[Cancer Research 60, 522-524, February 1, 2000]
© 2000 American Association for Cancer Research
High Frequency of K-ras Mutations in Biliary Duct Carcinomas of Cases with a Long Common Channel in the Papilla of Vater1
Eiji Hidaka,
Akio Yanagisawa2,
Makoto Seki,
Kouichi Takano,
Toshiaki Setoguchi and
Yo Kato
Departments of Pathology [E. H., A. Y., Y. K.], Surgery [M. S.], and Internal Medicine [K. T.], Cancer Institute, Tokyo 170-8455; and Department of Surgery I, Miyazaki Medical College, Miyazaki, 889-1692 [E. H., T. S.], Japan
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ABSTRACT
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The frequency of K-ras mutation in biliary duct
carcinomas in different locations and the relationship to the form of
the junction of the pancreaticobiliary duct (JPBD) are not understood
clearly. These points were investigated in the present study.
Thirty-seven biliary duct carcinomas in patients without anomalous JPBD
were investigated for K-ras mutations. Regarding
location, 12 were hilar, 4 in the upper, 11 in the middle, and 10 in
the lower portion of the duct. Furthermore, with 14 cases for which the
form of the JPBD could be confirmed by endoscopic retrograde
cholangiopancreatography or postoperative cholangiopancreatography,
division was made into two types: those with a long common channel (>5
mm) in the papilla of Vater (type 1, n = 4), and the other with a shorter or nonapparent common channel (type 2,
n = 10). The overall frequency of
K-ras mutation was 30%, the incidence gradually
increasing from upper to lower regions. K-ras mutations
were significantly more frequent in biliary duct carcinomas associated
with long common channels (P < 0.05).
These results suggest that a long common channel may bear a relation to
K-ras mutations in biliary duct carcinogenesis,
presumably through its influence on pancreatic juice regurgitation.
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Introduction
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Biliary tract carcinomas are relatively rare, but their prognosis
is poor. With application of molecular biology techniques,
K-ras mutations have been found to be frequent in various
tumors, especially in pancreatic carcinomas (1, 2, 3, 4, 5)
, even
at the step of mucous cell hyperplasia (6)
. With biliary
tract carcinomas, in contrast, there is no definite consensus regarding
K-ras mutations (3
, 7, 8, 9, 10, 11, 12, 13, 14, 15)
, although recent
studies demonstrated more frequent detection in gallbladder carcinomas
in patients with than without an
AJPBD3
(16
, 17)
. Therefore, it seems that K-ras
mutations might bear a relation with pancreatic juice. In the cases
without AJPBD, however, little information is available concerning the
relationship between K-ras mutation and the form of the JPBD
or the tumor location.
In this study, we, therefore, concentrated attention on the JPBD and
point of origin in the bile duct of a series of bile duct carcinomas.
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Materials and Methods
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Tissue Samples.
Resected specimens of 37 biliary duct carcinomas (12 hilar, 4 upper, 11
middle, 10 lower) in patients without AJPBD were obtained from the
Cancer Institute Hospital. The tissues had all been fixed in formalin
and embedded in paraffin wax.
Types of Pancreaticobiliary Duct Junction.
In this study, the two types of JPBD were: one with a common channel
>5 mm in length (type 1), and the other with shorter (<5 mm) or a
nonapparent common channel (type 2) on endoscopic retrograde
cholangiopancreatography or postoperative cholangiopancreatography
(Fig. 1)
. Fourteen cases for which the form of the JPBD could be confirmed were
analyzed, 4 of type 1 and 10 of type 2.
DNA Extraction.
Carcinoma tissues were microdissected from 20-µm formalin-fixed
paraffin-embedded sections as described previously (6)
,
deparaffinized with xylene three times, cleared with ethanol twice,
completely dried, and digested with proteinase K. The resultant lysates
were used directly for the PCR.
PCR Amplification and Detection of K-ras Mutations.
The protocol used to analyze the tissue for point mutations in codon 12
of K-ras has been described in detail elsewhere
(6)
. DNA isolated from surgical materials was used for PCR
using primers A (5'-GGCCTGCTGAAAATGACTGA-3') and D
(5'-TAGCTGTATCGTCAAGGCAC-3'). The resulting PCR products were
dot-blotted onto seven different nylon membranes, and each of these
separate membranes was hybridized with allele-specific oligonucleotide
probe for the wild-type K-ras sequence or for one of six
possible activating point mutations in codon 12. Positive controls
included cloned wild-type and mutant sequences.
