Vol.
6, 13851388, April 2000
Marked Increase of Trypsin(ogen) in Serum of Linitis Plastica
(Gastric Cancer, Borrmann 4) Patients
Yasushi Ichikawa,1 Naohiko Koshikawa,
Satoshi Hasegawa, Takashi Ishikawa,
Nobuyoshi Momiyama, Chikara Kunizaki,
Masazumi Takahashi, Yoshihiro Moriwaki,
Hirotoshi Akiyama, Hiroyuki Yamaoka,
Shunsuke Yanoma, Akira Tsuburaya,
Yoji Nagashima, Hiroshi Shimada, and
Kaoru Miyazaki
Second Department of Surgery, Yokohama City University School of
Medicine [Y. I., S. H., T. I., N. M., C. K., M. T., Y. M., H. A., H. Y.,
H. S.], Kihara Institute for Biological Research, Yokohama City
University [N. K., K. M.], and Department of Pathology, Yokohama
City University School of Medicine [Y. N.], Yokohama 236-0004,
and Kanagawa Cancer Center, Kanagawa 241-0815 [S. Y., A. T.],
Japan
ABSTRACT
Linitis plastica, or Borrmann 4 gastric cancer, shows
very poor prognosis, and the reason has not been under-
stood. In the present study, we examined serum levels of
trypsin(ogen) in 44 gastric cancer patients, including 17
early gastric cancer, 18 non-Borrmann 4 advanced gastric
cancer, and 9 Borrmann 4 gastric cancer, by using the RIA
gnost Trypsin kit (Hoechst Japan, Tokyo, Japan), which was
expected to detect trypsin-1, trypsin-2, trypsinogen-1, and
trypsinogen-2 in sera. The trypsin(ogen) concentration was
much higher in the patients with linitis plastica than in the
other gross types of gastric cancer. Hypertrypsinemia was
identified in 60% of advanced gastric cancer cases. Lymph
node involvement, liver metastasis, or poorly differentiated
adenocarcinoma is an important factor of hypertrypsine-
mia. The serum trypsin(ogen) level in linitis plastica patients
was 3484.4 2319.7 ng/ml (mean SD), which was signif-
icantly higher not only than that of the early gastric cancer
(384.1 92.1) but also the stage IV gastric cancer patients
(578 440.4), excluding those with linitis plastica. The
elevated serum trypsinogen level in linitis plastica patients
may be related to the malignant behavior of this type of
cancer cell. Serum trypsin(ogen) of linitis plastica shows
significantly higher concentrations than do the other types
of advanced gastric cancer. Therefore, serum concentration
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
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 Second Depart-
ment of Surgery, Yokohama City University, School of Medicine, 3-9
Fukuura Kanazawa-ku, Yokohama 236-0004, Japan. Phone: 045-787-
2650; Fax: 045-782-9161.
Clinical Cancer Research 1385
of trypsin(ogen) might be a good marker of gastric cancer of
linitis plastica.
INTRODUCTION
Trypsinogen, a major proteolytic enzyme precursor pro-
duced by pancreatic acinar cells, is known to also be secreted by
cancer cells such as ovarian cancer (1), colon cancer (2), pan-
creatic cancer (3), and cholangiocarcinoma (4). The activated
form of trypsinogen, trypsin, plays an important role in the
pathophysiology of acute pancreatitis to degrade i.p. tissues, and
it may be involved in the extracellular matrix degradation by
invasive cancer cells (2).
Thus far, four types of trypsin have been identified: cati-
onic trypsin (trypsin-1), anionic trypsin (trypsin-2), meso-tryp-
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
brain (5).
Serum levels of anionic trypsinogen, trypsinogen-2, are
elevated not only in patients with acute pancreatitis but also in
patients with hepatic, biliary, or pancreatic malignancies such as
cancer of the ampulla Vateri, hepatocellular carcinoma, cholan-
giocarcinoma, and pancreatic cancer (4).
