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ICANCER RESEARCH 56. 1244-1249, March 15. 1996 Advances in Brief Motility-related Protein-1 (MRP-1/CD9) Reduction as a Factor of Poor Prognosis in Breast Cancer1 Masayuki Miyake,2 Keiji Nakano, Shin-ichi Itoi, Takashi Koh, and Toshihiko Taki Department of Thoracic Surgery and Department V of Oncology. Kitano Hospital, Tazitke Koftikai Medical Research Insiliate. 13-3. Katni\tiittacho. IM. M.. K. N.. S-i. !.. T. T.j. and Department of Pathology and Tumor Biology. Faculty of Medicine. Kyoto University, Kyoto 606 ¡T.KJ, Japan Abstract The application of reliable markers is of major importance for predict ing the prognosis of and instituting the appropriate postsurgical treatment of patients with breast cancer. Previously we showed that motility-related [unfein-1 iMKI'-l i. which is identical to CD9, regulates cell motility, and that cultured tumor cells transfected with MRP-1/CD9 cDNA have low motility and low mctastatic potential. In addition, MKP-1/CD9 immunoblotting and immunohistochemical study with breast cancer revealed that MRP-1/CD9 expression diminished as the clinical stage of a given breast cancer advanced and that the MRP-1/CD9 gene and protein expression in the metastatic lymph nodes was strikingly lower than in the primary breast cancers. In this study, we also investigated the expression of MRP1/CD9 by immunoblotting and immunohistochemical analysis in 143 freshly resected invasive ductal carcinomas of the breast: 52 tumors were stage I, 61 were stage II, and 30 were stage III. Tumors were classified as MRP-1/CD9 positive when a band intensity of >30% compared with positive control cells, ZR-75-30 were evaluated with the antibody M31-15, and those with intensities <30% as negative. Moreover, these results were ascertained by immunostaining. Tumor specimens classified as MRP-1/ CD9 positive using Western blotting had >50% of the cancer cells immunostained with M31-15, and those classified as MRP-1/CD9 reduced had <50% of the cancer cells immunostained with M31-15. There were 97 patients with MRP-I/CD9 positive tumors and 46 patients whose tumors had reduced MRP-I/CD9 levels. The disease-free rate of the former group of patients was strikingly higher than that of the latter (84.7% versus 51.4%, P < 0.001). Similarly, the overall survival rate was also signifi cantly different between the two groups (93.6% versus 69.6%, P = 0.004). Multivariate analysis with the Cox regression model indicated that MRP1/CD9 positivity correlated better with disease-free survival (P < 0.001) than that ated may Kilaku, Osaka 530 ical grade, and estrogen receptor status. Recently, the focus pertaining to factors of potential prognostic utility has turned to the expression of molecular markers. A few of these have been reported to be associated with poor prognosis, and their presence or levels may be an indication for adjuvant therapy. For example, the c-erbB-2 oncogene overexpression is found to be associated with shorter survival, particularly in breast cancer patients with involved lymph nodes (3-5). Mutations of the tumor suppressor gene f>53 also may be of prognostic importance, especially for patients with node-negative disease (6. 7). On the other hand, it has been reported that nm23 protein expression in breast cancer was associated with good prognosis and absence of lymph node métastases,but that low nm23 mRNA levels correlated with the presence of lymph node métastasesat surgery (8, 9). We have previously generated a murine MAb\ M3I-I5. that in hibits cell motility and established that the antibody recognizes the MRP-1.' a transmembrane glycoprotein (10). The amino acid se quence of MRP-1 is identical to that of CD9, a WBC differentiation antigen, reported in the same year (11). MRP-1/CD9 is expressed not only by hematopoietic cells, but also by most established cell lines derived from solid human tumors. However, its role in nonhematopoietic cells remains unknown. In efforts to elucidate the functions of MRP-1/CD9 in such cells, we transfected various types of cultured cells with plasmid constructs containing human MRP-I/CD9 cDNA (12). These experiments revealed that cell motility and growth were suppressed in the MRP-l/CD9-expressing cells. In addition, investi gations with mouse melanoma BL6 cells and the BALB/c-/i(i//i» mouse system disclosed that the metastatic potential of all transfor mants