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Cathepsin B, a Prognostic Indicator in Lymph Node-negative Breast Carcinoma Patients: Comparison with Cathepsin D, Cathepsin L, and Other Clinical Indicators¹

Tamara T. Lah², Miha erek, Andrej Blejec, Janko Kos, Ella Gorodetsky, Robert Somers and Ierachmiel Daskal

Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, 1000 Ljubljana, Slovenia [T. T. L., M. C., A. B.]; Department of Biochemical Research and Drug Design, KRKA, d.d. Novo mesto, 8000 Slovenia [J. K.]; and Breast Cancer Center [R. G. S.] and Department of Pathology and Laboratory Medicine [E. G., I. D.], Albert Einstein Medical Center, Philadelphia, Pennsylvania 19141

	ABSTRACT

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New prognosticators are needed for breast cancer patients after the initial surgical treatment to make therapeutic decisions that ultimately will affect their DFS. These consist of specific proteolytic enzymes including lysosomal endopeptidases. In this study, the activity and protein concentrations of cathepsins (Cats) D, B, and L were measured in 282 invasive breast tumor cytosols. These potential biological prognostic indicators were compared with other histopathological parameters, such as tumor size, lymph node involvement, tumor-node-metastasis stage, histological grade, DNA analysis, and steroid receptors. CatD protein concentration correlated with lymph node involvement. CatB and CatL levels correlated significantly with Scarf-Bloom-Richardson histological grade and were also higher in estrogen-negative tumors, and CatB was higher in larger tumors.

As prognostic markers, CatB concentration was significant for increased risk for recurrence in the entire patient population and specifically also in lymph node-negative patients as follows: high CatB concentration (above 371 µg/g) in tumor cytosols was significant (P < 0.00) for high risk of recurrence but was of only borderline prognostic significance (P < 0.06) for overall survival of all patients. In lymph node-negative patients, CatB (above 240 µg/g, P < 0.003) was highly significant for recurrence-free survival, followed by CatL (above 20 µg/g, P < 0.049) and CatD (above 45 nmol/g, P < 0.044) concentrations. For overall survival of node-negative patients, only CatB was a significant (P < 0.014) prognosticator. We conclude that CatB is useful as a prognostic indicator in lymph node-negative patients. This suggests that selective adjuvant therapy should be applied in this lower risk group of patients when high levels of CatB are determined.

	INTRODUCTION

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The early diagnosis of breast cancer has yielded a large increase in node-negative patients diagnosed with invasive carcinoma of the breast. This population consists of several subgroups of patients that vary in their propensity for recurrence after lumpectomy (1) . Therefore, there is a need for new biological prognostic indicators that would, alone or in combination with others, be sufficient to predict disease recurrence and, hence, the basis for supplemental treatment after local therapy.

Several new prognosticators, including aspartic and cysteine lysosomal proteases, also named Cats,³ have been suggested (2) . Although these share a similar biosynthetic pathway, lysosomal Cats differ structurally and biochemically, mostly in the specificity for substrates and their inhibition by exogenous and endogenous inhibitors. A large body of literature has accumulated, demonstrating that lysosomal proteinases—Cats D, B, and L—are involved in the process of cancer invasion and possibly facilitate metastatic pathways (3, 4, 5, 6) . Altered regulation and subcellular trafficking was observed in neoplastic tissues, and increased expression of mRNA, protein, and activity levels were reported for all three of the Cats. It is not known, however, if their up-regulation is correlated because Cats were investigated in tumors of different histological origins.

