Marked Increase of Trypsin(Ogen) in Serum of Linitis Plastica (Gastric Cancer, Borrmann 4) Patients

Vol. 6, 1385–1388, April 2000 Clinical Cancer Research 1385

Marked Increase of Trypsin(ogen) in Serum of Linitis Plastica (Gastric Cancer, Borrmann 4) Patients

Yasushi Ichikawa,1 Naohiko Koshikawa, of trypsin(ogen) might be a good marker of gastric cancer of Satoshi Hasegawa, Takashi Ishikawa, linitis plastica. Nobuyoshi Momiyama, Chikara Kunizaki, Masazumi Takahashi, Yoshihiro Moriwaki, INTRODUCTION Hirotoshi Akiyama, Hiroyuki Yamaoka, Trypsinogen, a major proteolytic enzyme precursor pro- duced by pancreatic acinar cells, is known to also be secreted by Shunsuke Yanoma, Akira Tsuburaya, cancer cells such as ovarian cancer (1), colon cancer (2), pan- Yoji Nagashima, Hiroshi Shimada, and creatic cancer (3), and cholangiocarcinoma (4). The activated Kaoru Miyazaki form of trypsinogen, trypsin, plays an important role in the Second Department of Surgery, Yokohama City University School of pathophysiology of acute pancreatitis to degrade i.p. tissues, and Medicine [Y. I., S. H., T. I., N. M., C. K., M. T., Y. M., H. A., H. Y., it may be involved in the extracellular matrix degradation by H. S.], Kihara Institute for Biological Research, Yokohama City invasive cancer cells (2). University [N. K., K. M.], and Department of Pathology, Yokohama City University School of Medicine [Y. N.], Yokohama 236-0004, Thus far, four types of trypsin have been identified: cati- and Kanagawa Cancer Center, Kanagawa 241-0815 [S. Y., A. T.], onic trypsin (trypsin-1), anionic trypsin (trypsin-2), meso-tryp- Japan sin (trypsin-3), and trypsin-4. Pancreatic juice contains cationic and anionic trypsinogen in the ratio of 2:1 and a little meso- trypsin. The cDNA for trypsinogen-4 was cloned from human ABSTRACT brain (5). Linitis plastica, or Borrmann 4 gastric cancer, shows Serum levels of anionic trypsinogen, trypsinogen-2, are very poor prognosis, and the reason has not been under- elevated not only in patients with acute pancreatitis but also in stood. In the present study, we examined serum levels of patients with hepatic, biliary, or pancreatic malignancies such as trypsin(ogen) in 44 gastric cancer patients, including 17 cancer of the ampulla Vateri, hepatocellular carcinoma, cholan- early gastric cancer, 18 non-Borrmann 4 advanced gastric giocarcinoma, and pancreatic cancer (4). cancer, and 9 Borrmann 4 gastric cancer, by using the RIA It has been reported previously that human gastric carci- gnost Trypsin kit (Hoechst Japan, Tokyo, Japan), which was noma cell lines also secrete trypsinogen (6, 7). Gastric cancer is expected to detect trypsin-1, trypsin-2, trypsinogen-1, and the leading cause of death attributable to cancer in Japan. trypsinogen-2 in sera. The trypsin(ogen) concentration was Surgical prognosis for advanced gastric cancers is improving by much higher in the patients with linitis plastica than in the excessive dissection of regional lymph nodes. However, the other gross types of gastric cancer. Hypertrypsinemia was 5-year survival rate of advanced gastric cancer, overall, is still ϳ identified in 60% of advanced gastric cancer cases. Lymph Ͻ50%. Moreover, the special gross-type linitis plastica, or Bor- node involvement, liver metastasis, or poorly differentiated rmann 4 gastric cancer, which shows a leather-bottle shape in adenocarcinoma is an important factor of hypertrypsine- upper gastrointestinal series, still has a very poor prognosis. mia. The serum trypsin(ogen) level in linitis plastica patients Although many factors of linitis plastica, such as growth factor ؎ ؎ was 3484.4 2319.7 ng/ml (mean SD), which was signif- secretion, hormonal properties, and plasminogen activity, have icantly higher not only than that of the early gastric cancer been investigated, the cause of its poor prognosis is unclear. In ؎ (384.1 92.1) but also the stage IV gastric cancer patients the present study, we examined serum levels of trypsin(ogen), ؎ (578 440.4), excluding those with linitis plastica. The including trypsin(ogen)-1 and trypsin(ogen)-2, in gastric cancer elevated serum trypsinogen level in linitis plastica patients patients. The concentration was much higher in the patients with may be related to the malignant behavior of this type of linitis plastica than in the other gross types of gastric cancer. cancer cell. Serum trypsin(ogen) of linitis plastica shows significantly higher concentrations than do the other types of advanced gastric cancer. Therefore, serum concentration MATERIALS AND METHODS Samples. Serum was obtained preoperatively from 44 gastric cancer patients (Table 1), including 9 with linitis plastica, during the years 1995–1997 at Yokohama City University Hospital and was kept at Ϫ80°C until use. Cancer gross classi- Received 4/30/99; revised 11/22/99; accepted 1/6/00. The costs of publication of this article were defrayed in part by the fication and staging were according to Japanese guidelines (8). payment of page charges. This article must therefore be hereby marked RIA. Serum levels of trypsin(ogen) were measured by a advertisement in accordance with 18 U.S.C. Section 1734 solely to classic simultaneous-addition, double-antibody RIA (9), using indicate this fact. the RIA gnost Trypsin kit (Hoechst Japan), with antihuman 1 To whom requests for reprints should be addressed at Second Depart- ment of Surgery, Yokohama City University, School of Medicine, 3-9 trypsinogen rabbit antibodies. The kits were used according to Fukuura Kanazawa-ku, Yokohama 236-0004, Japan. Phone: 045-787- the manufacturer’s instructions. Briefly, 0.1 ml of patient’s 2650; Fax: 045-782-9161. serum was incubated with 0.2 ml of antihuman trypsinogen