expressing MRP-1/CD9 was lower than that of the parent BL6 cells (12). This set of observations indicated that MRP-1/CD9 regu estrogen receptor, tumor, and lymph node status. Our data suggest low MRP-1/CD9 expression by tumors of the breast may be associ with poor prognosis. It is conceivable that testing for MRP-1/CD9 identify node-negative breast cancer patients who are at high risk for early disease recurrence. Introduction Several randomized clinical trials have shown that adjuvant che motherapy improves the survival of patients with breast cancer (1-3). However, because it is not clear which clinical characteristics deter mine the need for adjuvant chemotherapy, it is very difficult to distinguish patients for whom surgery alone is adequate from those who may benefit from both surgery and additional treatment. Hence, the search for sensitive and reliable prognostic tests is of cardinal importance since they would be valuable for identifying patients for whom intensive adjuvant therapy is warranted (3). The prognostic factors that may help predict recurrences of breast cancer include the number of involved lymph nodes, the size of the tumor, its histologReceived 12/19/95; accepted 2/6/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate (his fact. 1 Supported in part by Grants-in-Aid from the Ministry of Education in Japan and The lates cell motility, and that it is a receptor for negative signal ligands. More recently, to investigate whether the levels of MRP-1/CD9 in tumor tissues are valuable for predicting the clinical behavior of actual human cancer, we tested MRP-1/CD9 expression in invasive ductal carcinomas of the breast with immunohistochemical study and immu noblotting. Thus, we found that MRP-1/CD9 expression diminished as the clinical stage of a given breast cancer advanced (13). Besides, primary tumors in almost half of the cases had higher MRP-1/CD9 protein levels than their respective metastatic lymph nodes. These findings, obtained with immunohistochemical and immunoblotting methods, were corroborated by determining MRP-1/CD9 gene expres sion with a quantitative reverse transcription-PCR-based assay. Gene overexpression was not observed in any of the samples studied (13). Moreover, we have applied quantitative reverse transcription-PCR analysis to determine MRP-I/CD9 gene expression in non-small cell lung cancer and found that low MRP-1/CD9 gene expression by tumors of the lung may be associated with poor prognosis ( 14). Based on these results, in this report we present the findings of the prospec tive evaluation of MRP-1/CD9 protein expression in tumor tissues 3 The abbreviations used are: MAb. monoclonal antibody: MRP-1. motility-related protein-1; TM4SF. transmembrane 4 supertamily. Sagawa Foundation for Promotion of Cancer Research (M. M.). 2 To whom requests for reprints should be addressed. 1244 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1996 American Association for Cancer Research. MRP-I/CD9 AS A PROGNOSTIC FACTOR OF BREAST CANCER Table 1 Disease-free sun'ival and overall sumval rale of 143 patients with breas! cancer according to clinicopathological characteristics and MRP-ÃŒ/CD9status MRP-1/CD9 rateMRP-1 survival rateMRP-1 survival statusCharacteristicsAge -'ci77.659.558.285.284.474.546.771.371.750.033.393.373. -%)48.155.036.070.875.050.020.080.032.150.0086.752.921.451.4P0.005<0.001•C0.0010.0180.0110.0310.1670.010<0. +(<90.397.788.8100.096.792.476.2100.089.985.780.0100.093.378.393.6 +(77.993.077.895.090.380.855.692.979.362.580.090.983.766.784.7MRP-1 + of patients)2620252120161020212315161546Disease-free (no.53445641612794440g537451597MRP-1 (yr)<50g50Estrogen at surgery status—+Nodal receptor statusNONIN2Tumor statusT,T2TT.Pathological stageStage IStage IIStage IIITotal no. of patientsMRP-1 from 143 breast cancer patients for whom adequate clinicopatholog ical data were available. Immunoblot Analysis. Tissue samples containing only cancer cells were solubilized with 1% CHAPS. An aliquot of the soluble fraction was subjected to slab gel electrophoresis, followed by Western blotting and probing with the anti-MRP-1/CD9 MAb, rabbit antimouse IgG (y chain specific; Cappel, Malvern, PA) and I25l-labeled protein A (DuPont, Boston, MA); Refs. 16 and Materials and Methods Clinical Characteristics of the Patients. This study was carried out on 143 patients with invasive ductal carcinoma of the breast. All of them under