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© 2000 American Society for Clinical Oncology Detection of Circulating Tumor Cells by Reverse Transcriptase Polymerase Chain Reaction of Maspin in Patients With Breast Cancer Undergoing Conventional-Dose ChemotherapyFrom the Cattedra di Oncologia Medica ed Ematologia, Dipartimento di Oncologia ed Ematologia, Università di Modena, Modena, Italy. Address reprint requests to Massimo Federico, MD, Cattedra di Oncologia Medica, Università di Modena, Policlinico, Via del Pozzo 71, 41100 Modena, Italy; email federico{at}unimo.it
PURPOSE: To establish, in patients with breast cancer subjected to primary conventional chemotherapy and enrolled in a prospective study, the mobilizing effect of therapy on potentially neoplastic cells by means of a reverse transcriptase polymerase chain reaction (RT-PCR) assay for mRNA of maspin, a protein related to the serpin family of protease inhibitors. PATIENTS AND METHODS: Peripheral-blood samples were collected from 30 patients with histologically proven breast cancer before and 4 and 8 days after conventional chemotherapy for three consecutive courses. A total of 216 samples were screened for the presence of maspin mRNA by RT-PCR. RESULTS: Before therapy, all samples but one were negative. After chemotherapy, 11 patients (38%) had positive samples. No difference in the rate of positivity was observed between groups defined according to initial stage, type of chemotherapy, Ki-67–related proliferative activity, or CA 15.3 expression. CONCLUSION: Our results confirm that RT-PCR for maspin mRNA is a sensitive assay for the study of circulating potentially neoplastic mammary cells in patients with breast cancer. Moreover, our findings indicate a marked effect of conventional-dose chemotherapy on the mobilization of these cells in breast tumors. In our series of patients, this phenomenon does not seem to be associated with other known risk factors. Finally, the data suggest, without proving, an association between the presence of circulating maspin positive cells and a higher risk of disease progression. If this association could be confirmed, then the assay could have prognostic significance. However, larger confirmatory studies are necessary.
BREAST CANCER (BC) is by far the most common cancer affecting women. The prognosis of BC is strictly dependent on early diagnosis and appropriate treatment. With current diagnostic procedures, including self-examination and regular mammograms, up to 80% of tumors are detected at an early stage, thus providing the best opportunity of treating patients with conservative surgery and/or radiotherapy and limiting systemic therapy to only those patients with more aggressive disease. Despite its usual clinical presentation as a localized disease, 30% to 40% of these patients will have clinically evident metastatic disease within 10 years, suggesting that clinically occult micrometastatic disease is already present at the time of diagnosis. Thus detection of clinically occult sites of disease could be of value for better defining the prognosis and, probably, in planning more appropriate treatment strategies. In the past, various immunologic procedures, including immunocytology-, immunohistochemistry-, and immunofluorescence-based methods, have been used to detect systemic tumor cell contamination.1-5 More recently, a major advance in this area occurred with the advent of the reverse transcriptase polymerase chain reaction (RT-PCR), which makes possible the detection of as few as one tumor cell in 106 normal cells.6-10 In particular, RT-PCR for cytokeratin (CK) has been described as an assay system for detection of circulating tumor cells in patients with breast and prostate cancer.11-13 However, the specificity of CK has been recently questioned by various researchers who found nontissue-specific constitutive low-level expression of CK mRNA in either peripheral-blood mononuclear cells or bone marrow in a significant number of healthy donors.13-16 On the basis of our previous observations, maspin mRNA could represent a more valuable marker for detection of cancer cells in patients with BC. Maspin is a recently identified protein related to the serpin family of protease inhibitors and has been proposed as playing a role in human BC by means of a possible tumor-suppressing activity.17 Zou et al, however, stated that in tumor cells, the gene is not lost but downregulated. For this reason, we tested the possibility to detect trace amounts of maspin mRNA, otherwise undetectable, by RT-PCR and then to exploit this assay as a tool for identifying neoplastic BC cells. Using maspin mRNA amplification by RT-PCR, we found the presence of maspin in normal breast epithelial cells and in primary and metastatic BC cells, but not in the peripheral blood of healthy donors.18 Moreover, we found the presence of circulating maspin-positive cells, potentially neoplastic, in the peripheral blood of patients with BC undergoing conventional-dose chemotherapy. On the basis of these observations, we started a prospective study designed to assess a possible mobilizing effect of chemotherapy, delivered at standard doses, on mammary cells of possible neoplastic origin in patients with BC.