Table 1 Materials Gross typea n Stage n 018I19 2 7 II 2 3 9 III 7 4 (LP) 9IV16 51 Total 44 44 a Type 0, superficial, flat tumors; type 2, ulcerated type with clear margin; type 3, ulcerated type with infiltration; type 5, unclassified type. Staging refers to the classification by the Japanese Research Society for Gastric Cancer (8).

rabbit antibody and the same volume of 125I-labeled anti-trypsinogen antibody for 24 h at room temperature. After the first reaction, the sample was incubated with 0.1 ml of a goat antiserum to rabbit immunoglobulin for3hatroom temperature, and centrifugation was carried out at 1500 ϫ g for 15 min. After removal of the supernatant, radioactivity of the pellet was counted by a gamma counter. Quantification of serum trypsin- (ogen) was standardized by standard serum contained with seven different concentrations of human trypsin (0–1300 ng/ ml). When the serum concentration of trypsin(ogen) was over Fig. 1 Serum concentrations of trypsin(ogen) in preoperative gastric the upper range of the standard curve (1300 ng/ml), serum was cancer patients. Ⅺ, groups with normal concentrations of serum tryp- diluted by PBS and was reexamined. This kit is expected to sin(ogen); u, hypertrypsinemia groups. For the definition of early and measure trypsin-1, trypsin-2, trypsinogen-1, and trypsinogen-2. advanced gastric cancer, see the guidelines published by the Japanese Research Society for Gastric Cancer (8). The normal range of serum trypsin(ogen) levels of Japanese adults measured by RIA gnost Trypsin kit was 110–460 ng/ml (10), which was almost the same as the data obtained in England (138–406 ng/ml; Ref. 11). plastica showed values over the upper limit of serum trypsin(o- Statistics. Student’s unpaired t test or ␹2 analysis was gen). However, 100% of linitis plastica patients showed high used for comparison of serum trypsin(ogen) concentrations be- concentrations of serum trypsin(ogen). tween groups. Moreover, the averaged serum trypsin(ogen) level of linitis plastica patients was 3484.4 Ϯ 2319.7 ng/ml (mean Ϯ SD), RESULTS which was significantly higher than that of the early gastric Ϯ Serum trypsin(ogen) levels of 25 (56.8%) of 44 gastric cancer patients (384.1 92.1; Fig. 2) as well as that of the stage Ϯ cancer patients were within the normal range (110–460 ng/ml; IV gastric cancer patients as defined above (578 440.4), Fig. 1). Fifteen cases (60%) were early gastric cancer (defined excluding the linitis plastica patients. as carcinoma within mucosa or submucosa; Ref. 8), and among 17 patients of early gastric cancer, only 2 patients (13%) were DISCUSSION over the normal upper limit. Hypertrypsinemia is detected in ϳ60% of advanced gastric In contrast, 17 (63%) of 27 advanced gastric cancer pa- cancer, and this study shows that the sera of linitis plastica tients were over the upper limit (460 ng/ml) of the normal range patients contain a high level of trypsin(ogen). Gastric cancer is of serum trypsin(ogen), and 9 of them were linitis plastica a leading cause of death attributable to cancer in Japan. In patients. The ratio of the patients showing hypertrypsinemia was particular, linitis plastica, or Borrmann 4 gastric cancer, which significantly higher in the cases with lymph node metastases is a diffusely infiltrative carcinoma, shows significantly poor (P ϭ 0.00177) or with liver metastases (P ϭ 0.00505; Table 2). prognosis. In Japan, the cumulative 5-year survival rate of linitis However, peritoneal dissemination was not a significant factor plastica (0–13%) is far worse than that of the other types of for hypertrypsinemia (P ϭ 0.0898). Poorly differentiated ade- gastric cancer (20.2–50.3%; Ref. 12). Therefore, it is urgently nocarcinoma or signet ring cell carcinoma showed hyper- necessary to establish methods of early detection and effective trypsinemia more frequently than did well or moderately differ- therapy for linitis plastica. entiated adenocarcinoma. Biological properties of the linitis plastica gastric cancer Linitis plastica, a special gross type of advanced gastric are: (a) progressive fibrosis of tumor, so-called scirrhous carci- cancer, showed a much greater frequency of hypertrypsinemia. noma; and (b) quick, wide, and deep invasion of cancer cells in In stage IV gastric cancer, which was defined as direct invasion the stomach wall. Cancer cells of linitis plastica make surround- to other organs, para-aortic lymph node metastases, peritoneal ing fibroblasts synthesize collagen by some paracrine factors dissemination, or metastases to remote organs, according to secreted from cancer cells, such as epidermal growth factor (13) Japanese guidelines, only 60% of the cancers excluding linitis and transforming growth factors ␣ and ␤ (14). Mai et al. (15)