went mastectomy or quadrantectomy at the Department of Thoracic Surgery of the Kitano Hospital Medical Research Institute (Osaka, Japan) between May 1990 and December 1992. Complete clinical records of all of them were available, and the histopathological diagnoses were fully documented. The postsurgical pathological stage of each tumor was classified according to the TNM system (15). The salient clinical features of the patients are presented in Table 1. The median postoperative follow-up for the patients was 45.7, (range, 30-61) months. This report includes follow-up data as of July 1, 1995. Ten patients with intraductal carcinoma and nine with two or more forms of cancer were not included in the study. In addition, four women with stage IV disease were also excluded because even if their primary tumors are resected, such patients usually have cancer cells at the metastatic sites. This would make the disease-free survival analysis of these patients meaningless in the context of the present study. Eleven patients had quadrantectomy followed by immediate radiotherapy, and the other patients had no radiotherapy before recurrence. Adjuvant sys temic chemotherapy was given according to estrogen receptor status, meno pausa! status, and lymph node involvement. Node-positive or premenopausal patients («= 108) underwent treatment by oral 5-fluorouracil (200 mg/day) for 2 years, and, furthermore, 19 patients with N, disease were treated with six cycles of cyclophosphamide/Adriamycin. Sixty-two estrogen receptor-positive patients were treated with tamoxifen (20 mg/day) for 2 years or before recurrence. Twenty-two patients (postmenopausal node negative and receptor negative) did not have any further adjuvant treatment. We detected distant métastasesin 25 patients during the observation period, and among these 25 patients, 3 patients also had local recurrences. Twelve patients had locore- 17). The total protein content applied to the lanes was adjusted to the same concentration. Soluble extracts of the established human breast cancer cell line ZR-75-30 were used as positive controls. Band intensity was evaluated with densitometry. Immunohistochemical Assay. Since the MRP-1/CD9 antigen is not pre served in formalin-fixed paraffin-embedded tissues, we used frozen sections for these assays. They were performed using an antimouse IgG ABC kit (Vector Laboratories, Inc., Burlingame, CA) as described previously (13). Sections incubated with mouse myeloma SP2 supernatant served as negative reaction controls. Statistical Analysis. Overall survival curves and disease-free survival curves were constructed according to the Kaplan-Meier method (18). Com parisons were made with the log rank test (19). The SAS statistical package (SAS Institute, Cary, NC) was used for the Cox proportional hazards model; five factors (age at surgery, T stage, N stage, estrogen receptor status; and tumor MRP-1/CD9 status) were studied, and scores were assigned to each variable for the regression analysis (20). The MRP-1/CD9 expression and T stage, N stage, investigated using a frequency table. All P statistical analysis; a P value of <0.05 was significance. association between reduction in and estrogen receptor status was values are based on two-tailed considered to indicate statistical Results immediately embedded in optimum cutting temperature compound (Miles, Kankakee, IL) and frozen at -80°C. Frozen sections were cut on the cryostat Specimen Classification Based on Western Blotting Results. MRP-1/CD9 expression in breast tumor tissues was analyzed using Western blotting with MAb 31-15. The major Mr 25,000 MRP-1/CD9 band is usually accompanied by a minor Mr 28,000 band. The major band was readily evident in the immunoblots of the soluble fractions of the primary tumors that were classified as positive in the immunohistochemical assays, as described previously (Fig. 1; Ref. 13). By contrast, the band intensity was weak or entirely absent in the primary invasive ductal carcinomas that had reduced immunohistochemically detectable MRP-1/CD9. To classify the specimens on the basis of to a thickness of 6 ¿un,mounted on poly-D-lysine-coated slides, and either used immediately or stored at —¿ 80°Cfor no more than 2 weeks. To verify the Western blotting, the M, 25,000 band obtained with the positive control cells ZR-75-30 was set at 100%. The distribution of the ratio presence of cancer ceils, a frozen