Predictive Value of C-Erbb-2 and Cathepsin-D for Greek Breast Cancer Patients Using Univariate and Multivariate Analysis

Vol. 5, 815–821, April 1999 Clinical Cancer Research 815

Predictive Value of c-erbB-2 and Cathepsin-D for Greek Breast Cancer Patients Using Univariate and Multivariate Analysis

Andreas Scorilas, Julia Yotis, Charilaos Pateras, ever, none of the prognostic factors available at present is able Theoni Trangas, and Maroulio Talieri1 to determine the final outcome with certainty, and there is contradicting data in the literature on whether the newer prog- Departments of Virology and Biochemistry, “G. Papanikolaou” Research Center of Oncology [A. S., T. T., M. T.], Hormone Receptor nostic factors can be used to predict long-term survival. Unit [J. Y.], and Breast Cancer Clinic [C. P.], “St. Savas” Hospital, Insight into correlation between specific gene interactions Athens, 11522, Greece and the clinical behavior of tumors may provide new prognostic tools and may lead to new treatment strategies. Her2/neu (c-erbB-2 protein) shares an extensive homology with epidermal ABSTRACT growth factor receptor (1–3). High c-erbB-2 expression is an The value of various prognostic factors in breast cancer early feature of some breast tumors, present even at the nonin- patients has been determined in a number of studies. One vasive stage (4). Tumors with activated c-erbB-2 show several hundred thirty-eight Greek women were followed up over a characteristics of an aggressive phenotype (5–6). Although not 5-year period after surgery for breast cancer. Amplification generally considered to be correlated with axillary lymph node and overexpression of c-erbB-2 was found in 22.4% and status, c-erbB-2 activation is clearly associated with an in- 29.7% of the respective cases, and the concentration of total creased number of involved nodes, the presence of distant > cytosolic Cathepsin-D (CD) in 46.4% of them was high ( 60 metastases at diagnosis and in node-positive patients, early pmol/mg protein). The examined biological variables were recurrence, and death (6–11). The reason for failure to predict compared with standard clinicopathological prognostic fac- disease outcome in node-negative breast cancer is unclear. Pos- tors for the disease and related to early relapse (ER; before sibly, c-erbB-2 is involved in cell proliferation and, thus, confers 3 years), relapse-free survival (RFS; median, 5 years), and a growth advantage in the early stages and in local cancer. overall survival (OS; median, 5 years). It was found that To express its full potential in systemic disease, the gene high CD levels significantly shorten ER of both node-nega- may need to act in concert with other events, which also render ؍ tive and node-positive patients (P < 0.0001 and P 0.002, the cell capable of metastasizing. As shown, overexpression of respectively) and have prognostic value for RFS and OS of c-erbB-2 causes enhanced cell migration in human breast cancer ؍ ؍ node-negative patients (P 0.0012 and P 0.0288, respec- cells (12). One of the molecular mechanisms involved in the tively), but lose their value as relapse predictors for node- process of metastasis may be overproduction of proteases positive patients for periods longer than 3 years. Overex- that degrade the basement membrane and the extracellular pression of c-erbB-2 was found to be predictive for OS of matrix (13). ؍ ؍ node-positive and -negative patients (P 0.0048 and P The most extensively studied protease in human breast 0.0285, respectively), but its predictive power was weak for cancer is CD2, which was first identified as a 52-kDa estrogen- ؍ ؍ ER (P 0.0456) and RFS (P 0.0455) of node-negative dependent glycoprotein in MCF-7 cells (14). Several studies on patients and disappeared for node-positive patients. the prognostic value of CD in breast cancer have generally c-erbB-2 amplification offers minimal assistance to the pre- revealed a trend for poor survival if a high CD level has been diction. In conclusion, high CD concentration is indicative of detected (15–19). A combination of conventional and newer ER of patients, and c-erbB-2 overexpression correlates with tumor markers may identify patients with a worse prognosis OS of patients. within groups with a generally favorable prognosis and may lead to improved treatment modalities (20, 21). INTRODUCTION In the present report, we decided to extend our previous Postoperative treatment of women with breast cancer studies (22, 23) using a larger number of patients, as well as ranges from observation without further treatment to bone mar- univariate and multivariate Cox and LR analyses, to examine the row transplantation. These greatly different therapeutic deci- prognostic significance of CD and c-erbB-2 in relation to clin- sions are based on individually assessed risk for relapse. How- icopathological variables for the RFS and OS of Greek women with breast cancer. We focused our attention not only on long RFS, as most investigators have, but also on ER, in an attempt to assess the malignant potential of individual breast carcinomas and to speculate on new therapeutic schemes. Received 8/4/98; revised 12/3/98; accepted 1/5/99. 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 this fact. 1 To whom requests for reprints should be addressed, at Department of 2 The abbreviations used are: CD, cathepsin-D; ER, early relapse; OS, Pharmacology, Wayne State University, Gordon H. Scott Hall of Basic overall survival; RFS, relapse-free survival; LR, logistic regression; RR, Medical Sciences, 540 East Canfield Avenue, Detroit, MI 48201. Phone: relative risk; CI, confidence interval; EsR, estrogen receptor; PgR, (313) 577-1580; Fax: (313) 577-6739. progesterone receptor.