CatD was the first lysosomal protease shown to be related to survival of breast cancer patients (7) . The prognostic impact of CatD was later confirmed in many, but not in all studies (reviewed in Refs. 5 , 8 , 9 ). Different cutoff points were determined, possibly because of different methodologies using different antibodies to the CatD molecule. However, in studies using standardized assays, CatD level in breast cancer cytosols was found to be an independent prognostic parameter associated with occurrence of clinical metastases and shorter survival (5 , 8) . In our pilot studies on matched pairs of breast tumors and nonmalignant control tissues of the same breast, we demonstrated a significantly higher increase in cysteine Cats B and L, compared with the increase in the aspartic CatD at the activity and protein concentration levels (10, 11, 12) . We have also demonstrated prognostic significance for the relative increase in CatB and CatL in 60 patients (11) . This data were confirmed in a larger population of breast tumors (13 , 14) .

The aim of this study was to evaluate the levels of the three Cats in breast tumor cytosols of invasive carcinomas. Specifically, we aimed: (a) to correlate Cats at the activity and protein levels with clinical and histopathological parameters, which are the present standards by which to predict breast tumor progression; and (b) to compare their relative potential use to predict DFS and OS of breast cancer patients in the total population and in the subpopulation of lymph node-negative patients for recurrence of breast cancer.

	MATERIALS AND METHODS

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Materials.
The chemicals used were of analytical grade and were purchased from Sigma (Poole, United Kingdom). Papain (2x crystallized, type IV) and E-64 were purchased from Sigma. Peptide substrates and inhibitors were obtained from Bachem Biochemica Gmbh (Heidelberg, Germany).

Patient Selection.
Patients selected for this study were women, ages 35–89 years, and treated at Albert Einstein Medical Center from 1988 to 1993. Breast tumors were surgically removed and were processed for cytosolic immunoenzymatic and activity assays, as described below. Clinical staging was performed according to the TNM classification according to European Organization for Research and Treatment of Cancer recommendation (15) . Histological grading was performed according to the modified Bloom and Richardson method (16) . Tumor cytosols were prepared as described for the determination of steroid receptors (15) and stored at 70°C for 2–4 years before use for the analysis of Cats. Patients of higher stages (II, III, and IV) were treated postoperative by modified radical mastectomy or wide excision (lumpectomy). The patients were followed from 1988 until 1995.

Histology.
H&E- and van Gieson-stained slides were prepared from each tissue block, which were routinely fixed in neutral formalin and embedded in paraffin. The histological typing diagnosed 222 cases of infiltrating ductal carcinoma (IDC), 27 cases as invasive lobular carcinoma (LOB), 9 cases as mixed ductal and lobular carcinoma; 8 cases as medullary carcinoma (MED), 2 cases as mixed ductal and medullary carcinoma, and 14 cases as other types of ductal carcinoma (Table 1) .

CatB and CatL protein concentrations were analyzed immunochemically by double-sandwich ELISA kits (KRKA, d.d. Novo mesto, Slovenia), as suggested by the manufacturer. Purified native CatB and CatL were used as antigens and standards. The characteristics of the assays, linearity, and precision controls were reported previously (17) .

CatB.
Immunoselective sheep and rabbit polyclonal Abs (IgG) were used as capture and detection Abs, respectively. Tumor samples in 1:100 (v:v) dilution and control samples in 1:10 (v:v) were added to microtiter plate wells, and the assay was carried out as described. As internal controls, two pools of normal breast cytosols and tumor cytosols were prepared, aliquoted (50 µl), and frozen at -70°C before assay along with the samples.

CatL.
Sandwich ELISA sheep anti-CatL immunoselective IgG was used as a capture Ab and for detection as horseradish-peroxidase-conjugated Ab. Tumor tissue samples were diluted 1:5 (v:v), and control samples were diluted 1:2 (v:v). According to the manufacturer, the ELISA assay for CatL and CatB showed no cross-reactivity but recognized all of the molecular forms of the enzymes including the complexes with cystatins.