Thirty patients with histologically proven BC who were scheduled to receive at least three courses of conventional chemotherapy were registered for the study. Additional criteria for inclusion were informed consent and no treatment in the previous 6 months. One patient was subsequently excluded from the study because baseline assessment could not be performed. The main characteristics of the 29 assessable patients with BC are listed in Table 1. Twenty-one patients had limited disease and eight had advanced disease. Twenty patients were treated with cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, and fluorouracil 600 mg/m2 (CMF), all administered intravenously on days 1 and 8. At the end of six courses of CMF, 15 patients with estrogen receptor–positive tumors (tested immunocytologically) received tamoxifen 20 mg/d. Five patients with estrogen receptor–negative tumors did not receive any further therapy after CMF. One patient with high-risk limited disease was treated with four consecutive courses of doxorubicin 75 mg/m2 every 3 weeks followed by four courses of CMF and, after chemotherapy, tamoxifen. Eight patients with advanced disease were treated with six courses of anthracycline-based chemotherapy (doxorubicin 75 mg/m2 or epidoxorubicin 90 mg/m2). Treatment was repeated at 3-week intervals for a total of six courses. After chemotherapy, three patients received tamoxifen and three were treated with anastrozole. In two nonresponder patients, a second-line chemotherapy with taxoids was administered. During the study period, the use of hematopoietic growth factors was not allowed.
Samples of heparinized peripheral blood were collected 30 minutes before and 4 and 8 days after chemotherapy administration for three consecutive courses (Fig 1). Peripheral-blood mononuclear cells (PBMCs) were immediately prepared by centrifugation on Ficoll-Hypaque density gradient (Ficoll-Hypaque; Pharmacia, Uppsala, Sweden). Total RNA was extracted according to the guanidinium thiocyanate-phenol-chloroform standard method.19
cDNA Synthesis and Nested PCR We evaluated the RNA transcript encoding for maspin as a marker for mammary carcinoma cells using a specific RT-PCR–based assay recently described by Luppi et al.18 One microgram of total RNA was reverse transcribed according to the manufacturer’s instructions in 1 x RT buffer (Gibco BRL Life Technologies, Gaithersburg, MD) supplemented with 0.01 mol/L dithiothreitol (DTT), 200 units of cloned Moloney murine leukemia virus RT (Gibco BRL), 1 mmol/L of each dNTP (Pharmacia), and 200 units of RNAse inhibitor (Boehringer, Mannheim, Germany) in a final volume of 30 µL incubated at 42°C for 60 minutes. Primers designed from the coding sequence of the human maspin cDNA (GenBank accession no. U04313) and reported elsewhere were used for amplification. To increase the sensitivity of the RT-PCR assay, 2.5 µL of the primary amplified product was subjected to a second PCR under the same conditions for 35 cycles with already described nested primers. Distilled water, the entire reaction mixture without RT enzyme, and placenta DNA were used as negative controls and equaled or exceeded the number of samples assayed. To avoid false-positive results, the different procedures (extraction and purification, amplification, and gel electrophoresis) were physically separated. Moreover, extraction and amplification were performed in duplicate in experiments performed at different times. The assays were accepted only when confirmed in a second round. In all of the specimens tested for maspin expression, the presence of intact RNA, an adequate cDNA synthesis, and the absence of enzyme inhibitors were also confirmed by a single round of RT-PCR using human ABL gene sequence-specific PCR primers.20 The specificity of the amplified band was assessed by Southern blot analysis followed by hybridization with an internal specific probe. Thirty PBMCs and four bone marrow samples from healthy donors (partially already described) tested negative. As previously reported,18 10 primary and five metastatic BC samples tested positive, as well as five nonneoplastic mammary tissue samples. A mixing study performed using serial dilutions of MCF7 cells in normal PBMCs indicated that hybridization of the segment amplified after the first 35 cycles of amplification was able to recognize one positive cell of 106 negative cells.18
Statistical Analysis The comparison between maspin positivity and other prognostic factors was performed with a series of univariate analyses because of the small number of patients in each group. A multivariate analysis was not performed, because in univariate analysis, none of the tested variables resulted in statistical significance at the level of P = .05.
