Jpn J Clin Oncol 2006;36(10)638–642 doi:10.1093/jjco/hyl077 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 1 Department of Gastroenterological Surgery, Kobe University Graduate School of Medical Sciences, Kobe and 2 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 PATIENTS 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: firstname.lastname@example.org 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). IMMUNOHISTOCHEMISTRY RESULTS 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 DISCUSSION 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 SW480 site CEA CA 19-9 p53 K-ras BDC GBC BDC GBC expression mutation M 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 –118bp 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. 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