Jpn J Clin Oncol 2006;36(10)638–642
Double Cancer of Gall Bladder and Bile Duct not Associated with
Anomalous Junction of the Pancreaticobiliary Duct System
Hiiroshige Hori1, Tetsuo Ajiki1, Tsunenori Fujita1, Taro Okazaki1, Yasuyuki Suzuki1, Yoshikazu Kuroda1 and
Department of Gastroenterological Surgery, Kobe University Graduate School of Medical Sciences, Kobe and
Department of Surgical and Molecular Pathology, Dokkyo University School of Medicine, Shimotsuga-gun,
Received February 15, 2006; accepted June 14, 2006; published online August 14, 2006
Background: Simultaneous double cancers of the biliary tract are rare. Most of them are thought
to be associated with pancreaticobiliary maljunction (PBM); however, the characteristics of
tumours without PBM are still unclear.
Methods: Histology, immunoreactivity with carcinoembryonic antigen, carbohydrate antigen 19-9
and p53 and mutations in the K-ras gene were examined in tumours resected from cases of
simultaneous double cancers of the biliary tract.
Results: Four cases of simultaneous double cancers of the biliary tract were identified among 108
patients with biliary tract cancer (3.7%). None of the four cases associated with PBM, and the
results of histological, immunohistochemical and genetic examinations differed between the bile
duct and gall bladder cancers in each case.
Conclusion: Even when they do not associate with PBM, double cancers in the biliary tract are
more likely to be the result of multicentric development.
Key words: double cancer – gall bladder – bile duct – K-ras – p53
INTRODUCTION (CEA) and carbohydrate antigen (CA) 19-9 can also be used to
characterize biliary tract tumours (11–13).
Simultaneous double cancers in the biliary system are rare.
We have treated four cases of simultaneous double cancers
Most are thought to be associated with pancreaticobiliary
of the biliary tract without PBM. In this study, we compared
maljunction (PBM) owing to the action of the same carcinogen
several characteristics of these tumours, including immuno-
on the mucosa of the entire extrahepatic biliary system (1,2).
histochemical evaluation of CEA, CA 19-9 and p53 over-
With regard to biliary cancer cases without PBM, the presence
expression, as well as identification of mutations in K-ras.
of simultaneous double tumours poses the question of whether
The results reported below suggest that most double cancers
differentiation between independent primary cancers has
of the biliary tract have multicentric development, even in
occurred or different cancer foci have metastasized from a
the absence of PBM.
single tumour. From a clinical viewpoint, differentiation
between these events is important since these two mechanistic
origins imply different stages of disease, as well as different
subsequent treatments and prognoses. PATIENTS AND METHODS
In order to track the origin of multiple cancers, altered steady
state levels of p53 polypeptide or the presence or absence of
mutations in K-ras can function as objective diagnostic Of the 52 gall bladder and 56 bile duct cancer cases that
adjuncts (3–8), since these abnormalities are due to genetic were operated on at our hospital between 1980 and 2005,
changes found frequently in a wide variety of malignancies, four cases of simultaneous double cancers of the biliary
including biliary tract carcinomas (9,10). The patterns of tract were identified. Histological diagnoses of these cases
immunohistochemical staining for carcinoembryonic antigen were based upon mapping examination of each entire biliary
tract. Among these 108 biliary cancers, there were 12 cases
with PBM; however, none of the four cases with simultaneous
double cancers displayed anomalous junction of pancreati-
For reprints and all correspondence: Tetsuo Ajiki, Department of cobiliary duct system. The presence of PBM was confirmed
Gastroenterological Surgery, Kobe University Graduate School of Medical
Sciences, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. by endoscopic retrograde cholangiopancreatography (14).
E-mail: email@example.com Assessments of clinicopathological factors and staging were
# 2006 Foundation for Promotion of Cancer Research
Jpn J Clin Oncol 2006;36(10) 639
in accordance with the General Rules for Surgical and SW 480 colon cancer cells for use as a positive control, as
Pathological Studies on Cancer of Biliary Tract (15). described previously (20).