section from each specimen was stained immediately with H&E. One-half of a given tumor sample containing only of each specimen to the control is shown in Fig. 2. There was a clear difference among them with regard to intensity at the point of 3040%. Patient samples with a band intensity of >30% were classified gional recurrences. After recurrence, locoregional tumors or lymph nodes were principally resected and then these patients were treated with radiotherapy. Patients with distant métastaseswere treated with more effective adjuvant chemotherapy, including cisplatin and pirarubicin. Surgical Specimens. One-half of each freshly resected tumor tissue was cancer cells was used for Western blotting. 1245 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1996 American Association for Cancer Research. MRP-1/CTO AS A PROGNOSTIC FACTOR OF BREAST CANCER levels (Figs. 4 and 5). The disease-free survival rates were 84.7% and 51.4% (P < 0.001), respectively (Table 1). Moreover, tumors with reduced MRP-1/CD9 expression of N0 and N, disease or with T, and T2 disease were associated with progressively reduced disease-free survival (Table 1). The overall survival rate of patients with MRP-1/ CD9-positive tumors was 93.6% (Table 1) and that of the individuals whose tumors had reduced MRP-1/CD9 was 69.6% (P = 0.004). Prognostic Value of MRP-1/CD9 Status. The variables used in the Cox regression analysis are shown in Tables 2 and 3. The esti mated prognostic value of each variable in relation to disease-free survival and overall survival among the 143 patients is expressed as a P value. Four variables (MRP-1/CD9 status, estrogen receptor status, N stage, and T stage) were found to be significant prognostic factors of survival. MRP-1/CD9 had the most significant P value for diseasefree survival (P < 0.001), and the second with respect to overall survival (P = 0.0084). Other variables (body weight, serum albumin, 1 2345678 carcinoembryonic antigen, progesterone receptor status, postoperative hormonal therapy, and chemotherapy) had no relationship with MRP-1/CD9 status or its prognostic value (data not shown). Fig. 1. Immunoblot analysis of MRP- 1/CD9 expression in breast tumors. Lane I, breas! cancer cell line ZR-75-30 (positive control); Lanes 2, 4, and 6-8. primary invasive ductal carcinomas with reduced MRP-1/CD9 levels; Limes 3 and 5. primary invasive ductal carcinomas with high MRP-1/CD9 levels. Discussion as MRP-1/CD9 positive, and those with intensities <30% as negative. Of the 143 primary breast cancers studied, 97 (67.8%) were classified as MRP-1/CD9 positive. There were 46 cases (32.2%) with negative MRP-1/CD9 expression. Immunohistochemical Detection of MRP-1/CD9 in Breast Cancer Tissues. In primary breast cancers classified as MRP-1/CD9 positive, expression resembled that of normal glands and benign fibroadenomas (Fig. 3A) since immunostaining was intense and was seen uniformly at the cell surface membrane (Fig. 3B). There were 46 cases (32.2%) with reduced MRP-1/CD9 expression (Fig. 3C). Im munostaining of most of these tumors was heterogeneous, and the staining pattern along cell junctions was not linear, but granular. In each instance, MRP-1/CD9 expression in metastatic lymph nodes was lower than that in the corresponding primary tumor (Fig. 3D). All immunostained sections were examined by two pathologists who had no knowledge of the patients' clinical status. At least 200 tumor cells were counted per X40 field. Positive tumor cells were stained equivalent to normal breast glands and benign fibroadenoma tumor cells. When >50% of the carcinoma cells in a given specimen were positively stained, the sample was classified as MRP-1/CD9 positive, and when <50% were stained, the sample was classified as reduced. Overall, the immunoblotting results agreed well with those of the imniunohistochemical assays, and, in seven cases of discrepancy, the results of Western blotting were used in specimen classification. Relationship between Tumor MRP-1/CD9 Immunoreactivity and Known Prognostic Factors. Analysis of the results obtained with the 143 breast carcinomas tested revealed no statistically signif icant relationship (x2) between MRP-1/CD9 expression and the pa tients' age at surgery or estrogen and progesterone receptor status, Prevention of metastasis is among the most important problems in the treatment of patients with malignant tumors (21. 