MATERIALS AND METHODS Steroid Receptors. EsRs and PgRs were assayed by the Patients. Tumor specimens from 138 patients (mean age, charcoal method, as previously described (30, 31). The cutoff 59 years; range, 24–89 years) with no signs of distant metas- level used was 10 fmol/mg cytosolic protein (results evaluated tasis and who underwent surgery for primary breast cancer from by quality control assessment; Ref. 31). 1988–1992 [modified mastectomy, 52 patients (34.7%); breast- CD Assay. CD was assayed by an immunoradiometric conserving lumpectomy, 86 patients (62.3%)] at “Saint Savas” kit (Elsa Cath-D kit; CIS Bio International, Gif-sur-Yvette, France), according to the procedure described by the manufac- Hospital in Athens were evaluated in this study. Tumor speci- turer and elsewhere (31), in 1:40 and 1:80 dilution of the mens were submitted to the Laboratory of Hormone Receptors reconstituted cytosols, both in duplicate. for steroid receptor analysis. Women with positive lymph nodes Statistics. Cox and LR analyses were used to study the generally received adjuvant chemotherapy (48 patients); one association between CD or c-erbB-2 against patient and disease hundred two patients received adjuvant (tamoxifen) therapy, characteristics. The Cox proportional hazard model was used for whereas 113 patients were irradiated. Median follow-up for univariate and multivariate analyses (32). For multivariate anal- patients was 60 months. A computerized database containing ysis, the forward LR test was used. RRs and 95% CIs are updated clinicopathological information was available for sta- presented only for retained variables, significant in multivariate tistical analysis. analysis. Tumor size and differentiation grade are continuous Tumor Sample Processing. Tumor tissue was stored in variables, with scores 1–3. liquid nitrogen. Samples were processed as described previously (22, 23). Tissue was pulverized in the frozen state and homogenized in 5 ml of cytosol buffer [10 mM Tris, 1.5 mM EDTA, 5 RESULTS mM NaMolybdate (pH 7.4), and 5 mM DTT]. The homogenates The frequency of c-erbB-2 amplification measured in 138 underwent centrifugation at 40,000 rpm for1hat4°C, and the primary breast tumors was 22.4% and that of overexpression cytosols were kept at -80°C for later processing. The same was 29.7%, whereas 46.4% of tumors had high concentrations Ն cytosols were used for hormone receptors and for CD assays. of CD ( 60 pmol/mg protein; Table 1). With a median fol- DNA was isolated from 100 mg of tumor tissue, minced finely, low-up of 5 years, 55.7% of patients had a RFS and 71.7% of patients had an OS, whereas 71% of all patients experienced and dispersed in 1 ml of 2xTNE [20 mM Tris (pH 8.0), 300 mM only short (3 years) RFS (Table 1). The patients examined were NaCl, and 20 mM EDTA] containing 0.5% SDS and digested divided in subgroups according to survival (ER, RFS, and OS) with proteinase K (100 ␮g/ml) at 37°C. After repeated phenol, and according to nodal status (negative and positive). phenol/chloroform, and chloroform/isoamyl alcohol extractions, intact genomic DNA was pooled after precipitation with 2 volumes of ethanol. RNA was isolated from frozen samples, ground to a fine powder in liquid nitrogen, and subsequently homogenized in an acid guanidine thiocyanate- phenol- chloro- Table 1 Distribution of patients on the basis of the factors examined form solution according to Chomczynski et al. (24). Southern No. of patients (total 138 Factor tumor samples) % blotting of ECO-R1-digested DNA was performed by standard techniques (25, 26). The integrity of the RNA was confirmed by Age formaldehyde-agarose gel electrophoresis. Northern blotting Յ50 yrs 39 28.3 51–65 yrs 54 39.1 was performed according to Thomas (27). Equal amounts of Ͼ ␮ 65 yrs 45 32.6 DNA (20 g) were slot blotted on nylon membranes (Hybond c-erbB-2 amplification 31 22.4 ϩ N ; Amersham Corp.). RNA (20 ␮g) was slot blotted according c-erbB-2 overexpression 41 29.7 to Maniatis et al. (28). CD 64 46.4 Ն Oncogene Detection. To determine c-erbB-2 overex- 60 pmol/mg protein EsR positive 111 80.4 pression or amplification, blots were hybridized to c-erbB-2 by PgR positive 118 85.5 a5Ј end labeling procedure using[␥-32P]ATP. The probe used Lymph node positive 63 45.6 was human oligonucleotide Pr 2 (Oncogene Science No ON Tumor stage T 45 32.6 112). The hybridization was performed according to the instruc- 1 T 78 56.5 tions of the manufacturer and others (29). Briefy, the blots were 2 T3/4 15 10.9 washed at high stringency (0.3 ϫ standard sodium saline cit- Grade rate), autoradiography with intensifying screens was performed I 10 7.2 for 2–4 days at -70°C using kodak X-OMAT 100 films, and II 90 65.2 III 36 26.1 autoradiograms were scanned with a Bio-Rad video densitom- Xa 2 1.5 eter 620. DNA and RNA extracted from normal breast tissue Menopausal status (obtained during mastectomy of cancer patients from areas pre/peri 36 26.1 distant to cancer) were used as negative controls. The values post 102 73.9 3 years RFS 98 71 obtained for c-erbB-2 by densitometer scanning were normal- RFSb 77 55.7 ized to values derived from ␤-actin. To determine amplification OSb 99 71.7 or overexpression, the ratios obtained were compared with av- a X, status unknown. erage values derived from normal samples. b Median follow-up, 5 years.

Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. Table 2 Univariate and multivariate analysis in 63 breast cancer patients with lymph node metastases Downloaded from ERa (before 3 years) RFSb (median, 5 years) OSb (median, 5 years) Univariate Multivariate Univariate Multivariate Univariate Multivariate Factor PrPrRR 95% CI PrPrRR 95% CI PrPrRR 95% CI CD 0.002 0.3421 0.0083 0.1431 2.33 1.15–4.67 NS NS NS NS c-erbB-2 NSc NS NS NS 0.0048 0.3452 0.0243 Ϫ0.1742 2.54 1.17–5.50

clincancerres.aacrjournals.org overexpression c-erbB-2 NS NS NS NS 0.1830 NS amplification EsR 0.0367 Ϫ0.1921 NS NS NS NS NS PgR 0.0453 Ϫ0.1342 NS NS NS NS NS Grade 0.0215 0.2431 NS NS NS NS NS Tumor stage 0.0123 0.2873 0.0077 0.1601 2.76 1.31–5.81 0.0815 NS 0.0010 0.3926 0.0069 Ϫ0.2747 3.21 1.49–6.89 Age NS NS 0.0345 Ϫ0.0956 NS 0.0170 Ϫ0.1373 NS Menopausal NS NS 0.0258 Ϫ0.1213 0.0456 Ϫ0.11 0.54 0.35 –0.82 NS NS status a LR analysis. b Cox regression analysis. c NS, not significant (P Ͼ 0.05). on September 28, 2021. © 1999American Association for Cancer Research.

