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Clinical Cancer Research Vol. 5, 815-821, April 1999
© 1999 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

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, Theoni Trangas and Maroulio Talieri1

Departments of Virology and Biochemistry, "G. Papanikolaou" Research Center of Oncology [A. S., T. T., M. T.], Hormone Receptor Unit [J. Y.], and Breast Cancer Clinic [C. P.], "St. Savas" Hospital, Athens, 11522, Greece


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The value of various prognostic factors in breast cancer patients has been determined in a number of studies. One hundred thirty-eight Greek women were followed up over a 5-year period after surgery for breast cancer. Amplification and overexpression of c-erbB-2 was found in 22.4% and 29.7% of the respective cases, and the concentration of total cytosolic Cathepsin-D (CD) in 46.4% of them was high (>= 60 pmol/mg protein). The examined biological variables were compared with standard clinicopathological prognostic factors for the disease and related to early relapse (ER; before 3 years), relapse-free survival (RFS; median, 5 years), and overall survival (OS; median, 5 years). It was found that high CD levels significantly shorten ER of both node-negative and node-positive patients (P < 0.0001 and P = 0.002, respectively) and have prognostic value for RFS and OS of node-negative patients (P = 0.0012 and P = 0.0288, respectively), but lose their value as relapse predictors for node-positive patients for periods longer than 3 years. Overexpression of c-erbB-2 was found to be predictive for OS of node-positive and -negative patients (P = 0.0048 and P = 0.0285, respectively), but its predictive power was weak for ER (P = 0.0456) and RFS (P = 0.0455) of node-negative patients and disappeared for node-positive patients. c-erbB-2 amplification offers minimal assistance to the prediction. In conclusion, high CD concentration is indicative of ER of patients, and c-erbB-2 overexpression correlates with OS of patients.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Postoperative treatment of women with breast cancer ranges from observation without further treatment to bone marrow transplantation. These greatly different therapeutic decisions are based on individually assessed risk for relapse. However, none of the prognostic factors available at present is able to determine the final outcome with certainty, and there is contradicting data in the literature on whether the newer prognostic factors can be used to predict long-term survival.

Insight into correlation between specific gene interactions 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 growth factor receptor (1, 2, 3) . High c-erbB-2 expression is an early feature of some breast tumors, present even at the noninvasive stage (4) . Tumors with activated c-erbB-2 show several characteristics of an aggressive phenotype (5, 6) . Although not generally considered to be correlated with axillary lymph node status, c-erbB-2 activation is clearly associated with an increased number of involved nodes, the presence of distant metastases at diagnosis and in node-positive patients, early recurrence, and death (6, 7, 8, 9, 10, 11) . The reason for failure to predict disease outcome in node-negative breast cancer is unclear. Possibly, c-erbB-2 is involved in cell proliferation and, thus, confers a growth advantage in the early stages and in local cancer.

To express its full potential in systemic disease, the gene may need to act in concert with other events, which also render the cell capable of metastasizing. As shown, overexpression of c-erbB-2 causes enhanced cell migration in human breast cancer cells (12) . One of the molecular mechanisms involved in the process of metastasis may be overproduction of proteases that degrade the basement membrane and the extracellular matrix (13) .

The most extensively studied protease in human breast cancer is CD2 , which was first identified as a 52-kDa estrogen-dependent glycoprotein in MCF-7 cells (14) . Several studies on the prognostic value of CD in breast cancer have generally revealed a trend for poor survival if a high CD level has been detected (15, 16, 17, 18, 19) . A combination of conventional and newer tumor markers may identify patients with a worse prognosis within groups with a generally favorable prognosis and may lead to improved treatment modalities (20 , 21) .

In the present report, we decided to extend our previous studies (22 , 23) using a larger number of patients, as well as univariate and multivariate Cox and LR analyses, to examine the prognostic significance of CD and c-erbB-2 in relation to clinicopathological 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.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
Tumor specimens from 138 patients (mean age, 59 years; range, 24–89 years) with no signs of distant metastasis and who underwent surgery for primary breast cancer from 1988–1992 [modified mastectomy, 52 patients (34.7%); breast-conserving lumpectomy, 86 patients (62.3%)] at "Saint Savas" Hospital in Athens were evaluated in this study. Tumor specimens were submitted to the Laboratory of Hormone Receptors for steroid receptor analysis. Women with positive lymph nodes generally received adjuvant chemotherapy (48 patients); one hundred two patients received adjuvant (tamoxifen) therapy, whereas 113 patients were irradiated. Median follow-up for patients was 60 months. A computerized database containing updated clinicopathological information was available for statistical analysis.

