Int. J. Cancer: 67,492-497 (1996) 0 1996 Wiley-Liss, Inc. I Publicationof the InternationalUnion Against Cancer Publicationde I Union InternationaleContre le Cancer EXPRESSION OF CATHEPSIN E IN PANCREAS: A POSSIBLE TUMOR MARKER FOR PANCREAS, A PRELIMINARY REPORT Takeshi A z u M A ~Masamichi .~, HIRAI',Shigeji I T d , Kenji YAMAMOTO?, R. Thomas TAGGART~, Takao MATSUBA4, Kiyoshi YASUKAWA4,Kouji U N d , Takanobu HAYAKUMO~ and Masatsugu NAKAJIMA5 lSecond Department of Internal Medicine, Fukui Medical School, Fukui 910-11, Japan; 2Department of Pharmacology, Kyushu University,Faculty of Dentistry, Fukuoka 812, Japan; 3Divisionof Genetics, Children's Hospital of Buffalo, Buffalo, NY 14209, USA; 4 T ~ kResearch y~ Center, Tosoh Corporation, Hayakawa, Kanagawa 252; sDepartment of Gastroenterology, Kyoto Second Red Cross Hospital, Kyoto 602, Japan. Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells. Cathepsin E (CTSE) is a nonsecretory, intracellular, but non-lysosomal proteinase found in the highest concentration in the superficial epithelial cells of the stomach. The aims of our study were to examine the expression of CTSE in the pancreas, t o establish an assay system of CTSE and to evaluate the diagnostic usefulness of CTSE in the pancreatic juice. Eleven patients with pancreatic ductal adenocarcinoma, 10 with mucin-producing adenoma, 3 with intraductal papillary hyperplasia and 43 with chronic pancreatitis were examined. Surgically resected pancreatic tissues were subjected t o irnmunohistochemistry for CTSE. Pancreatic juice was collected from the patients and subjected t o sandwich ELISA and Western analysis for detecting CTSE. Positive staining for CTSE was observed in pancreatic ductal adenocarcinoma by immunohistochemistry. CTSE was also expressed in mucin-producing adenoma, intraductal papillary hyperplasia and mucinous hyperplasia. CTSE in the pancreatic juice was present in 8 of I I patients with pancreatic ductal adenocarcinoma. 5 of 10 patients with mucin-producing tumor, I of 3 patients with intraductal papillary hyperplasia and 4 of 43 patients with chronic pancreatitis. The detection frequency of CTSE in the pancreatic juice was significantly higher in the patients with pancreatic ductal adenocarcinoma than in the patients with chronic pancreatitis. Our findings suggest that the expression of CTSE is associated with the pathogenesis of pancreatic ductal adenocarcinoma, that CTSE in the pancreatic juice seems t o be a useful marker for a definitive diagnosis and that CTSE may be expressed at a relatively early stage of multistep carcinogenesis in pancreatic lesions. o 1996 Wiley-Liss,Inc. The incidence of pancreatic cancer continues to increase in industrialized Western countries (Silverberg and Lubera, 1989). Pancreatic carcinoma has the lowest 5-year survival rate. Late diagnosis, high recurrence rates and a characteristic tumor biology with regard to growth behavior are responsible for the fact that most tumors of the pancreas, when diagnosed, have spread beyond the reach of surgical cure. Only 10-20% of the patients have a resectable tumor at the time of diagnosis and a chance of being cured by surgery (Gudjonsson, 1987; Warshaw and Castillo, 1992). Early diagnosis of pancreatic cancer is presently the only means to increase the proportion of patients detected at resectable and curable tumor stages (Warshaw and Castillo; 1992). During the past few years, great efforts have been made to render the pancreas visible with new o r improved imaging techniques. It can still be difficult to distinguish between pancreatic cancer and chronic pancreatitis, since the two diseases may share many clinical and radiologic characteristics (Kawai et al., 1987). A wide variety of tumor markers have been proposed for use in the diagnosis of pancreatic cancer. These include the serum levels of tumorassociated antigens, enzymes and hormones (Steinberg et al.. 1986; Friess et al., 1993; Warshaw et al., 1990; Robles-Diaz et al., 1991). Interestingly, Sessa et al. (1990) have reported that ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells. Cathepsin E (CTSE) is one of four immunologically distinct groups of aspartic proteinases found in the human gastric mucosa. In addition to CTSE, the gastric aspartic proteinases include pepsinogen A, pepsinogen C and cathepsin D. Unlike other gastric aspartic proteinases, CTSE is a non-secretory, intracellular, but non-lysosomal proteinase found in the highest concentration in the superficial epithelial cells of the stomach (Samloff et al., 1987). In the present study, we have described the expression of CTSE in pancreatic ductal adenocarcinomas, established an assay system of CTSE and evaluated the diagnostic