Clin Biochem, Vol. 24, pp. 491-496, 1991 Printed in the USA. All rights reserved.

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Increased Plasma Cathepsin D Concentration in Hepatic Carcinoma and Cirrhosis but Not in Breast Cancer J.P. BROUILLET, 1 B. HANSLICK, 3 T. MAUDELONDE, 1 M.T. PIVAT, 3 J. GRENIER, 4 F. BLANC, 3 and H. ROCHEFORT 1'2 1Laboratoire de Biologie Cellulaire et Hormonale, CHRU Maternite, 13 Avenue du Pr. Grasset, 34059 Montpellier, Cedex, France; 2Unite Hormones et Cancer (U 148 INSERM), 60 Rue de Navacelles, 34090 Montpellier, France; 3Service de Medecine Interne E, CHRU Saint-Eloi, 2 Avenue Bertin Sans, 34059 Montpellier, Cedex, France; "Centre Paul Lamarque/Val d'Aurelle, 34094 Montpellier, Cedex, France Using a sandwich enzyme-linked immunoassay, plasma total cathepsin D concentration was assayed in 40 breast cancer patients and 84 patients with various liver diseases and compared to that of 52 normal subjects. There were no significant variations found in breast cancer patients related to tumor size, node invasiveness or metastases. In normal women, cathepsin D levels were slightly but not significantly increased in the luteal phase and in pregnancy. By contrast, plasma cathepsin D concentration was significantly increased in 70-75% of patients with liver disease (cirrhosis, hepatocarcinoma, hepatitis), but not in those with liver steatosis. Cathepsin D was independent of most of the plasma hepatic function tests and was correlated with alpha-fetoprotein in cirrhosis and with alpha-fucosidase in primary hepatocellular carcinoma. We conclude that plasma cathepsin D is not a useful marker in breast cancer. However, since the cellular level of this protease is associated with risk of metastasis in breast cancer, clinical follow-up will be required to test whether high cathepsin D plasma concentration has any prognostic value in liver cirrhosis and primary hepatocarcinoma.

KEY WORDS: cathepsin D; immunoassay; circulating marker; breast cancer; cirrhosis; liver cancer.

are also increased in some proliferative ductal mastopathies (5). Cathepsin D circulates in plasma mostly as the precursor form (52 kDa) (6). Since pro-cathepsin D is overexpressed and secreted in excess in breast cancer cells, its assay might be useful as a circulating marker of breast cancer to follow the progression of the disease. Moreover, since cathepsin D gene expression is differentially regulated by sex-steroid hormones in uterine (7-9) and mammary tissues (9-11), we expected to find that circulating levels would vary according to the hormonal status of the patients. Finally, like most circulating plasma proteins, cathepsin D is also produced by the liver (6). We also assayed plasma cathepsin D in a large range of liver diseases to evaluate its potential interest in hepatology. We report on the prognostic value of plasma cathepsin D in 83 patients with liver disease.

Introduction

Methods

POPULATION C

athepsin D is an aspartyl acid protease found in all eukaryotic cells which accumulates in lysosomes in its active form (34 kDa + 14 kDa). Part of the 52 kDa molecular weight precursor is secreted following overexpression of the cathepsin D gene (1). Using monoclonal antibodies, we have shown that total cathepsin D (52 kDa, 48 kDa and 34 kDa) concentration in the cytosol of primary breast cancer is an important prognostic parameter which is correlated with relapse and metastasis-free survival (2-4). Tissue cathepsin D levels

Correspondence: H. Rochefort, U 148 INSERM, 60 Rue de Navacelles, 34090 Montpellier, France. Manuscript received January 2, 1991; revised August 7, 1991; accepted August 12, 1991.

The control population included 58 healthy subjects. Breast cancer patients of different TNM stages numbered 48 and were from the Centre Anticancereux Paul Lamarque (Montpellier, France). Eightythree liver disease patients, including 20 with primary hepatocellular carcinoma (PHC), 7 with secondary metastatic liver carcinoma, 43 with liver cirrhosis, 8 with hepatitis and 5 with benign steatosis were from the Departement de Medecine Interne (CHU, Montpellier, France). PHC diagnosis was based on liver biopsy in 14 cases or high plasma alpha-fetoprotein (AFP) (->500 ng/mL) with lesions visible on ultrasound or liver scan in 6 cases. Diagnosis of secondary metastatic liver carcinoma was by liver imaging in 3 cases or by the association of liver scan images with the histological evidence of primary tumors in 4 cases. All cirrhosis cases were

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histologically proven after biopsy. The following serum assays were performed for each patient with liver disease: activities of aspartate aminotransferase (AST), alanine aminotranferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl-transferase (GGT), alphafucosidase (AFU), concentrations of prothrombin, bilirubin, albumin and AFP. In the cirrhotic group, the Child-Pugh grade (12) was evaluated to determine the severity of liver disease, and alcohol withdrawal was noted in alcoholic cirrhosis.