Of the 30 patients, 29 were assessable for the mobilizing effect of the first course of therapy, 24 for the first two courses, and 14 for all three courses. Two hundred sixteen samples were analyzed for the maspin transcript. A representative example of maspin RT-PCR assay is shown in Fig 2. Before chemotherapy, samples from all but one patient tested negative for the presence of maspin mRNA. The one patient who tested positive had stage IIIB disease. Interestingly, all eight patients with metastatic disease tested negative. On chemotherapy, maspin mRNA–positive cells (hereon referred to as maspin-positive) were recruited in a total of 11 patients (39%). Eight patients (29%) who had tested negative before chemotherapy tested positive after the first course of chemotherapy, and three patients (10%) who had tested negative tested positive after the second course. In the patient who tested positive for circulating maspin-positive cells at baseline, further maspin-positive cells were detected in peripheral blood only after the third course of therapy. The cumulative probability of positivity after the three courses was 50% (Fig 3). Seven patients had at least two positive tests (median, three positive tests; range, two to four positive tests), and five tested positive only once (Table 2). No statistically significant correlations between maspin positivity and other well-established prognostic parameters were found, including stage of disease, proliferative activity, and CA 15.3 serologic levels (Table 3). Of relevance, a similar percentage of positivity was observed in patients with advanced or limited disease (three of eight patients and nine of 21 patients, respectively). Comparable rates of positivity were also observed in the group of patients who were treated with CMF and anthracyclines.
After a median follow-up of 27 months (range, 7 to 38 months), four patients with advanced disease (two in the group of maspin-positive and two in the group of maspin-negative patients) showed signs of disease progression. In addition, four relapses were observed in the group of patients with limited disease who were treated with adjuvant therapy. Two relapses were observed among nine maspin-positive patients, both while receiving maintenance therapy with tamoxifen. Two relapses were registered also among 12 maspin-negative patients, one while receiving hormonal therapy with tamoxifen and the second 6 months after completing tamoxifen therapy. Among 21 patients who were treated in the adjuvant setting, the 2-year failure-free survival rate was 78% in maspin-positive and 87% in maspin-negative patients (P = not significant [NS]). Among eight patients with metastatic BC, the 2-year overall survival rate was 33% in maspin-positive and 60% in maspin-negative patients (P = NS).
Tumor cell contamination of bone marrow and peripheral blood was observed in patients with different tumors, including cancer of the lung, prostate, esophagus, and breast.1,2,13,21,22,26 In BC patients, immunocytochemical (ICC) methods have been performed extensively, using one or more monoclonal antibodies directed against surface glycoproteins and, more often, cytoskeletal intermediate filaments (CKs). The clinical use of these assays has been hampered by serious concerns about specificity and sensitivity of the assays. In fact, the antibodies used in the ICC reactions, being directed against tissue- but not tumor-specific antigens, may cross-react with normal cells.23,24 Despite an often high sensitivity, the ICC tests may still lead to false-negative results if the number of cells evaluated is inadequate. Moreover, the real metastatic potential of the cells identified in this way is still a debated issue. This issue has been addressed using clonogenic assays, which are laborious and time-consuming. Recently, the RT-PCR method has been suggested as a rapid screening procedure that showed a sensitivity 10- to 100-fold higher than routine immunologic methods. In particular, CK RT-PCR has been reported as an assay system for detection of circulating tumor cells in patients with BC.11 Some authors reported a specificity of up to 97% and a sensitivity of 10-5 to 10-7 in the detection of tumor cells in peripheral blood or bone marrow. However, using primers specific for CK, false-positive findings in blood and bone marrow from noncarcinoma patients have been reported. In particular, it has been reported that RT-PCR methods detected an illegitimate expression of CK-19 mRNA in peripheral-blood cells in a significant number of healthy controls.15 Similarly, Zippelius et al14 have shown a constitutive low-level expression of CK-18 mRNA in normal BM cells using the RNA template-specific PCR assay. Thus, due to the variable levels of expression of CK mRNA in epithelial tissues or to ectopic expression of small amounts of epithelial mRNA in mesenchymal cells, the specificity of CK RT-PCR assays is still largely unsatisfying. For this reason, we have looked at the possible use of maspin mRNA as a further marker of mammary cells in BC patients. Until now, we did not find maspin mRNA in any of the peripheral-blood and bone marrow samples analyzed as controls. This behavior suggests that this assay might prove to be a more specific approach for the study of breast tumor cell contamination.18 