usefulness of the CTSE in the pancreatic juice. MATERIAL AND METHODS Subjects Sixty-seven consecutive patients, in whom pancreatic disorders were diagnosed by ultrasound, computed tomography, endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic ultrasound, were studied. Forty-three patients had chronic pancreatitis (27 men and 16 women, age range 42-85 years, mean 61.8 years). All chronic pancreatitis patients were followed up for 2 years after the testing, and the presumption of the absence of malignant pancreatic disease was verified if there was no new or further evidence suggesting carcinoma. Eleven had pancreatic ductal adenocarcinoma (5 men and 6 women, age range 54-70 years, mean 61.3 years), 10 had mucin-producing tumor (4 men and 6 women, age range 48-78 years, mean 66.0 years) and 3 had intraductal papillary hyperplasia (2 men and 1woman, age range 50-69 years. mean 62.3 years). These patients underwent surgery, and the final diagnosis was proved by histology. We categorized the mucinproducing tumor as belonging to the group of diseases that can b e detected clinically by the pooling of mucus produced by tumors inside the main or branch ducts of the pancreas, thus causing dilatation of the main or branch ducts (Kozuka et a/., 1979). In the present study all mucin-producing tumors were adenomas. Immunohistochemistry for CTSE Surgically resected human pancreatic tumors were subjected to immunohistochemistry to identify CTSE. All specimens were fixed in formalin and embedded in paraffin. The antisera against CTSE purified from the human erythrocyte membranes were raised in rabbits as described previously (Saku et al., 1990). Immunohistochemical staining was performed by the avidin-biotin-peroxidase complex technique (Vectastain ABC kit, Vector, Burlingame, CA) described by Hsu et al. (1981). All incubations were done at room temperature. Each section (4 k m thick) was immersed in 0.3% H 2 0 2in methanol for 30 min to destroy endogenous peroxidase activity, washed for 10 min with 0.05 M Tris-buffered saline, pH 7.5, and then hTo whom correspondence and reprint requests should be addressed, at the Second Department of Internal Medicine, Fukui Medical School, Matsuoka-cho, Yoshida-gun, Fukui 910-11, Japan. Fax: (81) 776-61-8110. Received: Janualy 22, 1996 and in revised form April 2, 1996. CATHEPSIN E IN PANCREAS TUMORS 493 incubated for 30 min with normal goat serum to block Statistical analysis non-specific Ig binding sites. The goat serum was removed, and Comparisons of the detection frequencies of CTSE in the the slides were incubated overnight with 1:1,000 dilution of the pancreatic juice among the patient groups were made by the x2 rabbit polyclonal antiserum specific for CTSE. This was test, with significance assigned to values below p < 0.05. followed by a 30-min wash in Tris-buffered saline, a 30-min Comparisons of the ratio of CA19-9 to secretory protein incubation with biotinylated goat anti-rabbit IgG, a repeat concentration in the pancreatic juice among the patient groups wash in Tris-buffered saline, and a 30-min incubation with were made by Student’s t test for unpaired samples, with avidin DH-biotinylated horseradish peroxidase. After a final significance assigned to values below p < 0.05. wash in Tris-buffered saline, the slides were incubated for 2 min in a solution containing 0.05% diaminobenzidine and 0.015% H202.Nuclear staining was carried out with hematoxyRESULTS lin. The specificity of the immunohistochemical reaction was lrnrnunohistochernical analysis for CTSE established first by showing that the non-immune rabbit serum CTSE was expressed in pancreatic ductal adenocarcinomas did not reveal positive staining. In addition, the specificity of the antibody was evaluated by absorption controls. The diluted examined in the present study. CTSE was stained diffusely in antibody was incubated with the recombinant human gastric the cytoplasm of the carcinoma cells (Fig. la). Normal ductal CTSE in concentrations of up to 10 nmol/ml for 24 hr at 4°C cells were not stained (Fig. lb). CTSE was also expressed in mucin-producing adenomas, intraductal papillary hyperplasia and then applied to the tissue section. and mucinous hyperplasia (Fig. lc, d ) . In absorption tests, the Enzyme-linked irnrnunosorbent assay for CTSE staining of CTSE was inhibited by the recombinant human Serum was obtained from patients after an overnight fast, CTSE. and was stored at -80°C until analyzed. Pancreatic juice was collected from patients during ERCP with an injection of CTSE in the serum and the pancreatic juice secretin and was stored at -80°C until analyzed. CTSE in the Figure 2 shows the standard curve of the ELISA. CTSE at a pancreatic juice was