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B L O O D COLLECTION PROCEDURE

IMMUNO-ENZYMATIC ASSAY OF PLASMA CATHEPSIN D

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MISCELLANEOUS TECHNIQUES

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2 5 0

c~FUCOSIDASE ( n K a t / L )

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Figure 3-Quantitative correlation between cathepsin D concentration and AFP or AFU concentrations in primary hepatocarcinoma and cirrhosis patients. Pearson's least-squares method was used to compute linear regressions, and the significance levels were determined with the t-test. Correlation was statistically significant between cathepsin D and AFU in primary hepatocarcinoma (p = 0.004) and between cathepsin D and AFP in cirrhosis (p = 0.002). Other correlations were not significant, n = number of patients.

shown). At a 10 pmol/mL cut-off level, cathepsin D status was correlated in PHC and cirrhosis with other enzyme tests (AST, ALT), weakly in cholestasis (only with ALP and GGT), but not with tumor markers such as A F P or with AFU, another lysosomal enzyme (Table 1). However, when cirrhosis and PHC were considered separately, cathepsin D concentration was correlated with A F U in PHC and A F P in cirrhosis (Figure 3). The absence of correlation with A F P in PHC is worth noting. There were two patients with a high cathepsin D level and normal AFP, which suggests that cathepsin D m a y complement A F P for detecting PHC when A F P is low. The postsurgery clinical follow-up of patients with PHC (only 16 were included) did not show any correlation between survival and plasma cathepsin D level after 18 months, suggesting that this m a r k e r is not a prognostic p a r a m e t e r in PHC. Currently, the clinical follow-up of patients with cirrhosis is too short and the frequency of liver cancer secondary to cirrhosis too low to draw any

In breast cancer patients plasma cathepsin D concentrations were not significantly increased compared to normal subjects. In some cases it was slightly decreased. This contrasts with the fact that cathepsin D is often secreted in excess in breast cancer tumors, not only in cell lines (16) b u t also in pleural effusions (17). The pro-cathepsin D secreted by breast cancer cells does not therefore contribute to plasma levels of cathepsin D, suggesting that it does not reach the blood and m a y act mainly locally. Conversely, it could be secreted into the blood but masked for antibody recognition by other interacting molecules. Whatever the mechanism, these studies indicate that this cathepsin D ELISA is not useful as a plasma m a r k e r in breast cancer. Moreover, the slight increase following progestin t r e a t m e n t or at late pregnancy suggests that some

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CLINICAL BIOCHEMISTRY,VOLUME 24, DECEMBER 1991

conclusions on the prognostic value of cathepsin D in liver cirrhosis. Discussion

P L A S M A CATHEPSIN D IN BREAST C A N C E R A N D LIVER DISEASES TABLE 1 Correlation of Cathepsin D Status in Primary Hepatocarcinoma and Cirrhosis with Other Hepatic Tests

Number of Patients

P AST

0.008

normal increased ALT 0.045 normal increased ALP 0.009 normal increased Bilirubin 0.297 normal intermediate increased GGT

9 7

9 37

13 3

24 21

12 4

17 29

8 5 3

13 22 10

5 11

2 44

2 7 4

5 12 22

2 3 6

7 12 15

14 1

28 11

8 8

21 23

0.009

normal increased Prothrombin 0.252 low intermediate normal Albumin

Normal Cathepsin High Cathepsin D ( 10 pmol/mL) pmol/mL)

0.830

low intermediate normal AFP 0.084 < 500 - 500 AFU 0.87 normal increased

AST, ALT and GGT were defined using a cut-off level of 25 IU/L; ALP, prothrombin level, albumin and bilirubin using cut-off levels of 170 IU/L, 45 and 60%, 30 and 37 g/L and 20 and 50 umol/L, respectively: AFP and AFU were defined using cut-off levels of 500 ng/mL and 100 nkat/L, respectively. Some tests are missing in a few patients; thus, the total number of patients for each parameter is variable.

AFU, another lysosomal enzyme proposed as a PHC m a r k e r (19). However, AFU was measured as enzymatic activity, while cathepsin D was measured by antibodies, which m a y explain this discrepancy. Moreover, our immunoassay measured total cathepsin D, including both precursor and m a t u r e forms (13,14). Previous studies of 6 patients with diverse types of hepatitis or cirrhosis showed t h a t only pro-cathepsin D was increased in plasma when compared to 5 normal subjects (6). The biological significance of this increase in liver disease (cytolysis, cell proliferation or both) is not quite clear. It seems to be associated with increased liver cytolysis. However, since mainly the 52 kDa pro-cathepsin D form was recovered in plasma, it could be the result of increased secretion of pro-cathepsin D by liver cells. Cathepsin D is also induced by growth factors in breast cancer cells (11) and in the uterus (8). It is associated with liver regeneration and m i g h t be mitogenic in vivo in the liver (20) and in breast cancer cells (21). Moreover, the IGF II gene is, like the cathepsin D gene, located at the extremity of the short arm of chromosome 11 (22,23); its mRNA level is high in PHC tissues (24) and deletions of chromosome 11 have been identified as a potentially tumorigenic mechanism in PHC (25). An interesting hypothesis is t h a t a high plasma cathepsin D level might be a useful prognostic m a r k e r in liver cirrhosis for detecting proliferative precancerous nodules. More research is required to test this hypothesis, including the clinical follow-up of patients described in this study. Acknowledgements This work was supported by INSERM, the CHRU of Montpellier, and Sanofi Laboratories. We are grateful to C. Esnault and M. Chenon for technical assistance and to M.J. Circhirillo for typing the manuscript. References

progestin-responsive tissues, such as uterus (7-9), m a y contribute to circulating cathepsin D levels. By contrast, cathepsin D levels might be import a n t in liver disease since they can be increased as much as 10-fold in plasma of PHC and cirrhosis patients. This increase is in agreement with the two-fold increase of tissue cathepsin D activity previously reported in 6 h u m a n hepatomas compared to normal h u m a n liver (18). Cathepsin D plasma levels seem to be independent of other tests or tumor markers routinely used in hepatology. For instance, in PHC it is only slightly correlated with

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CLINICAL BIOCHEMISTRY, VOLUME 24, DECEMBER 1991