Table 3 Univariate and multivariate analysisa in 75 breast cancer patients without lymph node involvement ERa (before 3 years) RFSb (median, 5 years) OSb (median, 5 years) Univariate Multivariate Univariate Multivariate Univariate Multivariate Factor PrPrRR 95% CI PrPrRR 95% CI PrPrRR 95% CI CD 0.0001 0.4265 0.0037 0.3167 6.63 3.78–11.58 0.0012 0.3514 0.0042 0.2606 5.13 2.96–8.92 0.0285 0.2147 0.0104 0.1924 4.99 2.88–8.49 c-erbB-2 0.0456 0.1692 NSc 0.0455 0.1104 NS 0.0084 0.3779 0.0301 0.2655 6.93 4.98–11.13 overexpression c-erbB-2 NS NS NS NS 0.045 0.1213 NS amplification EsR 0.0406 Ϫ0.185 NS 0.044 Ϫ0.124 NS 0.0024 Ϫ0.434 NS PgR NS NS NS NS NS NS Grade 0.0306 0.2153 0.0276 0.166 3.5 2.12–5.77 NS NS NS NS Age NS NS NS NS 0.0139 Ϫ0.325 NS Tumor stage 0.0103 0.2946 NS 0.014 0.1643 0.0180 0.1242 2.36 1.45–3.93 0.0142 0.2443 0.0423 0.1544 2.81 1.70–4.63 lnclCne Research Cancer Clinical Menopausal status NS NS NS NS 0.0443 Ϫ0.131 NS a LR analysis. b Cox regression analysis. c NS, not significant (P Ͼ 0.05). 817 818 c-erbB-2 and CD in Breast Cancer

Fig. 1 Cox regression analysis of RFS (A) and OS (B) curves stratified by high concentrations (Ն60 pmol/mg protein) of CD status; median follow-up was 60 months. Bold line, group of patients with high CD.

Statistical Analysis of 63 Patients with Positive Lymph conflicting results concerning its true role in prognosis (38–40). Nodes. For the ER of patients with lymph node involvement, Various investigators have suggested that the presence of CD in the contribution of high CD levels, grade, and tumor stage is stromal and inflammatory cells has a causative association with positive and that of EsR and PgR levels are negative. The most invasiveness (41, 42). Also, the prognostic value of c-erbB-2 in accurate prediction for ER is based on CD and grade (P ϭ breast carcinomas has been controversial. The findings of the 0.0023 and P ϭ 0.00077, respectively); for RFS, however, only present work are in agreement with our previous studies (18, 22, age and menopausal status have an impact, whereas CD and 35) and with those of others (15–17), confirming the adverse c-erbB-2 overexpression are not significant. OS is negatively effects of high CD levels on the clinical outcome of breast associated with c-erbB-2 overexpression and amplification. cancer patients. According to Razumovic et al. (43), the CD, as Nevertheless, the best prediction is based on a combination of measured by the immunoradiometric method, represents the c-erbB-2 overexpression and tumor stage (P ϭ 0.0243 and P ϭ cumulative result of CD content in both carcinoma cells and 0.0069, respectively; Table 2). stromal macrophages. Parametric Analysis of 75 Patients with Negative According to the present study, CD has a significant prog- Lymph Nodes. As shown in Table 3, for the ER of patients nostic value for ER, especially of node-negative patients (Table without lymph node involvement, the outcome correlates posi- 3), in agreement with previous studies (41, 42, 44), although tively with CD, c-erbB-2 overexpression, grade, and tumor recent data (45), also obtained via the immunoradiometric tech- stage, and correlates negatively with EsR. By multivariate anal- nique, conclude that CD is of doubtful value in predicting risk ϭ ysis, CD (P 0.0037) was found once more to be the most of ER or death for patients with newly diagnosed invasive breast potent prognostic variable for ER, and the prediction could be cancer. The prognostic relevance of CD for RFS (Fig. 1A)is improved by considering the grade. RFS is negatively associated decreased if node-negative patients only are considered, in with CD, c-erbB-2 overexpression, and tumor stage and posi- agreement with a recent meta-analysis (39), and disappears if tively associated with EsR. Multivariate analysis for RFS once node-positive patients only are regarded, which, nevertheless, again points to CD (P ϭ 0.0042) as the most important variable, contradicts results by Duffy et al. (16). The CD measurement by followed by tumor stage. OS is negatively associated with CD, multivariate analysis does not contribute to precision of predic- c-erbB-2 overexpression, and amplification, as well as tumor tion for OS either in the case of node-positive (Table 2) or stage, and positively associated with EsR, age, and menopausal node-negative (Table 3) patients (Fig. 1B). In addition, we status. In multivariate analysis, the most accurate prediction for noticed that CD gives a better prediction for RFS of stages I and OS results from the combination of CD, c-erbB-2 overexpres- II (data not shown), which has been reported by others also (37, sion, and tumor stage. 38). However, for stages III and IV patients, it does not improve the precision of the prediction. All this controversy in the DISCUSSION literature is obviously due to different measurement methods The necessity for new prognostic subgroups in breast can- and the use of different antibodies. Therefore, the standardiza- cer patients is well recognized. CD and c-erbB-2 are two relation of techniques is of paramount importance. In this study and tively new biological markers. The lysosomal protease CD, first in previous analyses (18, 35), we noticed that CD is the only one described by Westley and Rochefort (33), has been shown to of the examined variables with a statistically negative correla- correlate with prognosis of breast cancer in numerous studies tion for locoregional recurrence. Another interesting observation (15, 16, 18, 19, 22, 23, 34–37). However, other studies give of this study is that in the Greek population we find a substantial