22). Despite various types of antitumor defense mechanisms, the malignant tumor cells that invade tissue boundaries and move through the host's cellular and extracellular matrix barriers resist the shear stresses arising in the vascular bed, the frictional forces arising between their peripheries, and the vessel walls. In addition, they traverse capillaries that are generally rigid and whose diameter is smaller than the tumor cells. It has been shown that motility of cancer cells is one of the essential cellular functions closely related to the metastatic process (21, 22). We have previously demonstrated that motility of M AC 10 cells, derived from a human adenocarcinoma of the lung, is inhibited by MAb M31-15, and that this antibody recognizes MRP-1, a transmem brane glycoprotein (10). We also determined that the sequence of MRP-1 is identical to that of CD9. the sequence of which was reported in the same year by another laboratory (11). Because disruption of cell to cell contacts induces tumor cell invasion and métastases,it is of interest in the present context that MAbs to CD9 elicit an enhance ment of Fc-independent heterotypic adhesion of pre-B cell lines to bone marrow stromal fibroblasts, but not to bone marrow stroma (23). Moreover, these antibodies specifically induce homotypic adhesion of pre-B lymphocytes and augment neutrophil adherence to the endothe- (N) 30-1 s u 20- o. i— o IM tumor size, or postoperative hormonal therapy and chemotherapy. However, MRP-1/CD9 expression was associated with lymph node status (P = 0.045). Thus, whereas 24.7% of the patients with N0 stage disease had reduced expression, low MRP-1/CD9 was found in 37.2% of the N, stage patients and 52.6% of those with N2 stage. Association of Tumor MRP-1/CD9 Status with Disease-free and Overall Survival. Comparing survival among the 143 patients re vealed that the disease-free and overall survival rates for the 97 patients with MRP-l/CD9-positive tumors differed significantly from those of the 46 individuals whose tumors had reduced MRP-I/CD9 25- 15- o .o g 5- -ñUil 0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-9090-100 100< r;.) Ratio of western blotting value Fig. 2. Distribution of the ratio of Western blotting values of tumor specimens:the control cell line ZR 75-30. 1246 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1996 American Association for Cancer Research. MRP-1/CD9 AS A PROGNOSTIC FACTOR OF BREAST CANCER B •¿ •¿ . ~ •¿ 4 . V Fig. 3. Immunohistochemical staining of human breast tissues with the MRP-l/CD9-specific MAb M31-15. Avidin-biotin-peroxidase complex procedure. A, the cell surface in a benign fibroadenoma was strongly stained, ß.positive immunostaining of an invasive ductal carcinoma of the breast. C. invasive ductal carcinoma with reduced MRP-1/CD9 expression. D. metaslatic lymph node corresponding to the invasive carcinoma shown in B. Note that staining intensity is lower than in the primary tumor. A-D, X 150. S 100-ça -75 c¿ go 80-b 1^~~V, around 15 members that are variously expressed by leukocytes and a variety of mammalian tissues, as well as on the surface of two types of parasites (26). Although their precise functions are still unknown, current data largely suggest that TM4SF proteins are involved in the regulation of cell development, proliferation, activation, and motility. The melanoma-associated antigen ME491/CD63 is an interesting member of this superfamily. It is preferentially expressed in early stages of tumor progression, but expression declines significantly and sometimes disappears entirely in advanced, rapidly growing melano mas and metastatic melanoma cells (27). In addition, the predicted ~1"1 (n=97)v_1MRP-1/CD9MRP-1/CD9 positive 60-W 50-<B 40-^ 30-S =46)P<0.0010 reduced (n 20-I 10-f*S O "*"T-^1 10 20 30 40 5060 7 \7v)100-£ 97)~^~ Months after Surgery 90-(2 Fig. 4. Disease-free survival in 143 patients with invasive ductal carcinoma of the breast ¡nrelation to the tumor MRP-I/CD9 status (P < 0.001). P value was determined with the log rank test. positive (n = ^i_^MRP-1/CD9 80-n|60-i (n=46)P reduced Hum probably caused by an adhesion-promoting activation event within the endothelial cells (24). These findings would suggest a direct participation of CD9 in signal transduction. Since the MAb to MRP- 1/CD9 inhibits the motility of various types of cancer cells, it is conceivable that the glycoprotein affects motility and invasiveness by modulating the levels of cellular adhesion (10, 13). MRP-1/CD9 belongs to the TM4SF, which generally has four highly conserved hydrophobic domains that are assumed to span the lipid bilayer of the cell membrane (25, 26). The TM4SF consists of 50-w 40-75 30-£20O = 0.004 10n -MRP-1/CD9 10 20 30 40 50 60 70 Months after Surgery Fig. 5. Overall survival of the 143 patients with invasive ductal carcinoma of the breast in relation to the tumor MRP-I/CD9 status (P = 0.004). 