assayed by sandwich ELISA. The capture antibody was a mouse anti-human gastric CTSE monoclonal concentration as low as 1.75 ng/ml could be detected. We also antibody (MAb) obtained from mouse ascites fluid (Samloff et examined the effects of exogenously added FCS or human al., 1987), and the second antibody was the polyclonal rabbit pancreatic juice obtained from normal patients on the ELISA anti-human CTSE antibody. Alkaline phosphatase-conjugated for CTSE. The presence of 10-100% (v/v) of FCS or pancreanti-rabbit IgG was used for detection in this assay, and the atic juice did not significantly alter the slope of the standard recombinant human gastric CTSE was used to create the curve or the level of the background. The intraassay coefficient standard curve (Yamada et al., 1994). Briefly, 96-well micro- of variation was 8.0% (n = 5 ) , and the interassay coefficient of plates were coated with the capture antibody (100 p1 of variation was 11.0% (n = 5). CTSE was not detected in the serum of the patients. In 500-fold dilution in PBS) at 4°C overnight. The plates were then washed with PBS and blocked with 2% BSA at 4°C pancreatic juice, CTSE was present in 8 of 11 patients with overnight. Serum or pancreatic juice samples (100 yl; if pancreatic ductal adenocarcinoma, 5 of 10 patients with necessary samples were diluted in PBS) were allowed to mucin-producing adenoma, 1 of 3 patients with intraductal remain at room temperature for 2 hr. The plates were washed papillary hyperplasia and 4 of 43 patients with chronic pancrewith PBS, and the second antibody (100 p1of 500-fold dilution atitis. The detection frequency of CTSE in the pancreatic juice in PBS) was added and allowed to stand at room temperature was significantly higher in the patients with pancreatic ductal for 2 hr. The bound antibodies were detected with sequential adenocarcinoma (72.7%) than in the patients with chronic incubation with alkaline phosphatase-conjugated goat anti- pancreatitis (9.3%) (x2 test, x2 = 20.39, p < 0.005) (Fig. 3). rabbit IgG and the enzyme substrate solution. The plates were Although quantitative analysis was not proper for CTSE in the washed with PBS, and the second antibody (100 pl of 500-fold pancreatic juice, in the CTSE-positive cases the ratio of CTSE dilution in PBS) was added and allowed to stand at room to secretory protein concentration in the pancreatic juice was temperature for 2 hr. The bound antibodies were detected higher in the patients with pancreatic ductal adenocarcinoma with sequential incubation with alkaline phosphatase-conju- than in the patients with chronic pancreatitis, mucingated goat anti-rabbit IgG and the enzyme substrate solution. producing adenomas or intraductal papillary hyperplasia. Only The plates were read on an ELISA reader at 405 nm. one patient with chronic pancreatitis had a higher ratio than The concentration of CA19-9 in the pancreatic juice was 30.0 pgl yg. In contrast, 5 of 11patients with pancreatic ductal measured by a CA19-9 radioimmunoassay kit (Centocor, adenocarcinoma had values higher than 30.0 pg/ pg (Fig. 3). Malvern, PA) according to the manufacturer’s instructions. Protein in the pancreatic juice was measured by the Bradford CA19-9 in the pancreatic juice method using a Bio-Rad protein assay (Richmond, CA). The The ratio of CA19-9 to secretory protein concentration is concentrations of CTSE and CA19-9 in the pancreatic juice shown in Figure 4. The mean of the ratio was higher in the were expressed as pg/pg protein and U/yg protein, respecpatients with pancreatic ductal adenocarcinoma [41.45 2 76.88 tively. U/pg (mean % SD)] than in the patients with chronic pancreatitis (4.15 2 8.66 Ulyg), in the patients with mucinWestern blot analysis The pancreatic juice collected from the patients with pancre- producing adenomas (2.25 f 1.86 U/p,g) and in the patients atic cancer was also subjected to SDS-PAGE, which was with papillary hyperplasia (2.29 r 2.46 U/kg). However, the performed following the procedure of Laemmli (1970). For difference was not significant. immunoblotting, proteins electrophoresed on SDS-PAGE gels were transferred to nitrocellulose using the transblot appara- Western blot anaIysis of CTSE in pancreatic juice tus (BioRad, Richmond, CA) according to the company’s By Western blot analysis, CTSE was also detected in the specifications. The transferred proteins were probed with the pancreatic juice from the patients who were positive in the mouse anti-human CTSE MAbs. Goat anti-mouse IgG coupled ELISA. The molecular size of the CTSE in the pancreatic juice to horseradish peroxidase was used as the second antibody of the patients was 84 kDa, which was larger than that of CTSE followed by development using the ECL system ( h e r s h a m , purified from gastric mucosa and the recombinant CTSE (82 Aylesbury, UK) according to the company’s specifications. kDa) (Fig. 5). 