventional induction chemotherapy with two cycles of ifosfamide and epirubicin followed by two cycles of stem cell-supported, high-dose ifosfamide (total dose of 12,000 mg m–2), epirubicin (180 mg m–2), and carboplatin (900 mg m–2). Carboplatin was not included for the first five patients. Another 17 patients received a D-HDCT regimen with three conventional cycles of doxorubicin and docetaxel followed by one cycle of high-dose ifosfamide (12,000 mg m–2), etoposide (1,500 mg m–2), and carboplatin (1,500 mg m–2) and one cycle of high-dose cyclophosphamide (6,000 mg m–2) and thiotepa (800 mg m–2). For T-HDCT, one cycle of induction chemotherapy with paclitaxel, ifosfamide and epirubicin was followed by two cycles of stem cell-supported high-dose ifosfamide, epirubicin and carboplatin and one cycle of high-dose paclitaxel (180 mg m–2), etoposide (1,500 mg m–2), and thiotepa (600 mg m–2) [25,26]. Seventeen patients were scheduled to receive only one cycle of high-dose cyclophosphamide (6,000 mg m–2), carboplatin (900 mg m–2), and thiotepa (500 mg m–2) following induction chemotherapy with three cycles of doxorubicin and docetaxel. In 8 of 48 patients (17%) enrolled in the D-HDCT trial, therapy was discontinued after the first cycle of HDCT because of adverse events or disease progression. Twelve of 55 patients (22%) enrolled in the T-HDCT trial received only one or two cycles of HDCT for the same reasons.
After each cycle of induction chemotherapy, R-metHuG-CSF (filgrastim, 300 µg day-1 s.c.; Neupogen®, Amgen Inc.; Thousand Oaks, CA; http://www.amgen.com) was administered to accelerate neutrophil recovery and mobilize progenitor cells. Peripheral blood stem cell collection began after at least one cycle of induction chemotherapy when CD34+ cells could be measured after nadir. The leukapheresis procedure has been published in detail elsewhere [27].
Tumor Specimens and Immunohistochemistry
Tumor tissue was fixed in buffered formalin for at least 24 hours, and subsequently embedded in paraffin and cut into tissue sections of approximately 2 µm. These sections were deparaffinated by incubation at 37°C for 24 hours. After incubation at 50°C for another 30 minutes, the sections were immersed in xylene for 2 x 10 minutes and then rehydrated in ethanol at graded concentrations (100%, 96%, 70% each for 5 minutes). Prior to immunohistochemical staining, the tissue sections were subjected to microwaves for 10 minutes at a high energy setting. After the addition of 50 ml of distilled H2O, 7 more minutes of high microwave energy were applied after adding a diluted antigen retrieval buffer (DAKO; Glostrup, Denmark; http://www.dako.dk). The slides were allowed to cool at room temperature for a minimum of 20 minutes, and the sections were then stained using an automated immunohistochemical technique (Biotek TechMate, Biotek Solutions; Newport Beach, CA; http://www.qmlabs.com), strictly adhering to the staining protocol. In brief, primary antibodies were applied for 30 minutes, followed by an indirect streptavidin biotin method with 30 minutes of secondary goat-anti-mouse antibody and 45 minutes of streptavidin biotin conjugate. The following primary antibodies were used (clones in brackets, all reagents by DAKO): Her2/neu [A0485], Ki67 [MIB-1], p53 [DO7], Bcl-2 [124], estrogen receptor (ER) [1D5], progesterone receptor (PR) [PR88].
The antibody staining was recorded as the percentage of positive tumor nuclei for Ki67 and p53. Bcl-2 and Her2/neu immunoreaction was scored from 0 to 3, considering merely cell membrane staining. ER and PR staining was evaluated for staining intensity and number of positive nuclei [28]; receptor positivity was assumed when the semiquantitative score was at least 3 (out of a maximum of 12 points).