1247 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1996 American Association for Cancer Research. MRP-I/CD9 AS A PROGNOSTIC FACTOR OF BREAST CANCER Table 2 Value of five variables in predicting disease-free survival of 143 patients with breast cancer VariablesMRP-1/CD9-+Age interval-1.4769 ß score010101123401295% SE Wald's X2 Hazard ratio 0.109-0.4790.1456 0.3781 15.2535 0.228 0.560-2.390-1.6012 0.3703 0.1546 1.157 0.086-0.4710.6842 0.433 13.6761 0.202 1.348-2.9160.7155 0.1969 12.0755 1.982 10.7729 2.045 Confidence (yr)<50g50Estrogen receptor-+Tumor statusT,T2TjT4Nodal statusN„N,N2Assigned 0.218 1.334-3.136P<0.0010.69420.00020.00050.0010 Table 3 Value of five variables in predicting overall xunival of 143 patients with breast cancer VariablesMRP-1/CD9-+Age (yr)<50SSOEstrogen x2-1.4083 score0101011234012ß SE Wald's interval0.245 ratio 6.9566-0.1422 0.5339 0.086-0.6960.867 0.0699-1.3385 0.5381 0.302-2.4900.262 4.28870.8360 0.6463 0.074-0.9312.307 9.02120.7262 0.2784 1.337-3.9812.067 Confidence receptor-+Tumor statusTIT,T3T4Nodal statusNON,N2Assigned 0.3153 5.303795%Hazard product encoded by KAII, a recently described metastasis suppressor gene of prostate cancer, is identical to R2 and C33, which also belong to the TM4SF (28-30). It is thought that decreased KAI1 expression may be involved in the malignant progression of prostate cancer (28). These observations on other systems are very consistent with our data that the level of MRP-1/CD9 expression is inversely related to the clinical stage of a given cancer of the breast. Thus, MRP-l/CD9-positive tumors were evident in 73% of the patients whose tumors were in stages I or II, but only in 50% of patients with stage III tumors. As the present results indicate, reduction in MRP-1/CD9 expression by primary invasive ductal carcinomas of the breast was associated with poor prognosis. The link between diminished MRP-1/CD9 and poor prognosis was independent of tumor stage, lymph node status, and estrogen receptor status. Reduction in tumor MRP-1/CD9 expres sion was strongly associated with an increased risk of recurrence among the patients with N0 and N, stage disease and with T, and T2 tumors. However, the interpretation of these findings has to be made with caution in view of the relatively short postoperative follow-up (30-61 months) of the patients. Adjuvant endocrine therapy and chemotherapy have signifi cantly improved the disease-free survival of patients with early stage breast cancer (1-3). However, the role of adjuvant chemo therapy after mastectomy, particularly in women whose disease is confined to the breast, has remained unresolved. It is possible that patients with MRP-l/CD9-positive tumors may not require adju vant chemotherapy after mastectomy. By contrast, patients whose tumors have reduced MRP-1/CD9 levels, even if confined to the 1.114-3.835P0.00840.79150.03840.00270.0213 breast, have a poor prognosis, and may therefore benefit from adjuvant treatment. It is conceivable that testing tumors for MRP1/CD9 status, in combination with other molecular and biochem ical assays, may improve the prognostic evaluation of breast cancer patients and enhance the clinician's ability to prospectively iden tify patients who will have early disease recurrence require adjuvant chemotherapy. and who will Acknowledgments We thank Dr. Masato Vagita for critical editorial help with the manuscript, Tatsuya Hirai for the technical assistance, Drs. Shinji Sawada and Tadashi Oobayashi for the histopathological examination of the tumor samples, and Dr. Isao Horiguchi for statistical analysis. References 1. Early Breast Cancer Trialists' Collaborative Group. Effect of adjuvant tamoxifen and of cytotoxic therapy on mortality in early breast cancer: an overview of 61 random ized trials among 28,896 women. N. Engl. J. Med., 319: 1681-1692, 1988. 2. Early Breast Cancer Trialists' Collaborative Group. 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