Statistical Analysis.
Categorical variables were analyzed using the Fisher’s exact
probability test. Ps of <0.05 were considered significant.
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Results
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K-ras mutations were detected in 11 of 37 samples
(30%; Fig. 2
), at frequencies of 17% (2 of 12) in hilar, 25% (1 of 4) in upper,
27% (3 of 11) in middle, and 50% (5 of 10) in lower biliary duct
lesions, respectively (Fig. 3)
. The incidence, thus, gradually increased from upper to the lower
biliary duct.
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Fig. 2. A dot blot hybridization analysis of K-ras
mutations in biliary duct carcinoma. Top,
left, letters (a-g) represent positive
controls. Sample numbers 1–12 were hilar, 13–16 upper, 17–27 middle,
and 28–37 lower portion tumors. GGT is the wild type, and the others
are mutant types.
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Fig. 3. Frequencies of K-ras mutation in tumors at
different locations. The data for gallbladder carcinomas are from our
previous report. A gradual increase in frequency is evident from the
upper to lower regions. CBD, common bile duct;
GB, gallbladder; Panc, pancreas;
Du, duodenum.
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Four point mutation types were found in this study: GGT to GAT in six
cases, to GTT in three cases, and to GCT and TGT each in one case (Fig. 2)
. No relationship between the type of mutation and the location was
evident.
The frequencies of the K-ras mutation in type 1 and type 2
cases were 75% (3 of 4) and 10% (1 of 10), respectively (Table 1
; P < 0.05, Fisher’s exact test). In type 1,
GGT to GAT mutations were detected in two cases, and to TGT in one
case. In type 2, the single mutation was a GGT to GAT.
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Discussion
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The results of our present investigation clearly showed that
K-ras mutations are more frequently detected in cases with a
long common channel in the region of the papilla of Vater. Furthermore,
the frequency of K-ras mutations gradually increased from
the upper to lower bile duct, suggesting that the JPBD form and the
location might have a relation with K-ras mutation in
biliary duct carcinoma.
According to previous studies, K-ras mutations are very
frequent in pancreatic carcinomas and gallbladder carcinomas associated
with AJPBD (1, 2, 3, 4, 5
, 16)
. Moreover, mutations were detected
even in mucous cell hyperplasia of pancreas and in noncancerous
epithelium of the gallbladder and common bile duct with AJPBD (6
, 17
, 18) . In light of those findings, it might be presumed that
K-ras mutation has a relation to pancreatic juice exposure.
In the biliary tract system, reflux of pancreatic juice into the bile
duct is influenced by the function of Oddi’s sphincter and the form of
the JPBD (19)
. It has been reported that a longer common
channel in the papilla of Vater might predispose to regurgitation of
pancreatic juice into the bile duct (20)
. The high
frequency of K-ras mutations found in biliary duct
carcinomas is associated with a long common channel; the present study
is, therefore, very indicative with regard to the role of pancreatic
juice.
As for the site of origin in the bile duct, our data are in line with
the report of Motojima et al. (3)
that
K-ras mutations were more frequent in lower than middle or
upper biliary duct carcinomas. These results again support a role for
pancreatic juice.
In the future, to confirm this hypothesis, analysis of the relationship
between the concentration of amylase in bile juice and K-ras
mutations is needed. Furthermore, the relationship with function of
Oddi’s sphincter should be investigated.
In conclusion, the form of the long common channel in the papilla of
Vater seems linked to K-ras mutations in biliary duct
carcinomas.
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ACKNOWLEDGMENTS
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We are grateful to Yuzo Sakai and Yurika Tada (Cancer Institute,
Tokyo, Japan) for technical assistance.
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FOOTNOTES
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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 Grants-in-Aid from the Ministry of
Education, Science, Sports and Culture and the Ministry of Health and
Welfare of Japan, as well as the Vehicle Racing Commemorative
Foundation. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, Cancer Institute, 1–37-1
Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan. Phone:
81-3-5394-3865; Fax: 81-3-5394-3923; E-mail: a-yanagi{at}ims.u-tokyo.ac.jp
3 The abbreviation used is: AJPBD, anomalous
junction of the pancreaticobiliary duct.
Received 11/ 8/99.
Accepted 12/ 9/99.
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ABSTRACT
Introduction