Tissue sections from paraffin-embedded tissue were de-paraf-
finized and incubated individually with anti-CEA antibody CLINICOPATHOLOGICAL FINDINGS AND FOLLOW-UP
(Nichirei, Tokyo, Japan), anti-CA 19-9 antibody (Japan Four cases of simultaneous double cancer of the biliary tract
Turner, Tokyo, Japan) or anti-p53 antibody CM1 (1 : 2000, were identified among 108 patients with biliary tract cancers
Novocastra Laboratory, Newcastle, England). Pre-treatment (3.7%). The clinicopathological findings from these four cases
before staining was performed in double-distilled water by are summarized in Table 1. All cases were preoperatively
heating the immersed slides in a microwave oven for diagnosed as bile duct carcinomas, and no gall bladder carcin-
10 min. Immunohistochemical staining was performed using oma was detected in any case before surgery. Biliary drainage
labelled streptavidin–biotin complexes, as described previ- was performed for Cases 1 and 3. Pancreatoduodenectomy
ously (16). Immunohistochemical evaluation was based on with regional lymphadenectomy was performed in three
both the distribution (percentage of positive cells) and the cases (Cases 1–3), whereas bile duct resection and cholecys-
intensity of staining, as described previously (17). The patterns tectomy was performed in the fourth (Case 4). By mapping the
of CEA and CA 19-9 immunostaining were classified into entire biliary tract, the tumours were separated completely
three types (apical, cytoplasmic and stromal type) in accor- (Fig. 1). There were intramucosal lesions both in the gall
dance with previous reports (18,19). p53 protein expression bladder and bile duct tumours in all four cases. Dysplastic
was evaluated by the intensity and distribution of immuno- lesions around cancer lesions were seen in the gall bladder
staining, as reported previously by the authors (16). In the of two cases (Cases 2 and 4), but among the four cases, no
present study, p53 staining was confined to the nuclei of cancer hyperplastic or metaplastic lesions were seen. Although three
cells, but not to those of dysplastic lesions. cases (Cases 2–4) were classified with similar histological
subtypes (pap and tub1), Case 1 was the only case to display
DNA EXTRACTION AND ANALYSIS OF K-RAS MUTATION different histological subtypes (tub1 and por) in double biliary
cancers and to have regional lymph node metastases. Histology
DNA was extracted from paraffin-embedded tissue by of these lymph node metastases identified the cells as well-
microdissection, and DNA from dissected tissues was differentiated tubular adenocarcinoma. The patient defined as
extracted using the DNA isolator PS kit (Wako Pure Chemical, Case 1 has survived >5 years after resection, whereas patients
Osaka, Japan) according to the manufacturer’s protocol. A described as Cases 2, 3 and 4 died of recurrence within 3 years
two-step polymerase chain reaction (PCR)/restriction after surgery.
enzyme-based method was used to identify mutations in Serum levels of CEA and CA19-9 are shown in Table 2. Val-
K-ras at codon 12. Sequences for respective sets of ues for Case 1 and 3 were obtained after biliary drainage. A
mismatched primers targeting codon 12 were described mild elevation of CEA or CA 19-9 was seen in Cases 1, 2 and 3.
previously (20). For codon 12, the presence of either wild-
type or mutant sequence was distinguished by fragments of
IMMUNOHISTOCHEMICAL AND GENETIC FINDINGS
100 or 129 bp, respectively, by digestion to completion with
the restriction enzyme Mva I (Takara, Kyoto, Japan). DNA A representative figure depicting immunohistochemical
encoding a mutation at codon 12 of K-ras was isolated from examination of CEA, CA19-9 and p53 is shown in Fig. 2.
Table 1. Clincopathological features of four cases of double cancers of the biliary tract
Case Year Age Sex Tumour site Tumour size (mm) Gross feature Histological type, depth ly v n Stage Prognosis
1 1987 77 F BD, middle 15 · 12 Flat tub1, ss + À +* III Alive, 77 mo
GB, fundus-body 35 · 20 Nodular tub3, ss + À
2 1990 70 M BD, middle-upper 75 · 19 Papillary pap, fm À À À I Dead, 34 mo
GB, body-neck 20 · 10 Flat tub1, m À À
3 1992 62 F BD, middle 25 · 15 Nodular tub1, se + + À III Dead, 18 mo
GB, fundus-body 50 · 50 Flat tub1, ss À À
4 2000 62 F BD, lower-upper 70 · 20 Papillary tub1, ss + + À II Dead, 32 mo
GB, fundus-body 10 · 10 Papillary pap-tub1, mp À À
BD, bile duct; GB, gall bladder; tub1, well-differentiated tubular adenocarcinoma; tub3, poorly differentiated tubular adenocarcinoma; pap, papillary adenocar-
cinoma; m, mucosal layer; mp, proper muscle layer, ss; subserosal layer; se, serosa exposed invasion; fm, fibromuscular layer; ly, lymphatic invasion; v, venous
invasion; n, lymph node metastasis; *positive for #8, #12b1 and #12c; mo, months.