494 AZUMA ETAL. FIGURE1 - Immunohistochemical staining for CTSE. CTSE was stained diffusely in the cytoplasm in the pancreatic ductal adenocarcinoma (a). Normal ductal cells were not stained (b). The positive staining was also observed in the mucinous cell hyperplasia (c) and intraductal papillary hyperplasia (d). Scale bar = 10 km. OD 405nm I 0.5t FIGURE 2 - Standard curve of the ELISA for CTSE. Each point was the mean of quintuple determinations. DISCUSSION Our immunohistochemical analysis showed that all human pancreatic ductal adenocarcinomas expressed CTSE. Sessa et al. (1990) also reported that most pancreatic ductal carcinomas express one or more antigens normally found in gastrointestinal epithelial cells, and that the antigens expressed most commonly, M1 (an antigen associated with the peptide core of much in gastric superficial-foveolar cells) and CTSE, were each found in over 90% of the 88 differentiated ductal cell carcinomas. Both antigens are constituents of normal gastric superficial-foveolar cells. Therefore, we examined whether CTSE could serve as a marker for pancreatic ductal carcinomas. The inherent problems for characterization of CTSE have involved the difficulty in purification of the protein and the inability to assay its activity distinct from other aspartic proteinases. The lower concentration of this proteinase compared with that of the pepsinogens also contributed to the relatively slow progress in the characterization of the enzyme. Previously, we successfully isolated human CTSE cDNA clones (Azuma et al., 1989) and established a CTSE production system by the methylotrophic yeast Pichia pastoris (Yamada et al., 1994). Using recombinant human CTSE, we established an assay system for CTSE. CTSE was not detected in the serum of the patients with pancreatic ductal adenocarcinoma. By contrast, CTSE in the pancreatic juice was present in 72.7% (8/11) of the patients with pancreatic ductal adenocarcinoma. Although quantitative analysis of the CTSE in the pancreatic juice was difficult in the present study, the increased detection frequency of CTSE in the pancreatic juice was significantly higher in the patients with pancreatic ductal adenocarcinoma than in the patients with chronic pancreatitis. Several reports have described the usefulness of CA19-9 detected in pancreatic juice. Chen et al. (1989) reported that the CA19-9 content in the pancreatic juice was significantly CATHEPSIN E IN PANCREAS TUMORS 495 CTSE ( P Q I 114 160 50 140 120 40 100 80 30 60 40 20 20 0 n-11 FIGURE 3 - Ratio of CTSE to secretory protein concentration in the pancreatic juice. CTSE in the pancreatic juice was present in 8 of 11 (72.7%) patients with pancreatic ductal adenocarcinoma, 5 of 10 (50%) patients with mucin-producing adenoma, 1 of 3 (33.3%) patients with intraductal papillary hyperplasia and 4 of 43 (9.3%) patients with chronic pancreatitis. increased in pancreatic cancer patients, whereas in chronic pancreatitis patients it was normal, indicating that it is a specific and valuable tumor marker in the differential diagnosis of pancreatic cancer. In contrast, Schmiegel et al. (1985) have shown that although the average juice CA19-9 concentration was significantly higher in patients with pancreatic cancer than in chronic pancreatitis patients, there was considerable overlap among the groups. Tatsuta et al. (1985) found that assay of CA19-9 pancreatic juice concentration was not useful for the diagnosis of pancreatic cancer, since no significant difference was noted between the pancreatic cancer and chronic calcified pancreatitis groups. However, Malesci et al. (1987) observed that although the absolute CA19-9 juice concentration was not likely to discriminate between patients with pancreatic cancer and those with benign chronic pancreatic disorders, when the CA19-9itotal protein concentration ratio was calculated, the ratio in only 1 of 22 pancreatic cancer cases overlapped with those found in non-neoplastic disorders. Additionally, they have proposed that the measurement of the CA19-9itotal protein concentration ratio in the pancreatic juice may be an accurate diagnostic marker for pancreatic adenocarcinoma. In the present study there was considerable overlap among the groups of patients, although the ratio of CA19-9 to secretory protein concentration was higher in the patients with pancreatic ductal adenocarcinoma than in the patients with chronic pancreatitis. The definitive diagnosis between pancreatic cancer and pancreatitis remains difficult. CTSE in the pancreatic juice may be a