Enzyme Activity Assays.
CatBa was determined using the modified method of Barret and Kirschke (18) as described previously (10) . Briefly, 20 µl of the supernatant were added to 280 µl of the 400 mM phosphate buffer (pH 6.0), containing 4 mM EDTA/Na2 and 1.2 mM DTT and incubated for 10 min at 37°C. The substrate Z-Arg-Arg-AMC (100 µl) was added to final concentration of 60 µM and was further incubated for 150 min. The reaction was terminated by the addition of 500 µl of 1-mM ice-cold iodoacetic acid. In the blank assays, running in parallel, the sample solution was replaced by the addition of bidistilled water, whereas the control assays contained 1-mM final concentration of E-64. Fluorescence of the product 7-AMC was read at an excitation wavelength of 370 nm and emission wavelength of 460 nm using Perkin-Elmer spectrophotometer LS5 (Norwalk, CT). One EU represented the amount of the enzyme releasing one µmol of 7-AMC per ml. Specific activity was expressed in EU per g total protein in the sample.

CatL Activity.
CatLa was measured using Z-Phe-Arg-AMC as a substrate. To discriminate between CatB and CatL activities, the hydrolysis of Z-Phe-Arg-AMC was measured in the presence and absence of 0.5 µM final concentration of Z-Phe-Phe-CHN₂, the latter inhibiting CatL but not CatB as described previously (10) . In brief, 100 µl of the sample was preincubated for 30 min at 37°C in the activation buffer, i.e., 340 mM acetate buffer (pH 4.2) containing 2 mM DTT and 1 mM EDTA/Na2. After the addition of the substrate, 100 µl of Z-Phe-Arg-AMC (final concentration 100 µM) additional incubations were carried out in the presence and absence of 100 µl Z-Phe-Phe-CHN₂ for 180 min. The reaction was terminated by the addition of 500 µl of ice-cold 1 mM iodoacetic acid. In the blank assay running in parallel, the sample solution was replaced by bidistilled water. EUs were calculated as described above for CatB, after correcting for blanks and controls, and the specific activity was expressed in EU/g protein in the sample.

Steroid Hormone Receptors and Protein Concentration.
ERs and progesterone receptors were measured according to the protocol of BYK-Diagnostica, using dextran-assay with radiolabeled ligands. Tumors with receptor concentrations above 10-fmol/mg protein were considered receptor-positive. Protein was determined with Bio-Rad protein assay, using BSA as standard protein.

Tumor DNA content and S-phase fraction were determined as described by Camplejohn et al. (19) . Tumor homogenates were filtered, incubated with propidium iodide and analyzed on an EPICS V Dual Color Flow Cytometer (Coultronics, Hialeah, FL).

Statistical Analysis.
The data were analyzed using the SPSS statistical package, CA (20) . Nonparametric tests were used for comparative analysis of biochemical parameters, such as Pearson and Spearman rank test. Kruskal-Wallis one-way ANOVA test and ² tests were used to compare the biochemical variables with clinical and histopathological parameters of the disease. Survival analysis was calculated by the Kaplan-Meier product limit method (21) . DFS and OS were considered as the period from the operation of the primary tumor until the date of relapse or death of the malignant disease. Analysis of DFS and OS were also carried out by the Cox analysis (22) . Cutoff values for continuous variables were calculated using isotonic regression analysis and comparison between the randomly selected samples (training set) and validation set resulted in the cutoff value, which was used for the whole sample population. The cutoff values were tested on two independent, randomly selected sets of patients, and the closest cutoff point was confirmed in the total and lymph node-negative patients population.

	RESULTS

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Patients.
Tissues from primary breast tumors, obtained from 210 lumpectomies, 69 mastectomies and 3 biopsies were included in the study. The characteristics of patients treated at Albert Einstein Medical Center in Philadelphia are listed in Table 1 . Most patients were diagnosed for invasive ductal carcinoma (n = 222) and for invasive lobular carcinoma (n = 27). About half of the tumors (n = 132) were smaller than 2 cm, 132 were lymph node-negative and 195 tumors were lower stages (I and IIA). Patients were followed-up for 100 months with median follow-up of 45 months. Adjuvant therapy (chemotherapy, hormone, radiation therapy or a combination) was administered to all of the patients. Of 282 patients, 81 (28.7%) experienced relapse within 0.9–100 months with a median period of 32 months. The median follow-up of 201 patients without evidence of disease was from 10 to 99 months with median, 48 months. Forty-eight (17%) patients died with median survival time from 0.9 to 88.5 months (median, 29.9 months), whereas 3 patients died of other reasons and were censored as other patients at last follow-up.