640 Double cancers of the biliary tract
staining and gene abnormalities, the histological types of
the tumours were different (tub1 and por). In contrast, double
tumours from Case 2, 3 or 4 were similar histologically, but
immunohistochemical staining patterns and K-ras abnormali-
ties were different.
There are two competing hypotheses to explain the
pathogenesis of double cancers of the biliary tract: independent
primary lesions (multicentric) or metastasis of the original
cancer. Surgeons and pathologists have established several
criteria to differentiate between primary tumours and metas-
tases according to macroscopic appearance and histological
characteristics (21,22). Many double simultaneous cancers
have been detected clinically in cases with anomalous pancre-
aticobiliary ductal unions (1,2). Fujii et al. (23) reported that
62.5% of synchronous double cancers of biliary tract and 100%
of metachronous double cancers of biliary tract were asso-
ciated with PBM. Biliary cancer cases with PBM are thought
to develop multicentrically, in part owing to the influence of
pancreatic juice reflux on the mucosa of the entire biliary tract
(24). Furthermore, Fahim et al. (25) reported that intraductal
spread occurred in only $4% of biliary tract carcinomas. Both
Figure 1. Diagram of the biliary tract in Cases 1–4. Bars show the positions of of these findings support the hypothesis that double carcino-
cancer lesions in each case.
mas of the biliary tract tend to derive from different primary
lesions. In reality, however, determining whether double
cancers are metastases or independent tumours can prove
Table 2. Serum tumour markers of double cancers of the biliary tract difficult. Immunohistochemical and genetic results from the
four cases of double biliary tract carcinomas presented here
Case CEA (ng/ml)* CA19-9 (U/ml)** suggest that, even in the absence of PBM, multicentric
1 3.7 50 neoplastic development is common in the biliary tract double
2 6.0 65 cancers than previously thought.
3 2.1 44 Genetic information from multiple neoplastic lesions pro-
4 4.6 23
vides additional criteria for differentiating between diagnoses
of metastasis or independent primary neoplasms (3–8). Using
*normal range: <5.0, **normal range: <37. genetic markers, multifocal polyclonal processes have been
identified in lung (3,4) and head and neck cancers (8) (dem-
onstrating so-called ‘field cancerization’), whereas analyses of
The identity of a K-ras mutation is shown in Fig. 3. Immuno- p53 and c-erbB-2 expression in urothelial cancers revealed that
histochemical and genetic findings are summarized in multifocal carcinogenesis in the urothelium is generally due
Table 3. The tumours from Case 1 displayed two different to seeding or intraepithelial metastatic spread of the original
CEA patterns of immunostaining, but the same CA 19-9 cancer cells (6,7).
pattern. In contrast, patterns of CEA and CA 19-9 immuno- With regard to double biliary cancers, only one study reports
staining were distinctly different between tumour pairs the use of genetic assays as an adjunct of differential diagnosis
resected from Case 2, 3 or 4. Within each case, the two tumours and suggests its importance for analysing LOH in bile duct
examined from Cases 1 and 2 showed the same pattern of p53 double cancers (26). Although CEA or CA 19-9 staining
or K-ras, relative to each other, and the two tumours isolated patterns were informative in distinguishing double cancers,
from Cases 3 and 4 showed a different pattern of p53 or K-ras, ras or p53 are more likely to be important for differentiating
relative to each other. biliary tract double cancers, since CEA staining patterns have
been reported to shift from apical to cytoplasmic types, and
heterogeneous patterns of antigen localization may exist in one
Of the biliary cancer cases in this study, 3.7% (a relatively high Histologically, biliary tract cancers that occur in cases with
frequency) involved simultaneous double cancers of the biliary PBM frequently show hyperplasia (27); nonetheless, our
tract. On the basis of the histological type, protein staining cases showed no such lesions. In addition, K-ras mutation
and the presence of mutations in K-ras, the tumour pairs and p53 abnormalities are more frequently detected in
isolated from each of four cases were, in some ways, different. cancers with PBM than in those without PBM (28,29).