Fig. 2 Cox regression analysis of RFS and OS curves stratified by c-erbB-2 amplification and overexpression status: RFS as a function of cerbB-2 amplification (A) and overexpression (B); OS as a function of c-erbB-2 amplification (C) and overexpression (D); median follow-up was 5 years. “Normal,” two copy numbers of the proto-oncogene; “amplified,” 3–10 gene copies; “overexpressed,” 3–11 times; bold lines, group of amplified or overexpressed genes.

proportion of women with positive EsR (Ͼ78%) as well as PgR found in proliferating tumors. By contrast, overexpression of (Ͼ 80%).3 This may be due to the appearance of breast cancer c-erbB-2 does not show considerable differences in tumors with at a later age in these women or to prior parity; in any case, it distinct grade types. Moreover, 12 patients (8%) with c-erbB-2 needs further investigation. amplification did not display parallel overexpression of the The importance of c-erbB-2 amplification and overexpres- gene. Ten of the 12 patients had a long RFS. The existence of sion as a prognostic indicator in breast cancer has been exten- gene amplification without concurrent overexpression may be sively studied since the initial report of Slamon et al. (8), who due to transcriptional inhibition. The present data suggest that showed that c-erbB-2 amplification is an independent prognos- only c-erbB-2 overexpression (Fig. 2, B and D), but not its tic factor in node-positive patients. Although we previously (23) amplification (Fig. 2, A and C) has a prognostic value for breast showed that there is a positive relationship between c-erbB-2 cancer patients. These results are in agreement with Berns et al. amplification and overexpression, in the present study we found (46) and Kreipe et al. (47), but are in contrast to recent data (48, 22 patients (16%) having c-erbB-2 overexpression without am- 49) showing (by PCR) that c-erbB-2 amplification is a signifi- plification. Most of these patients had an ER or did not respond cant prognostic factor. Recently Beckmann et al. (50), using to chemotherapy. We also found differences in the relation immunohistochemistry, showed that c-erbB-2 gives no advan- between c-erbB-2 amplification and overexpression and clini- tage in predicting recurrence or survival in breast cancer pa- copathological characteristics (e.g., grade). Interestingly tients. Koscielny et al. (51), using an ELISA method to measure enough, most of the samples with c-erbB-2 amplification were c-erbB-2 oncoprotein in tumor cytosols, reported potential prog- grade II, which suggests that c-erbB-2 amplification is mainly nostic significance of low value, whereas Anan et al. (49), using fine-needle aspirates, assert that c-erbB-2 mRNA expression is useful in assessing the malignant potential of individual breast carcinomas. c-erbB-2 overexpression (Tables 2 and 3) does not 3 Unpublished data. seem to be predictive for ER. In contrast to CD, however, it has

small prognostic values for RFS if we examine the patients as a 11. Lonn, U., Lonn, S., Nilsson, B., and Stenkvist, B. Prognostic whole (data not shown), in agreement with the results of other significance of c-erbB-2 amplification in fine-needle biopsies of breast investigators (10, 52) who had used immunohistochemistry. cancer patients not operated at diagnosis. Breast Cancer Res. Treat., 39: 213–220, 1996. Nevertheless, the present study attributes substantial prognostic 12. Verbeek, A., Adriaansen-Slot, S. S., Vroom, T. M., and Rijksen, G. value to c-erbB-2 overexpression for OS regardless of nodal Overexpression of EGFR and c-erbB-2 causes enhanced cell migration status (Tables 2 and 3). in human breast cells and NIH3T3 fibroblasts. FEBS Lett, 425: 145– In conclusion, with this study we confirmed and extended 150, 1998. our previous results (22) showing that CD and c-erbB-2 over- 13. Rochefort, H. 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Andreas Scorilas, Julia Yotis, Charilaos Pateras, et al.

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