It has been reported previously that human gastric carci-
noma cell lines also secrete trypsinogen (6, 7). Gastric cancer is
the leading cause of death attributable to cancer in Japan.
Surgical prognosis for advanced gastric cancers is improving by
excessive dissection of regional lymph nodes. However, the
5-year survival rate of advanced gastric cancer, overall, is still
50%. Moreover, the special gross-type linitis plastica, or Bor-
rmann 4 gastric cancer, which shows a leather-bottle shape in
upper gastrointestinal series, still has a very poor prognosis.
Although many factors of linitis plastica, such as growth factor
secretion, hormonal properties, and plasminogen activity, have
been investigated, the cause of its poor prognosis is unclear. In
the present study, we examined serum levels of trypsin(ogen),
including trypsin(ogen)-1 and trypsin(ogen)-2, in gastric cancer
patients. The concentration was much higher in the patients with
linitis plastica than in the other gross types of gastric cancer.
MATERIALS AND METHODS
Samples. Serum was obtained preoperatively from 44
gastric cancer patients (Table 1), including 9 with linitis plas-
tica, during the years 19951997 at Yokohama City University
Hospital and was kept at 80C until use. Cancer gross classi-
fication and staging were according to Japanese guidelines (8).
RIA. Serum levels of trypsin(ogen) were measured by a
classic simultaneous-addition, double-antibody RIA (9), using
the RIA gnost Trypsin kit (Hoechst Japan), with antihuman
trypsinogen rabbit antibodies. The kits were used according to
the manufacturers instructions. Briefly, 0.1 ml of patients
serum was incubated with 0.2 ml of antihuman trypsinogen
1386 Serum Trypsin(ogen) Levels in Linitis Plastica Patients
Table 1 Materials
Gross typea n Stage
0 18 I 19
2 7 II
3 9 III
4 (LP) 9 IV
5 1
Total 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-tryp-
sinogen 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 for 3 h at room tempera-
ture, and centrifugation was carried out at 1500 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
the upper range of the standard curve (1300 ng/ml), serum was
diluted by PBS and was reexamined. This kit is expected to
measure trypsin-1, trypsin-2, trypsinogen-1, and trypsinogen-2.
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).
Statistics. Students unpaired t test or 2 analysis was
used for comparison of serum trypsin(ogen) concentrations be-
tween groups.
RESULTS
Serum trypsin(ogen) levels of 25 (56.8%) of 44 gastric
cancer patients were within the normal range (110 460 ng/ml;
Fig. 1). Fifteen cases (60%) were early gastric cancer (defined
as carcinoma within mucosa or submucosa; Ref. 8), and among
17 patients of early gastric cancer, only 2 patients (13%) were
over the normal upper limit.
In contrast, 17 (63%) of 27 advanced gastric cancer pa-
tients were over the upper limit (460 ng/ml) of the normal range
of serum trypsin(ogen), and 9 of them were linitis plastica
patients. The ratio of the patients showing hypertrypsinemia was
significantly higher in the cases with lymph node metastases
(P 0.00177) or with liver metastases (P 0.00505; Table 2).
However, peritoneal dissemination was not a significant factor
for hypertrypsinemia (P 0.0898). Poorly differentiated ade-
nocarcinoma or signet ring cell carcinoma showed hyper-
trypsinemia more frequently than did well or moderately differ-
entiated adenocarcinoma.
Linitis plastica, a special gross type of advanced gastric
cancer, showed a much greater frequency of hypertrypsinemia.
In stage IV gastric cancer, which was defined as direct invasion
to other organs, para-aortic lymph node metastases, peritoneal
dissemination, or metastases to remote organs, according to
Japanese guidelines, only 60% of the cancers excluding linitis
n
2
7
16
44
Fig. 1 Serum concentrations of trypsin(ogen) in preoperative gastric
cancer patients. , groups with normal concentrations of serum tryp-
sin(ogen); u, hypertrypsinemia groups. For the definition of early and
advanced gastric cancer, see the guidelines published by the Japanese
Research Society for Gastric Cancer (8).
plastica showed values over the upper limit of serum trypsin(o-
gen). However, 100% of linitis plastica patients showed high
concentrations of serum trypsin(ogen).