Although double tumours in Case 1 showed similar protein Although some tumours in these four cases showed K-ras
Jpn J Clin Oncol 2006;36(10) 641
Figure 2. Histological findings of Case 2 gall bladder carcinoma. (A) H & E staining, (B) CEA is localized with polarity, (C) CA19-9 is localized without polarity
in the cytoplasm, (D) nuclei of this cancer display extensive immunoreactivity with anti-p53 antibody.
Table 3. Immunohistochemical and genetic characteristics of double cancers
of the biliary tract
Case 2 Case 3 Case Tumour Staining pattern Gene abnormalities
CEA CA 19-9 p53 K-ras
1 BD Apical Cytoplasmic + À
GB Cytoplasmic Cytoplasmic + À
2 BD Negative Negative + +
GB Cytoplasmic Cytoplasmic + +
3 BD Cytoplasmic Stromal + À
GB Negative Cytoplasmic À +
4 BD Negative Cytoplasmic + À
GB Stromal Stromal À À
BD, bile duct; GB, gall bladder; +, positive; À, negative.
Figure 3. Detection of a codon 12 mutation in K-ras. In this representative
sample, the upper band results from Mva I digestion of PCR products from the
mutant allele and the lower band results from similar digestion of PCR products provide valuable data for differentiating multiple cancers
from wild-type allele. Mutant bands were seen in the gall bladder carcinomas of of the biliary tract. In the future, assays for mutations
Cases 2 and 3. M, fX174-Hae III DNA marker; GBC, gall bladder carcinoma; in other genes would increase the probability of identifying
BDC, bile duct carcinoma. accurately the origins of multiple lesions of the biliary
or p53 abnormalities, further studies with more cases will be
necessary to identify the importance of genetic information in
double cancers of the biliary tract without PBM. References
In conclusion, in conjunction with existing pathological 1. Todani T, Tabuchi K, Watanabe Y, Kobayashi T. Carcinoma arising in the
criteria, immunohistochemical and genetic analyses can wall of congenital bile duct cysts. Cancer 1979;44:1134–41.
642 Double cancers of the biliary tract
2. Ikoma A, Nakamura N, Miyazaki T, Maeda M. Double cancer of the 16. Ajiki T, Onoyama H, Asaka K, Fujimori T, Maeda S, Yamamoto M, et al.
gallbladder and common bile duct associated with anomalous junction of p53 protein expression and prognosis in gallbladder carcinoma and
pancreaticobiliary ductal system. Surgery 1992;111:595–600. premalignant lesions. Hepato-Gastroenterol 1996;43:521–6.
3. Noguchi M, Maezawa N, Nakanishi Y, Matsuno Y, Shimosato Y, 17. Yukawa M, Fujimori T, Maeda S, Tabuchi M, Nagasako K. Comparative
Hirohashi S. Application of the p53 gene mutation pattern for clinicopathological and immunohistochemical study of ras and p53 in flat
differential diagnosis of primary versus metastatic lung carcinomas. and polypoid type colorectal tumours. Gut 1994;35:1258–61.
Diagn Mol Pathol 1993;2:29–35. 18. Hamada Y, Yamamura M, Hioki K, Yamamoto M, Nagura H, Watanabe K.
4. Sozzi G, Miozzo M, Pastorino U, Pilotti S, Donghi R, Giarola M, et al. Immunohistochemical study of carcinoembryonic antigen in patients with
Genetic evidence for an independent origin of multiple preneoplastic and colorectal cancer: correlation with plasma carcinoembryonic antigen
neoplastic lung lesions. Cancer Res 1995;55:135–40. levels. Cancer 1985;55:136–41.
5. Oda T, Tsuda H, Scarpa A, Sakamoto M, Hirohashi S. Mutation pattern of 19. Sasaki R. Immunohistochemical study of cancer-associated carbohydrate
the p53 gene as a diagnostic marker for multiple hepatocellular carcinoma. antigens in carcinoma of the biliary tract. Jpn J Gastroenterol 1989;
Cancer Res 1992;52:3674–8. 90:1976–88.
6. Lunec J, Challen C, Wright C, Mellon K, Neal DE. c-erbB-2 amplification 20. Ajiki T, Fujimori T, Onoyama H, Yamamoto M, Kitazawa S, Maeda M, et al.
and identical p53 mutations in concomitant transitional carcinomas of K-ras gene mutation in gall bladder carcinomas and dysplasia. Gut
renal pelvis and urinary bladder. Lancet 1992;339:439–40. 1996;38:426–9.