Biochemical Characteristics: Distribution and Correlation.
The median values of Cats activity (CatBa, CatLa) and concentration [CatD], [CatB], [CatL] in tumor cytosols are presented in Table 2 . These biological variables were correlated by Pearson method (Table 2 .) and by Spearman rank correlation test (not shown) with similar significance. The expression of both cysteine proteinases correlated at activity (r = 0.69) and protein concentrations (r = 0.60). This suggests a similar mechanism of regulation of the two-cysteine proteinases in breast tumors. Low but significant correlation was found between CatB activity and CatB protein concentration but not between CatLa and CatL protein concentration. All of the other coefficients of correlation were below 0.5. This was not considered a high correlation.

Correlation of Biochemical Parameters with Patients’ Survival.
Kaplan-Meier univariate analysis (using log-rank test) was used to evaluate significance (P) of the differences between the two survival curves for DFS and OS. These analyses were performed on entire patient populations (Table 4a) and on lymph node-negative patients (Table 4b) . Increased CatBa and protein level were significantly related to DFS (Fig. 1, A and B , respectively). CatB had a borderline prognostic significance for OS. The latter was similar to CatD, which was of borderline significance for the DFS. It is noteworthy that the optimized cutoff value for DFS and OS were close although not always identical. CatL had no prognostic impact. In lymph node-negative patients, prognostic impact of Cats increased, particularly for DFS (Table 4b) . CatB protein had the highest prognostic impact (Fig. 2B) , followed by CatD. CatB activity (Fig. 2A) , and CatL protein concentration. Of all these indicators, CatB had also an impact on OS.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Prognostic significance of CatB for DFS in entire population of patients. Survival analyses for CatB in relationship to the recurrence of the DFS in a total of 282 patients were performed by the Kaplan-Meier method. The cut-off levels were determined by isotonic regression on two sets of patients (described in "Materials and Methods") at 66 EU/g of cytosolic protein (A) and 371 µg/g of cytosolic protein (B). Significance (P) between the curves was below 0.05 (see also Table 4a ).

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Prognostic significance of CatB for DFS and OS in lymph node-negative patients. Survival analyses for CatB in relationship to the recurrence of the DFS in the lymph node-negative population of 134 patients were performed by the Kaplan-Meier method. The cut-off levels were determined by isotonic regression on two sets of patients (described in "Materials and Methods") at 67 EU/g of cytosolic protein (A) and 240 µg/g of cytosolic protein (B). Significance (P) between the curves was below 0.05 (see also Table 4b ).

	DISCUSSION

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This study confirms our previous findings that in human breast tumors and in transformed breast epithelial cells, (10, 11, 12 , 23 , 24) , lysosomal aspartic and cysteine proteinases are independently regulated. Significant correlation was noted only between CatB and CatL activity and protein levels, indicating a possible common mechanism of their up-regulation in tumors. Further evidence for separate regulation of the cysteine and aspartic proteinases can be inferred from their unique correlation with clinical and histopathological parameters of breast tumor progression (Table 3) .

The present status of the reported data is equivocal and somewhat contradictory when Cats are compared with histopathological parameters.

In the present study, CatD was significantly higher in lymph node-positive patients, as reported by Garcia et al. (4) and others (5) . On the other hand, no correlation with positive lymph node involvement was seen with CatB and CatL. Similar results were reported by Thomssen et al. (13) . Contrary to the above findings, Foekens et al. (14) reported a correlation between Cats B and L with positive lymph nodes, whereas Budihna et al. (25) reported inverse correlation between CatB and lymph node positivity. At present, there is no consensus on the precise relationship between CatB, CatL and lymph node status. The above discordance is extended to the correlation between ER status, histological grade, and tumor size.