Table 2 Hypertrypsinemia s-trypsina n(ϩ)n(Ϫ) Ͼ460 15 2 ] ␹2 ϭ 9.7719 110–460 10 15 P ϭ 0.00177 s-trypsin H(ϩ)H(Ϫ) Ͼ460 5 13 ] ␹2 ϭ 7.858 110–460 0 25 P ϭ 0.00505 s-trypsin P(ϩ)P(Ϫ) Ͼ460 6 12 ] ␹2 ϭ 2.877 110–460 3 22 P ϭ 0.0898 s-trypsin poor, sig well, mod Ͼ460 14 5 ] ␹2 ϭ 7.503 110–460 8 17 P ϭ 0.006 a s-trypsin, serum concentration of trypsin-1/trypsinogen-1 (ng/ ml). n(ϩ), patients with lymph node metastasis; n(Ϫ), patients without lymph node metastasis; H(ϩ), patients with liver metastasis; H(Ϫ), patients without liver metastasis; P(ϩ), patients with peritoneal dissemination; P(Ϫ), patients without peritoneal dissemination; poor, poorly differentiated adenocarcinoma; sig, signet ring cell carcinoma; well, Fig. 2 Serum concentrations of trypsin(ogen) according to the his- well-differentiated adenocarcinoma; mod, moderately differentiated ad- topathological depth of gastric cancer and those of linitis plastica (LP). E enocarcinoma. The normal upper limit is 460 ng/ml. , mean and SD. The concentration in the LP group is significant higher than that of the other three groups (P Ͻ 0.05). m, mucosa; sm, submucosa; mp, muscularis propria; ss, subserosa; and se, serosa; si, direct tumor invasion of other organs or structures around the stomach. reported that linitis plastica patients showed high concentrations of serum tissue plasminogen activator, urokinase-type plasminogen activator, and their inhibitor, plasminogen activator inhibitor, which might support progressive fibrolysis before scirrhous a high concentration of serum trypsin(ogen) was detected in formation (15). Urokinase-type and tissue plasminogen activa- gastric cancer patients, especially in linitis plastica. The serum tors, as well as trypsinogen/trypsin, are members of matrix trypsin(ogen) concentration in linitis plastica was much higher serine proteinases. Urokinase-type and tissue plasminogen acti- than that in the other types of stage IV gastric cancer. Interest- vators activate plasminogen to plasmin, which shows proteolytic ingly, STKM-1 (6), which highly secretes trypsin, was derived activity toward various extracellular proteins and can activate from pleural effusion of a linitis plastica patient (we could not latent forms of some matrix metalloproteinases. Trypsin more determine whether MKN 28, which was from lymph node strongly degrades extracellular matrix proteins and more effec- metastases of moderately differentiated adenocarcinoma, was tively activates the latent forms of matrix metalloproteinases derived from linitis plastica). than plasmin. Serum trypsin(ogen) in linitis plastica patients is probably Trypsin was considered to be specifically secreted from derived from the cancer cells. In fact, Ohta et al. (18) reported pancreatic acinar cells; thus, it was expected to become a more in their immunohistochemical study that this trypsin(ogen) oc- specific marker of acute pancreatitis than amylase. However, curred in 92% of the scirrhous type of advanced gastric cancer recent studies have shown that trypsinogen is expressed in cases but only 25% of the intestinal type. Linitis plastica is the various normal human tissues (16) as well as in vascular endo- most