useful marker for such distinction. However, our work is a preliminary report in a small number of patients. Further analysis with larger numbers remains necessary. Multistep models of tumorigenesis, i e . , colon cancer, are based on a defined progression from atypical hyperplasia and dysplasia through benign tumors to malignant invasive tumors (Vogelstein et al., 1988). Changes in the ductal epithelium, ranging from papillary hyperplasia and atypia to carcinoma in 10 . . m adenocarcin=1 1 n=10 n=43 FIGURE 4 - Ratio of CA19-9 to secretory protein concentration in the pancreatic juice. There was considerable overlap among the groups, although the ratio was higher in the patients with pancreatic ductal adenocarcinoma than in the patients with chronic pancreatitis. situ, are frequent in cases of pancreatic ductal adenocarcinoma (Chen et al., 1985; Cubilla and Fitzgerald, 1975; Morohoshi et af., 1989). The multistep carcinogenesis theory in the case of pancreas has been also proposed to occur via c-Ki-ra5 oncogene activation. The development of human pancreatic ductal adenocarcinoma has been shown to be associated with the c-Ki-rus oncogene activating mutation at high frequencies, ranging from 75 to 100% (Almoguera et al., 1988; Smit et nl., 1988; Tada et al., 1991). Mutation has been also shown to be a frequent event in pancreatic ductal adenocarcinomas induced by the carcinogen N-nitroso bis(2-oxopropy1)amine in Syrian golden hamsters. The mutations were detected in 26% of hyperplasia, 46% of papillary hyperplasia, 76% of carcinoma in situ and 80% of adenocarcinomas (Cerny et al., 1992). These findings suggest that mutation is an early event in pancreatic multistep carcinogenesis. In the present study, CTSE in the pancreatic juice was detected in the patients with mucinproducing adenomas (5 of 10). and in the patients with intraductal papillary hyperplasia (1 of 3). By immunohistochemistry, CTSE was also expressed in mucin-producing adenomas, intraductal papillary hyperplasia and mucinous cell hyperplasia of ductal epithelium. These findings suggest that expression of CTSE is an important event in the pathogenesis of pancreatic ductal adenocarcinoma and that CTSE expression is an event occurring at a relatively early stage of multistep carcinogenesis in the pancreas lesions in a similar way as with the c-Ki-ras mutation. CTSE is an intracellular aspartic proteinase consisting of 2 identical subunits with a molecular mass of about 42 kDa. 496 AZUMA E T A L FIGURE 5 -Western blot analysis of CTSE in the pancreatic juice. The molecular size of the CTSE in the pancreatic juice of the patient with ductal adenocarcinoma was 84 kDa (lane 2), which was larger than that of the recombinant CTSE (82 kDa) (lane I). CTSE is known to be a glycoprotein and is considered to contain oligosaccharide chains of both the high mannose and the complex types (Yamamoto et al., 1978; Yonezawa et al., 1990; Finley and Kornfeld, 1994; Fowler et al., 1995). There is suggestive evidence that CTSE is synthesized as a high molecular mass precursor and subsequently processed to the mature form by the autocatalytic release of a NH2-terminal prosegment. This suggests that CTSE is initially synthesized on membrane-bound ribosomes and then transported to the Golgi complex in a manner similar to lysosomal enzymes (Athauda et al., 1991). Previously, we have reported the expression of human CTSE cDNA in Chinese hamster ovary (CHO) cells and the intracellular localization and processing of the recombinant enzymes; we suggested that the recombinant CTSE was initially synthesized on membrane-bound ribosomes as an N-glycosylated preproenzyme and that, after cleavage of the signal segment, the 90 kDa proenzyme was proteolytically processed to the intermediate (84 kDa) and mature (82 kDa) forms by the transport system (Tsukubaet al., 1993). Finley and Kornfeld (1994) also examined the subcellular localization and targeting of CTSE using murine L cells and monkey Cos 1 cells that were transfected with CTSE cDNA, and reported that the intracellular location of CTSE was the endoplasmic reticulum. An interesting question is why CTSE is present in the pancreatic juice of the patients with ductal adenocarcinoma at a high frequency. CTSE is an intracellular proteinase and is not secreted from cells. In this study, the molecular weight of the CTSE in the pancreatic juice (84 kDa) was larger than that of the mature form (82 kDa), and the mature form was not detected in the pancreatic juice. These findings suggest that the processing of the enzyme expressed in pancreatic ductal tumors is different from that in normal gastric superficial epithelial cells. 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