Moreover, the averaged serum trypsin(ogen) level of linitis
plastica patients was 3484.4 2319.7 ng/ml (mean SD),
which was significantly higher than that of the early gastric
cancer patients (384.1 92.1; Fig. 2) as well as that of the stage
IV gastric cancer patients as defined above (578 440.4),
excluding the linitis plastica patients.
DISCUSSION
Hypertrypsinemia is detected in 60% of advanced gastric
cancer, and this study shows that the sera of linitis plastica
patients contain a high level of trypsin(ogen). Gastric cancer is
a leading cause of death attributable to cancer in Japan. In
particular, linitis plastica, or Borrmann 4 gastric cancer, which
is a diffusely infiltrative carcinoma, shows significantly poor
prognosis. In Japan, the cumulative 5-year survival rate of linitis
plastica (0 13%) is far worse than that of the other types of
gastric cancer (20.250.3%; Ref. 12). Therefore, it is urgently
necessary to establish methods of early detection and effective
therapy for linitis plastica.
Biological properties of the linitis plastica gastric cancer
are: (a) progressive fibrosis of tumor, so-called scirrhous carci-
noma; and (b) quick, wide, and deep invasion of cancer cells in
the stomach wall. Cancer cells of linitis plastica make surround-
ing fibroblasts synthesize collagen by some paracrine factors
secreted from cancer cells, such as epidermal growth factor (13)
and transforming growth factors and (14). Mai et al. (15)
 Clinical Cancer Research 1387

Table 2 Hypertrypsinemia
s-trypsina n() n()
460 15 2] 2 9.7719
110460 10 15 P 0.00177
s-trypsin H() H()

460 5 13 2 7.858
]
110460 0 25 P 0.00505
s-trypsin P() P()

460 6 12 2 2.877
]
110460 3 22 P 0.0898
s-trypsin poor, sig well, mod

460 14 5 2 7.503
]
110460 8 17 P 0.006
a
s-trypsin, serum concentration of trypsin-1/trypsinogen-1 (ng/
ml). n(), patients with lymph node metastasis; n(), patients without
lymph node metastasis; H(), patients with liver metastasis; H(),
patients without liver metastasis; P(), patients with peritoneal dissem-
ination; P(), patients without peritoneal dissemination; poor, poorly
differentiated adenocarcinoma; sig, signet ring cell carcinoma; well,
well-differentiated adenocarcinoma; mod, moderately differentiated ad-
enocarcinoma.
reported that linitis plastica patients showed high concentrations
of serum tissue plasminogen activator, urokinase-type plasmin-
ogen activator, and their inhibitor, plasminogen activator inhib-
itor, which might support progressive fibrolysis before scirrhous
formation (15). Urokinase-type and tissue plasminogen activa-
tors, as well as trypsinogen/trypsin, are members of matrix
serine proteinases. Urokinase-type and tissue plasminogen acti-
vators activate plasminogen to plasmin, which shows proteolytic
activity toward various extracellular proteins and can activate
latent forms of some matrix metalloproteinases. Trypsin more
strongly degrades extracellular matrix proteins and more effec-
tively activates the latent forms of matrix metalloproteinases
than plasmin.
Trypsin was considered to be specifically secreted from
pancreatic acinar cells; thus, it was expected to become a more
specific marker of acute pancreatitis than amylase. However,
recent studies have shown that trypsinogen is expressed in
various normal human tissues (16) as well as in vascular endo-
thelial cells of cancer tissues (17). It was also reported that
human pancreatic cancer cell lines (18), which had been derived
from pancreatic ductal cells but not acinar cells and ovarian
carcinoma (1), also secreted trypsinogen or tumor-associated
trypsinogen; and they might correlate to invasion (19) of cancer
cells, leading to peritoneal dissemination or liver metastases
(18). A high concentration of trypsinogen also was detected in
the serum of pancreatic cancer patients and cyst fluid of ovarian
carcinoma patients (1, 20).