Flow Cytometry
Cell cycle analysis was performed in the cells obtained from tumor tissues. DNA flow cytometry was used as described elsewhere [29,30]. Briefly, tumor samples were minced thoroughly with scissors. The nuclei were extracted at room temperature by incubation in acid pepsin (3,000 units/mg, Serva; Heidelberg, Germany; http://www.serva.de), dissolved in 100 ml of 0.9 NaCl containing 0.25% hydrochloric acid, and carefully stirred for 20 minutes. After 30 seconds of sedimentation, 0.5 ml of the supernatant cell suspension was suspended in 1 µg/ml 4',6-diamindino-2-phenylindole dissolved in tris-buffer pH 7.8. Minimum incubation time was 30 minutes. Flow cytometry was carried out using a PAS II flow cytometer equipped with a high-pressure mercury lamp (Partec; Münster, Germany; http://www.partech.com/index.html) using the following filters: KG 1, BG 38, and UG 1 for excitation, TK 420 as dichromic mirror, and GG 435 as barrier filter. A flow rate of about 100 counts/second was maintained by adjusting the vacuum. DNA histograms of at least 10,000 counts were plotted. The DNA index of aneuploid cells was expressed as the relative modal DNA value of the aberrant peak in relation to the diploid peak. Normal human lymphocytes with a coefficient of variation of 1.0 were used to calibrate the diploid peak. The cell-cycle-phase distribution patterns of the diploid and aneuploid tumors were calculated using the Multi-cycle® software package (Phoenix Flow Systems; San Diego, CA; http://www.phnxflow.com) after adjustments were made for debris and aggregation (nuclear doublets and triplets).
Apheresis Products
Data on the CD34+ cell dose were collected from laboratory and patient records. The absolute number of CD34+ cells harvested and finally transfused was determined and calculated as previously described [31].
A total of 194 apheresis products collected from 50 patients was assessed for the presence of tumor cells using an immunostaining method; 1 x 106 mononuclear cells were transferred onto glass slides using a cytocentrifuge. A cocktail of four monoclonal epithelial-specific antibodies (BM7, BM8 against MUC1, 5D3 against CK8, 18, 19, and HEA125 against human epithelial antigen) was used for immunostaining as described elsewhere [32,33].
In a subgroup of 12 patients, CD34+ cells were selected from apheresis products using immunomagnetic beads with the Baxter Isolex 300 SA Magnetic Cell Separation Systems (Baxter Immunotherapy; Irvine, CA; http://www.baxter.com). CD34+ cells were enriched and released by chymopapain, resulting in a median purity of 95% (range 82%-99%) and a median recovery of 80% (range 27%-132%). Five apheresis products contained epithelial cells, whereas the CD34+ cell fraction selected was free of tumor cells.
Statistical Analysis
PFS and OS were taken as clinical outcome variables. PFS was measured from the date of first induction chemotherapy until the time of progression, death or last contact. OS was calculated from the date of first induction chemotherapy to death or the date of the last patient contact. Survival curves were estimated using the Kaplan-Meier product limit method [34]. Univariate and multivariate analyses were performed to identify risk factors associated with PFS and OS. Differences between the survival curves were compared using the log-rank test [35]. Multivariate analysis was performed using the Cox regression model with stepwise analysis (p-values for entry = 0.15 and for removal = 0.05) [36]. All tests were performed using the statistical software package SAS, Version 6.11 (SAS Institute Inc.; Cary, NC, 1995; http://www.sas.com).
The following risk factors were examined in a univariate analysis: combined ER and PR status (positive versus negative), disease-free interval calculated from initial diagnosis to diagnosis of metastases (2 versus >2 years), number of metastatic sites (2 versus >2), expression of Her2/neu (score 3+ versus 0-2+), proportion of p53+ (<50% versus 50%) and Ki67+ cells (<50% versus 50%), Bcl-2 immunoreactivity (score 2-3+ versus 0-1+), SPF (<4.9% versus 4.9%), ploidy (<1.5 versus 1.5), and CD34+ cell dose (7.8 x 106 CD34+ stem cells per kg [median] versus >7.8). Variables that were significant in the univariate analysis were included in a multivariate analysis as stated above.
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RESULTS |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionConclusionReferences |
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Univariate and Multivariate Analysis
Her2/neu overexpression (score 3+) was found in 33 of 75 patients tested (44%). Rates of 50% p53+ tumor cells were observed in 14 of 53 patients (26%); 50% Ki67+ tumor cells were detected in 15 of 41 patients (37%). Positive staining for bcl-2 (score 2+ and 3+) was observed in 14 of 40 patients (35%). DNA-hyperploidy (DNA-index 1.5) was observed in 29 of 51 patients (57%) and an SPF >4.9% in 23 of 46 patients (50%) (Table 2).
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shows the Kaplan-Meier graph of PFS in patients with hyperploidy versus patients with diploid tumor cells. A poorer OS was associated with negative ER and PR (p = .002) and more than two metastatic sites (p = .003). We found no correlation between Her2/neu, p53, Ki67, bcl-2, SPF, and PFS or OS (Table 3).
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). In a more homogeneous subgroup of patients who received a uniform induction and stem cell mobilizing chemotherapy with doxorubicin and docetaxel (n = 34), a low number of infused stem cells (<4.95 x 106 CD34+ cells [median] versus >4.95 x 106) was a significant predictor of a shorter PFS (p = .004) and OS (p = .0007) (Figs. 3 and 4).