7. Habuchi T, Takahashi R, Yamada H, Kakehi Y, Sugiyama T, Yoshida O. 21. Warren S, Gates O. Multiple primary malignant tumors: a survey of the
Metachronous multifocal development of urothelial cancers by literature and a statistical study. Am J Cancer 1932;16:1358–414.
intraluminal seeding. Lancet 1993;342:1087–8. 22. Moertel CG. Multiple primary malignant neoplasms: histological
8. Nees M, Homann N, Discher H, Andl T, Enders C, Herold-Mende C, et al. perspectives. Cancer 1977;40:1786–92.
Expression of mutated p53 occurs in tumor-distant epithelia of head and 23. Fujii T, Kaneko T, Sugimoto H, Okochi O, Inoue S, Takeda S, et al.
neck cancer patients: a possible molecular basis for the development of Metachronous double cancer of the gallbladder and common bile duct.
multiple tumors. Cancer Res 1993;53:4189–96. J Hepatobiliary Pancreat Surg 2004;11:280–5.
9. Wee A, Teh M, Raju GC. Clinical importance of p53 protein in gall bladder 24. Kobayashi S, Asano T, Yamasaki M, Kenmochi T, Nakagohri T, Ochiai T.
carcinoma and its precursor lesions. J Clin Pathol 1994;47:453–6. Risk of bile duct carcinogenesis of extrahepatic bile ducts in
10. Watanabe M, Asaka M, Tanaka J, Kurosawa M, Kasai M, Miyazaki T. Point pancreaticobiliary maljunction. Surgery 1999;126:939–44.
mutation of K-ras gene codon 12 in biliary tract tumors. Gastroenterology 25. Fahim RB, McDonald JR, Richards JC, Ferris DO. Carcinoma of
1994;107:1147–53. the gallbladder: a study of its modes of spread. Ann Surg 1962;156:
11. Albores-Saavedra J, Nadji M, Morales AR, Henson DE. Carcinoembryonic 114–24.
antigen in normal, preneoplastic and neoplastic gallbladder epithelium. 26. Ogawa A, Sugo H, Takamori S, Kojima K, Fukasawa M, Beppu T, et al.
Cancer 1983;52:1069–72. Double cancers in the common bile duct: molecular genetic findings with
12. Davis RI, Sloan JM, Hood JM, Maxwell P. Carcinoma of the extrahepatic an analysis of LOH. J Hepatobiliary Pancreat Surg 2001;8:374–8.
biliary tract: a clinicopathological and immunohistochemical study. 27. Hanada K, Itoh M, Fujii K, Tsuchida A, Hirata M, Ishimaru S, et al.
Histopathology 1988;12:623–31. Pathology and cellular kinetics of gallbladder with an anomalous
13. Kimura W, Miyata R, Takahashi T, Yamashiro M. Simultaneous junction of the pancreaticobiliary duct. Am J Gastroenterol 1996;
development of gall bladder and bile duct carcinomas with atypical 91:1007–11.
epithelium intervention: a case report. Jpn J Clin Oncol 1989;19:287–93. 28. Hanada K, Tsuchida A, Iwao T, Eguchi N, Sasaki T, Morinaka K, et al. Gene
14. The Japanese Study Group on Pancreaticobiliary Maljunction; the mutations of K-ras in gallbladder mucosae and gallbladder carcinoma with
Committee of JSPBM for diagnostic criteria. Diagnostic criteria of an anomalous junction of the pancreaticobiliary duct. Am J Gastroenterol
pancreaticobiliary maljunction. J Hepatobiliary Pancreat Surg 1999;94:1638–42.
1994;1:219–21. 29. Matsubara T, Funabiki T, Jinno O, Sakurai Y, Hasegawa S, Imazu H, et al.
15. Japanese Society of Biliary Surgery. General Rules for Surgical and p53 gene mutations and overexpression of p53 product in cancerous
Pathological Studies on Cancer of the Biliary Tract. 5th edn. Tokyo: and noncancerous biliary epithelium in patients with pancreaticobiliary
Kanehara 2003. maljunction. J Hepatobiliary Pancreat Surg 1999;6:286–93.