With respect to prognostic value, we found a borderline prognostic impact on either DFS or OS for CatD, confirming our previous reports on smaller patients population (10, 11, 12 , 24) and other reports (for reviews see 3 , 4 , 8 , 9 , 26 ). However, when our patients were stratified for lymph node negativity, CatD was significant for DFS. Recently, based on meta-analysis of a total of 2690 patients (27) , CatD was confirmed as an independent prognostic factor in lymph node-negative population both for DFS and OS.

Of the three Cats studied, the concentration of CatB was the most significant prognostic indicator for DFS. These results are in agreement with our previous studies (11 , 12) and partially with the two other clinical studies of Thomssen et al. (13) and Foekens et al. (14) . They reported that patients with high concentration of CatB or CatL in their primary tumors had a statistically significant higher risk for recurrence than patients with a low concentration of CatB or CatL. Also, as in the above studies (13 , 14) , our results confirm the higher prognostic significance of CatB for DFS compared with OS. With respect to the prognostic value of CatL, Thomssen et al. (13) have shown improved DFS and OS compared with CatB, whereas Foekens et al. (14) seem to indicate that CatB was equally predictive as CatL. In contrast to these, the present data shows that CatB is the preferred prognostic indicator for DFS and OS over CatL. The most important contribution of CatB appears to be in lymph node-negative patients. When these patients were analyzed separately from the total patient population, the prognostic significance of Cat concentration for DFS increased, suggesting a possible role in the invasive mechanism of breast carcinomas (for recent review see 28 ). In contrast, Foekens et al. (14) reported no prognostic advantage in either node-negative or node-positive patients. Thomssen et al. (13) on the other hand, reported increased significance for both Cats in node-positive patients.

The main difficulty in using this data in a practical manner in patient management is related to discrepancies in the mean levels and the lack of a universal cutoff value that would maximize the specificity and sensitivity of these assays. Several reasons may be responsible for these discrepancies: (a) the instability of the Cats, related to specimen preparation and storage conditions, especially temperature and reducing environment (14 , 29) ; (b) the specificity and sensitivity of the ELISA assays; (c) biological variations in different populations of patients (30) ; and (d) differences in the number of events and the lengths of follow-up (14) . Therefore, standardization of tumor tissue preparation, preservation protocols, and ELISA formats are needed before any clinical application of Cats as prognostic factors is undertaken. We suggest that given a standardized assay, tumor CatB concentration may be useful in discriminating the higher risk from the lower risk (such as lymph node-negative) patients. These findings may be useful for the development of new adjuvant treatment algorithms of patients after primary surgical resections.

	ACKNOWLEDGMENTS

 
We acknowledge the contribution of Dr. Joseph Tabachnick in preparing the tumor tissue bank.

	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 Supported by Albert Einstein Medical Center Council Grants 7040 and 8040 from the Albert Einstein Society and partially by Ministry of Science and Technology of Slovenia Grant L 3-8919.

² To whom requests for reprints should be addressed, at National Institute of Biology, Vena pot 111, 1000 Ljubljana, Slovenia. Phone: 386-61-123-5017; Fax: 386-61-123-5038; E-mail: tamara.lah{at}uni.lj.si

³ The abbreviations used are: Cat, cathepsin; CatBa, CatB activity; CatLa, CatL activity; AMC, 7-amino-4-methyl-coumarine; DFS, disease-free survival; E-64, L-epoxy succinylleucylamido (4-guanidine) butane; ER, estrogen receptor; OS, overall survival; TNM, Tumor-Node-Metastasis; EU, enzyme unit; Ab, antibody.

Received 6/ 3/99; revised 9/16/99; accepted 10/26/99.

	REFERENCES

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