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DISCUSSION |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionConclusionReferences |
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Rizzieri et al. recently reported longer survival in patients with hormone receptor-positive tumors who did not receive prior adjuvant chemotherapy, who achieved a long disease-free interval after primary therapy, and who had no visceral metastases [38]. A retrospective analysis of 1,188 patients treated with HDCT and ASCT for MBC identified age, low performance score, absence of hormone receptors, prior adjuvant chemotherapy, short initial disease-free interval, liver and central nervous system metastases, three or more sites of metastatic disease, and incomplete response to standard chemotherapy as factors associated with increased risk of treatment failure [39]. In our recently published analysis of prognostic factors in a subgroup of 76 MBC patients treated with D- or T-HDCT, a positive ER status, one or two metastatic sites, and a complete remission after HDCT were independent predictors of better survival [25].
Among the tumorbiologic parameters tested in the present analysis, only hyperploidy was associated with PFS. Patients with hyperploid tumor cells had a significantly better PFS than patients with a diploid tumor. This is in contrast to former observations that reported a poorer outcome in MBC patients with increased DNA content who received conventional chemotherapy [17,18]. The reason might be a higher proliferation rate of hyperploid tumor cells and, therefore, a greater sensitivity to HDCT than diploid tumors.
In contrast to their predictive value for outcome after conventional chemotherapy, we could not identify any significance for the markers Her2/neu, p53, and Ki67. Pts with increased Her2/neu expression had PFS and OS after HDCT similar to that of patients with normal expression. Data from the literature regarding this issue are inconclusive. Doroshow et al. found that Her2/neu overexpression is associated with earlier death [40], whereas Bezwoda et al. [41] reported that Her2/neu had no predictive value in MBC patients treated with HDCT. The results of another trial of this group, however, have recently been challenged [42]. In another series of 425 patients with MBC treated with HDCT, again, no association between outcome and Her2/neu expression measured by immunohistochemistry could be observed [43]. Nevertheless, our observation might indicate a favorable effect of HDCT in MBC patients with overexpression of Her2/neu who might have a worse prognosis when treated with conventional chemotherapy.
Gorin et al. recently analyzed the relationships between the CD34+ cell dose and long-term outcome after ASCT among 229 patients with acute leukemia [24]. In their study, patients received autografts with mafosfamide-purged marrow, and the stem cell dose before purging was the most important prognostic factor for survival. So far, the impact of the infused CD34+ cell dose has not been evaluated in patients with solid tumors.
In the present study we found an association between CD34+ cell dose and survival. With respect to all 120 patients treated with different protocols of HDCT, multivariate analysis showed that a higher total amount of CD34+ cells was associated with a significantly better OS (p = .01). This correlation was even more pronounced in a more homogeneous subgroup of patients that received a uniform induction chemotherapy before stem cell harvest (three cycles of doxorubicin and docetaxel), where a high number of infused stem cells was associated with both a favorable PFS (p = .004) and OS (p = .0007). It is possible that prior chemotherapy and/or irradiation might reduce the stem cell reserve and, hence, the number of CD34+ cells that can be harvested. In our patient population, however, there was no correlation between CD34+ cell dose and previous chemotherapy or radiotherapy. Furthermore, there was no difference in post-transplant morbidity and treatment-related mortality among patients with lower or higher numbers of reinfused stem cells. For patients with acute leukemia, it was suggested that a higher stem cell dose might accelerate engraftment, therefore lowering the rate of complications and improving survival. Furthermore, a high stem cell yield might be a surrogate marker for high-quality complete remission and, therefore, a good prognostic factor in itself. In our analysis, however, we observed no correlation between stem cell dose and hematopoietic reconstitution time.
At this point we do not have a satisfactory explanation for this surprising observation. One hypothesis is that there might be a faster reconstitution of cell-mediated immunity after reinfusion of more stem cells, leading to a better control of residual tumor cells. Unfortunately, we have no data on immune-reconstitution patterns of our patients following HDCT and ASCT.
The presence of tumor cells in apheresis products or the infusion of CD34-enriched apheresis products had no detectable influence on survival.
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CONCLUSION |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionConclusionReferences |
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2 metastatic sites to be independent prognostic factors for better survival. These parameters should be included into a comprehensive model to define the subgroup of MBC patients who might derive a long-term benefit from HDCT. Our observation might indicate a favorable effect of HDCT in MBC patients with overexpression of Her2/neu who have a worse prognosis when treated with conventional chemotherapy. Other parameters such as p53, Ki67, Bcl-2, and SPF were not found to be useful in our patient population.
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REFERENCES |
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