Tumor Sample Processing.
Tumor tissue was stored in 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 mM NaMolybdate (pH 7.4), and 5 mM DTT]. The homogenates underwent centrifugation at 40,000 rpm for 1 h at 4°C, and the cytosols were kept at -80°C for later processing. The same cytosols were used for hormone receptors and for CD assays. DNA was isolated from 100 mg of tumor tissue, minced finely, and dispersed in 1 ml of 2xTNE [20 mM Tris (pH 8.0), 300 mM NaCl, and 20 mM EDTA] containing 0.5% SDS and digested with proteinase K (100 µg/ml) at 37°C. After repeated phenol, 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- chloroform solution according to Chomczynski et al. (24) . Southern blotting of ECO-R1-digested DNA was performed by standard techniques (25 , 26) . The integrity of the RNA was confirmed by formaldehyde-agarose gel electrophoresis. Northern blotting was performed according to Thomas (27) . Equal amounts of DNA (20 µg) were slot blotted on nylon membranes (Hybond N+; Amersham Corp.). RNA (20 µg) was slot blotted according to Maniatis et al. (28) .

Oncogene Detection.
To determine c-erbB-2 overexpression or amplification, blots were hybridized to c-erbB-2 by a 5' end labeling procedure using[{gamma}-32P]ATP. The probe used was human oligonucleotide Pr 2 (Oncogene Science No ON 112). The hybridization was performed according to the instructions of the manufacturer and others (29) . Briefy, the blots were washed at high stringency (0.3 x standard sodium saline citrate), autoradiography with intensifying screens was performed for 2–4 days at -70°C using kodak X-OMAT 100 films, and autoradiograms were scanned with a Bio-Rad video densitometer 620. DNA and RNA extracted from normal breast tissue (obtained during mastectomy of cancer patients from areas distant to cancer) were used as negative controls. The values obtained for c-erbB-2 by densitometer scanning were normalized to values derived from ß-actin. To determine amplification or overexpression, the ratios obtained were compared with average values derived from normal samples.

Steroid Receptors.
EsRs and PgRs were assayed by the charcoal method, as previously described (30 , 31) . The cutoff level used was 10 fmol/mg cytosolic protein (results evaluated by quality control assessment; Ref. 31 ).

CD Assay.
CD was assayed by an immunoradiometric kit (Elsa Cath-D kit; CIS Bio International, Gif-sur-Yvette, France), according to the procedure described by the manufacturer and elsewhere (31) , in 1:40 and 1:80 dilution of the reconstituted cytosols, both in duplicate.

Statistics.
Cox and LR analyses were used to study the association between CD or c-erbB-2 against patient and disease characteristics. The Cox proportional hazard model was used for univariate and multivariate analyses (32) . For multivariate analysis, the forward LR test was used. RRs and 95% CIs are presented only for retained variables, significant in multivariate analysis. Tumor size and differentiation grade are continuous variables, with scores 1–3.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The frequency of c-erbB-2 amplification measured in 138 primary breast tumors was 22.4% and that of overexpression was 29.7%, whereas 46.4% of tumors had high concentrations of CD (>= 60 pmol/mg protein; Table 1Citation ). With a median follow-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 only short (3 years) RFS (Table 1)Citation . The patients examined were divided in subgroups according to survival (ER, RFS, and OS) and according to nodal status (negative and positive).


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  		Table 1 Distribution of patients on the basis of the factors examined

 
Statistical Analysis of 63 Patients with Positive Lymph Nodes.
For the ER of patients with lymph node involvement, the contribution of high CD levels, grade, and tumor stage is positive and that of EsR and PgR levels are negative. The most accurate prediction for ER is based on CD and grade (P = 0.0023 and P = 0.00077, respectively); for RFS, however, only age and menopausal status have an impact, whereas CD and c-erbB-2 overexpression are not significant. OS is negatively associated with c-erbB-2 overexpression and amplification. Nevertheless, the best prediction is based on a combination of c-erbB-2 overexpression and tumor stage (P = 0.0243 and P = 0.0069, respectively; Table 2Citation ).


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  		Table 2 Univariate and multivariate analysis in 63 breast cancer patients with lymph node metastases

 
Parametric Analysis of 75 Patients with Negative Lymph Nodes.
As shown in Table 3Citation , for the ER of patients without lymph node involvement, the outcome correlates positively with CD, c-erbB-2 overexpression, grade, and tumor stage, and correlates negatively with EsR. By multivariate analysis, CD (P = 0.0037) was found once more to be the most potent prognostic variable for ER, and the prediction could be improved by considering the grade. RFS is negatively associated with CD, c-erbB-2 overexpression, and tumor stage and positively associated with EsR. Multivariate analysis for RFS once again points to CD (P = 0.0042) as the most important variable, followed by tumor stage. OS is negatively associated with CD, c-erbB-2 overexpression, and amplification, as well as tumor stage, and positively associated with EsR, age, and menopausal status. In multivariate analysis, the most accurate prediction for OS results from the combination of CD, c-erbB-2 overexpression, and tumor stage.


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  		Table 3 Univariate and multivariate analysisa in 75 breast cancer patients without lymph node involvement

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The necessity for new prognostic subgroups in breast cancer patients is well recognized. CD and c-erbB-2 are two relatively new biological markers. The lysosomal protease CD, first described by Westley and Rochefort (33) , has been shown to correlate with prognosis of breast cancer in numerous studies (15 , 16 , 18 , 19 , 22 , 23 , 34, 35, 36, 37) . However, other studies give conflicting results concerning its true role in prognosis (38, 39, 40) . Various investigators have suggested that the presence of CD in stromal and inflammatory cells has a causative association with invasiveness (41 , 42) . Also, the prognostic value of c-erbB-2 in breast carcinomas has been controversial. The findings of the present work are in agreement with our previous studies (18 , 22 , 35) and with those of others (15, 16, 17) , confirming the adverse effects of high CD levels on the clinical outcome of breast cancer patients. According to Razumovic et al. (43) , the CD, as measured by the immunoradiometric method, represents the cumulative result of CD content in both carcinoma cells and stromal macrophages.

