Double Cancer of Gall Bladder and Bile Duct not Associated with by MikeJenny


									                                                                                                 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
Takahiro Fujimori2
 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,
Tochigi, Japan
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:                                            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
                                                                                  tumour (19).
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.
                                     placental DNA

                                                                                   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




                                                                                                                                          expression     mutation

                                                                                   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
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