The results reported here, besides confirming our previous findings, show that conventional-dose chemotherapy has a tumor cell–mobilizing effect into the peripheral blood in patients with primary BC. Although the mobilizing effect of high-dose chemotherapy, ranging between 5% and 50% in different conditions,25 was already reported, clinical issues concerning the role of recruitment of tumor cells into the peripheral blood exerted by conventional chemotherapy has not been clarified yet. In this prospective study, the presence of maspin-positive circulating cells was evaluated in a total of 29 chemotherapy-naive BC patients who underwent conventional chemotherapy without hematopoietic colony-stimulating factor support. Maspin RT-PCR was carried out on samples of blood at baseline and after each of the first three courses of chemotherapy at different times. In all but one prechemotherapy blood sample, maspin RT-PCR did not detect circulating tumor cells. When we analyzed the samples of patients after the first course of chemotherapy, maspin RT-PCR was positive in eight (29%) of 28 patients. The mobilizing effect was also evident in the subsequent courses of chemotherapy. However, in our experience, the risk of mobilization decreases with time. None of the 10 patients who tested negative after the first two courses changed to a positive test result after the third course of chemotherapy, although persistent circulating tumor cells were observed in patients who tested positive after the first course, which suggests a limited purging in vivo effect of the first courses of chemotherapy. Our data raise the intriguing possibility that there is a recruitment of BC cells into the peripheral blood after the first courses of chemotherapy. The timing of maspin mRNA assay could be critical for the detection of circulating tumor cells. Brugger et al25 suggested that mobilization of tumor cells in peripheral blood could be a transient phenomenon and occurs earlier than hematopoietic recovery. For this reason, we planned two assessments on days +4 and +8, after administration of each chemotherapy course. In seven patients who were maspin-positive on days +4 or +8, no positive assay was observed on day 0 of the next administration of chemotherapy. Thus high variability in timing of mobilization of circulating tumor cells was observed in our patients. A possible limitation of the present study is that tumor contamination of bone marrow was not assessed during the staging procedures, although a mobilization into peripheral blood of tumor cells was reported in bone marrow–negative women who had undergone peripheral-blood progenitor-cell priming.1,25 Our findings suggest that maspin RT-PCR analysis is an assay with high sensitivity in the detection of circulating BC cells in peripheral-blood samples and could be useful for evaluating tumor cell contamination in peripheral-blood progenitor-cell collection in patients with BC undergoing high-dose chemotherapy. There may be various clinical implications of the results of our study. First, using maspin RT-PCR assay, we confirmed the mobilization effects of tumor cells into peripheral blood by conventional chemotherapy. Second, the recruitment of tumor cells was observed at different times after chemotherapy administration and not only at the time of hematopoietic recovery.1,25,26 The prognostic significance of tumor cell involvement of bone marrow and peripheral-blood progenitor cells of BC patients is still debated. Ross et al,1 using ICC and clonogenic assay, showed that tumor cells in both BM and peripheral-blood progenitor cells seem to be capable of clonogenic growth in vitro, thus possibly participating in relapse. Vredenburgh et al,22 using ICC, had shown that contamination of the autologous marrow support was correlated with a shorter disease-free and overall survival in stage II-III patients. On the contrary, Cooper et al,26 using ICC, found no correlation with time to progression, sites of relapse, and overall survival. Despite the small number of patients and the relatively short follow-up period, we also investigated the possible prognostic significance of the presence of BC cells in peripheral blood, as detected with maspin RT-PCR. Four deaths occurred among eight patients with stage IV disease, and four relapses occurred among 21 patients with limited disease. The rate of failure was 33% (four of 12) and 24% (four of 17) for maspin-positive and maspin-negative patients, respectively (P = NS). The data presented here suggested that the detection of circulating BC cells could have prognostic significance, as it was associated with a higher risk of early relapse or disease progression for patients with limited or stage IV disease, respectively. The results of this study must be interpreted with caution, and larger studies with longer follow-up are required to definitely establish the clinical usefulness of the test.
Supported in part by grants from Ministero dell’Università e della Ricerca Scientifica e Technologica (60%), Associazione Italiana per la Ricerca sul Cancro (Milano), and Associazione "A. Serra" per la ricerca sul cancro (Modena, Italy). We thank Luigi Cafarelli for data collection and Maristella del Grande for manuscript preparation.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