According to the present study, CD has a significant prognostic value for ER, especially of node-negative patients (Table 3)Citation , in agreement with previous studies (41 , 42 , 44) , although recent data (45) , also obtained via the immunoradiometric technique, conclude that CD is of doubtful value in predicting risk of ER or death for patients with newly diagnosed invasive breast cancer. The prognostic relevance of CD for RFS (Fig. 1A)Citation is decreased if node-negative patients only are considered, in agreement with a recent meta-analysis (39) , and disappears if node-positive patients only are regarded, which, nevertheless, contradicts results by Duffy et al. (16) . The CD measurement by multivariate analysis does not contribute to precision of prediction for OS either in the case of node-positive (Table 2)Citation or node-negative (Table 3)Citation patients (Fig. 1B)Citation . In addition, we noticed that CD gives a better prediction for RFS of stages I and II (data not shown), which has been reported by others also (37 , 38) . However, for stages III and IV patients, it does not improve the precision of the prediction. All this controversy in the literature is obviously due to different measurement methods and the use of different antibodies. Therefore, the standardization of techniques is of paramount importance. In this study and in previous analyses (18 , 35) , we noticed that CD is the only one of the examined variables with a statistically negative correlation for locoregional recurrence. Another interesting observation of this study is that in the Greek population we find a substantial proportion of women with positive EsR (>78%) as well as PgR (> 80%).3 This may be due to the appearance of breast cancer at a later age in these women or to prior parity; in any case, it needs further investigation.



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

 
The importance of c-erbB-2 amplification and overexpression as a prognostic indicator in breast cancer has been extensively studied since the initial report of Slamon et al. (8) , who showed that c-erbB-2 amplification is an independent prognostic factor in node-positive patients. Although we previously (23) showed that there is a positive relationship between c-erbB-2 amplification and overexpression, in the present study we found 22 patients (16%) having c-erbB-2 overexpression without amplification. Most of these patients had an ER or did not respond to chemotherapy. We also found differences in the relation between c-erbB-2 amplification and overexpression and clinicopathological characteristics (e.g., grade). Interestingly enough, most of the samples with c-erbB-2 amplification were grade II, which suggests that c-erbB-2 amplification is mainly found in proliferating tumors. By contrast, overexpression of c-erbB-2 does not show considerable differences in tumors with distinct grade types. Moreover, 12 patients (8%) with c-erbB-2 amplification did not display parallel overexpression of the gene. Ten of the 12 patients had a long RFS. The existence of gene amplification without concurrent overexpression may be due to transcriptional inhibition. The present data suggest that only c-erbB-2 overexpression (Fig. 2, B and D)Citation , but not its amplification (Fig. 2, A and C)Citation has a prognostic value for breast cancer patients. These results are in agreement with Berns et al. (46) and Kreipe et al. (47) , but are in contrast to recent data (48 , 49) showing (by PCR) that c-erbB-2 amplification is a significant prognostic factor. Recently Beckmann et al. (50) , using immunohistochemistry, showed that c-erbB-2 gives no advantage in predicting recurrence or survival in breast cancer patients. Koscielny et al. (51) , using an ELISA method to measure c-erbB-2 oncoprotein in tumor cytosols, reported potential prognostic 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 2Citation and 3Citation ) does not 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 whole (data not shown), in agreement with the results of other investigators (10 , 52) who had used immunohistochemistry. Nevertheless, the present study attributes substantial prognostic value to c-erbB-2 overexpression for OS regardless of nodal status (Tables 2Citation and 3Citation ).



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  		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.

 
In conclusion, with this study we confirmed and extended our previous results (22) showing that CD and c-erbB-2 overexpression have an additive prognostic value for subgroups of breast cancer patients. Although presently available studies suggest that these biological factors might be helpful in making treatment decisions (20 , 21) , their clinical usefulness is still controversial (21 , 53) , and as with the assessment of hormone receptors, there are important unresolved technical issues, such as how to prepare the tissue, which reagents to use, and, most importantly, how to interpret the results.


   FOOTNOTES
 
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 Pharmacology, Wayne State University, Gordon H. Scott Hall of Basic Medical Sciences, 540 East Canfield Avenue, Detroit, MI 48201. Phone: (313) 577-1580; Fax: (313) 577-6739. Back

2 The abbreviations used are: CD, cathepsin-D; ER, early relapse; OS, overall survival; RFS, relapse-free survival; LR, logistic regression; RR, relative risk; CI, confidence interval; EsR, estrogen receptor; PgR, progesterone receptor. Back

3 Unpublished data. Back

Received 8/ 4/98; revised 12/ 3/98; accepted 1/ 5/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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