advanced gross type of scirrhous-type gastric cancers. thelial cells of cancer tissues (17). It was also reported that Serum concentrations of trypsin(ogen) in linitis plastica patients human pancreatic cancer cell lines (18), which had been derived all exceeded 1000 ng/ml, which is a much higher concentration from pancreatic ductal cells but not acinar cells and ovarian than those in the patients with acute pancreatitis (11). Massive carcinoma (1), also secreted trypsinogen or tumor-associated invasion of gastric cancer to the pancreas may release the trypsinogen; and they might correlate to invasion (19) of cancer pancreatic trypsin(ogen) into blood, increasing its serum level. cells, leading to peritoneal dissemination or liver metastases In this study, however, five of seven linitis plastica patients did (18). A high concentration of trypsinogen also was detected in not show invasion to the pancreas. On the other hand, there were the serum of pancreatic cancer patients and cyst fluid of ovarian three patients who later received pancreatoduodenectomy be- carcinoma patients (1, 20). cause of direct invasion of gastric cancer to the pancreas head. Koshikawa et al. (6) found that some human gastric cancer These patients were not classified as having linitis plastica and cell lines, e.g., STKM-1 and MKN 28, also secreted a trypsino- did not show high levels of serum trypsin(ogen). Moreover, one gen-like protein at high levels. More recently, it was found that linitis plastica patient, who received a curative operation, had a trypsin is expressed in many human cancer cell lines derived decrease of serum trypsin from 1800 to 360 ng/ml after the from the stomach, colon, and breast in culture and that in operation. He survived for 2 years and died from peritoneal stomach cancers the trypsin expression is higher in malignant, recurrence. His serum trypsin level re-increased to 1200 ng/ml noncohesive types than in the cohesive type (21). In this study, just before he died. For the six linitis plastica patients, noncu-

rative simple total gastrectomy was performed to arrest contin- trypsin in human cancer cell lines and cancer tissues, and its tight uous hemorrhage from cancer lesions. Serum trypsin(ogen) lev- binding to soluble form of Alzheimer amyloid precursor protein in els in three of the six cases markedly decreased, compared with culture. J. Biochem., 125: 1067–1076, 1999. preoperative values. The other three linitis plastica patients, 8. Japanese Research Society for Gastric Cancer. The General Rules for the Gastric Cancer Study, 12th ed. Kinbara Shuppan, Tokyo, Japan, whose serum trypsin(ogen) levels did not decrease after their 1993. operations, had massive para-aortic lymph node metastases or 9. Martin, M. J., and Landon, J. In: C. A. Pasternak (ed.), Radioim- massive peritoneal dissemination. Thus, an adequate decrease of munoassay in Clinical Biochemistry, p. 269. London, 1975. cancer volume by surgery resulted in a decrease of serum 10. 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Yasushi Ichikawa, Naohiko Koshikawa, Satoshi Hasegawa, et al.

Clin Cancer Res 2000;6:1385-1388.

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