Koshikawa et al. (6) found that some human gastric cancer
cell lines, e.g., STKM-1 and MKN 28, also secreted a trypsino-
gen-like protein at high levels. More recently, it was found that
trypsin is expressed in many human cancer cell lines derived
from the stomach, colon, and breast in culture and that in
stomach cancers the trypsin expression is higher in malignant,
noncohesive types than in the cohesive type (21). In this study,
Fig. 2 Serum concentrations of trypsin(ogen) according to the his-
topathological depth of gastric cancer and those of linitis plastica (LP).
The normal upper limit is 460 ng/ml. E, mean and SD. The concentra-
tion in the LP group is significant higher than that of the other three
groups (P 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.
a high concentration of serum trypsin(ogen) was detected in
gastric cancer patients, especially in linitis plastica. The serum
trypsin(ogen) concentration in linitis plastica was much higher
than that in the other types of stage IV gastric cancer. Interest-
ingly, STKM-1 (6), which highly secretes trypsin, was derived
from pleural effusion of a linitis plastica patient (we could not
determine whether MKN 28, which was from lymph node
metastases of moderately differentiated adenocarcinoma, was
derived from linitis plastica).
Serum trypsin(ogen) in linitis plastica patients is probably
derived from the cancer cells. In fact, Ohta et al. (18) reported
in their immunohistochemical study that this trypsin(ogen) oc-
curred in 92% of the scirrhous type of advanced gastric cancer
cases but only 25% of the intestinal type. Linitis plastica is the
most advanced gross type of scirrhous-type gastric cancers.
Serum concentrations of trypsin(ogen) in linitis plastica patients
all exceeded 1000 ng/ml, which is a much higher concentration
than those in the patients with acute pancreatitis (11). Massive
invasion of gastric cancer to the pancreas may release the
pancreatic trypsin(ogen) into blood, increasing its serum level.
In this study, however, five of seven linitis plastica patients did
not show invasion to the pancreas. On the other hand, there were
three patients who later received pancreatoduodenectomy be-
cause of direct invasion of gastric cancer to the pancreas head.
These patients were not classified as having linitis plastica and
did not show high levels of serum trypsin(ogen). Moreover, one
linitis plastica patient, who received a curative operation, had a
decrease of serum trypsin from 1800 to 360 ng/ml after the
operation. He survived for 2 years and died from peritoneal
recurrence. His serum trypsin level re-increased to 1200 ng/ml
just before he died. For the six linitis plastica patients, noncu-
1388 Serum Trypsin(ogen) Levels in Linitis Plastica Patients
rative simple total gastrectomy was performed to arrest contin-
uous hemorrhage from cancer lesions. Serum trypsin(ogen) lev-
els in three of the six cases markedly decreased, compared with
preoperative values. The other three linitis plastica patients,
whose serum trypsin(ogen) levels did not decrease after their
operations, had massive para-aortic lymph node metastases or
massive peritoneal dissemination. Thus, an adequate decrease of
cancer volume by surgery resulted in a decrease of serum
trypsin(ogen) levels.
Trypsin cannot be used as a sensitive marker of gastric
cancer, because 88% of early gastric cancer cases did not exceed
the normal range (Fig. 1). The serum level of trypsinogen-2,
which changes more sensitively than trypsinogen-1 in acute
pancreatitis (20) and cholangiocarcinoma (4) patients, should be
measured in comparison with trypsin(ogen) as the marker of
gastric cancer. The pathological meaning of the elevated serum
trypsinogen concentration in linitis plastica patients is still un-
clear. However, it seems likely that serum trypsin(ogen) will
become a good marker of linitis plastica.
ACKNOWLEDGMENTS
We thank N. Higashi (CIS Diagnostic K.K., Chiba, Japan) for
assay serum trypsin(ogen) in our initial study.
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