Pyloric gland adenoma of the cystic duct with malignant transformation: report of a case with a review of the literature

Background Pyloric gland adenoma consists of closely packed pyloric-type glands lined by mucus-secreting cells. To date, approximately 230 cases have been reported, mostly of gastric localization with a tumour size up to 3.5 cm and a mean age of occurrence around 70 years. Adenocarcinoma develops in about 40% of cases and may be difficult to detect due to relatively mild nuclear atypia. Case presentation We present the first case of a pyloric gland adenoma of the cystic duct in a 62-year-old male patient and demonstrate the clinicopathologic characteristics, including radiographic, molecular, and cytogenetic findings. The 2 cm-tumour developed in the cystic duct and protruded into the hepatic and common bile duct. On microscopic examination, it displayed closely packed pyloric-type glands, and focal architectural distortion with mild nuclear atypia. Immunohistochemically, it expressed MUC1, MUC5AC, MUC6 and p53, but not MUC2 and CD10. The Ki67-proliferation index was 25%. Furthermore, high-grade intraepithelial neoplasia was observed in the surrounding bile duct. We detected chromosomal gains at 7p, 7q11q21, 15q, 16p, 20, losses at 6p23pter, 6q, 18, and amplifications at 1q and 6p21p22 in the pyloric gland adenoma by comparative genomic hybridization. A KRAS codon 12 mutation (c.35G>T; p.G12V) was detected in the pyloric gland adenoma and in the adjacent dysplasia by sequencing analysis. The diagnosis of pyloric gland adenoma was established with transition into well-differentiated adenocarcinoma and high-grade biliary intraepithelial neoplasia. Conclusion Pyloric gland adenoma evolving in the cystic duct is a rare differential diagnosis of obstructive bile duct tumours. Other premalignant bile duct lesions may be associated. Due to the risk of developing adenocarcinoma, surgical resection should be performed.

Three cases of pyloric gland adenoma of the common bile duct have up to now been reported [7]. Here, we present the first reported case of pyloric gland adenoma evolving in the cystic duct, with transition into welldifferentiated adenocarcinoma, and associated high-grade intraepithelial neoplasia of the adjacent bile duct. The clinico-pathologic characteristics, including radiologic as well as molecular and cytogenetic findings, will be demonstrated with a review of the literature.

Case presentation
A 62-year-old male patient was admitted with a threeweek history of colic-like pain in the upper abdomen and jaundice. He had a metabolic syndrome (body mass index 45 kg/m 2 ) including a fatty liver disease with beginning fibrosis, and a history of smoking (25 pack years). Abdominal computed tomography (CT) revealed an approximately 3 × 2 cm polypoid mass lesion apparently located in the common bile duct and along the bifurcation into the cystic duct with consecutive dilation of the central intra-and extrahepatic bile ducts ( Figure 1A, B), gallbladder hydrops and cholecystolithiasis. Laboratory tests detected an increase in total bilirubin (1.4 mg/dL; normal ≤1.2 mg/dL), aspartate amino transferase (76 U/L; normal ≤35 U/L), alanine amino transferase (79 U/L; normal ≤45 U/L), and γ-glutamyl transferase (223 U/L; normal ≤55 U/L). Levels of alphafetoprotein (AFP) and carbohydrate antigen 19-9 (CA19-9) were within normal range (AFP 3 μg/L; normal <7 μg/L, and CA19-9 31 kU/L; normal <37 kU/L). As abdominal ultrasound showed a dilated common hepatic duct of up to 2 cm, endoscopic retrograde cholangiopancreatography (ERCP) was performed ( Figure 1C). It revealed a mass in the common hepatic duct and hematobilia. Via passage with a blocked balloon, material was obtained for histoxpathology. A stent was inserted into the bile duct, producing immediate bile drainage. After that intervention, the jaundice steadily declined and the cholestatic parameters normalized. The initial pathological diagnosis of the obtained tissue was a tubulo-villous adenoma of the bile duct.
After a four-week interval for weight reduction, ERCP was re-performed and the stent was removed. Fluoroscopic guidance with contrast application revealed the tumour in the middle part of the common bile duct. After obtaining biopsies from the tumour, a stent was inserted again for drainage until the operation. Endosonography located the tumour in the common bile duct ( Figure 1D) and the cystic duct, protruding into the infundibulum of the gallbladder. A suspect lymph node was detected between bile duct and cystic duct.
Histopathological examination revealed tubulo-papillary neoplastic proliferations and closely packed glandular structures with eosinophilic cytoplasm, round to oval nuclei and inconspicuous nucleoli. At the surface, small papillary proliferations were observed. Focally, marked architectural distortion with nuclear atypia, hyperchromatic nuclei with prominent nucleoli, and a back-to-back formation of stellar glands were present. Squamous morules were not  observed. The diagnosis of a pyloric gland adenoma with possible transition into well-differentiated adenocarcinoma was established and confirmed by reference pathology.
In the meantime the patient developed a thrombosis of the right cephalic vein after catheter infection and a non-ST-elevation myocardial infarction, which were treated non-interventionally with antibiotics and heparin for prolongation of the prothrombin time. Two weeks later, resection of the extrahepatic bile ducts including gallbladder with biliodigestive anastomosis was performed.
The resected specimen of the common bile duct, cystic duct with attached gallbladder, and common hepatic duct presented a 2.5 × 2 cm tumour with a gray-brown cut surface, developing in the cystic duct, and protruding through the bifurcation into both the common bile duct and common hepatic duct, with partial obstruction of the lumen ( Figure 2). The surrounding cystic duct showed epithelial cell proliferations in the mucosa with moderate and focal high-grade cellular atypia, outstretching into small branching bile duct ( The pyloric gland adenoma showed focal positive staining for MUC1, and negative staining for MUC2, as well as superficial staining for MUC5AC, and positive expression of MUC6 and VEGF (Figure 4). CD10 and CDX2 were not expressed. Nuclear expression of p53, focal p16, and p21 was observed. Proliferative activity was assessed by Ki67 and estimated at 25%. The high-grade intraepithelial neoplasia of the bile duct, in contrast, did not express MUC1, MUC2, and MUC5AC, but MUC6 and VEGF. Staining with CD10 and CDX2, p53, and p16 was negative, whereas p21 was only focally expressed. Proliferative activity was assessed by Ki67, and estimated at 10%. The diagnosis of a pyloric gland adenoma with focal high-grade intraepithelial neoplasia and transition into well-differentiated gastric-type adenocarcinoma associated with BilIN-3 of the cystic duct resembling gastric-type intraductal papillary neoplasm in areas with low-grade intraepithelial neoplasia was Figure 3 Microscopic findings of the pyloric gland adenoma and adjacent bile duct lesions. On microscopic view, the pyloric gland adenoma arose in the cystic duct (right) and displayed a papillary, intraluminal growth pattern with protrusion into the common bile duct (left) (A, H&E, x40). Closely packed pyloric type glands were lined by cuboidal to columnar mucus-secreting cells (B, x100) with focal architectural distortion (C, x100) and high-grade dysplasia with nuclear atypia, indicating transition into well-differentiated adenocarcinoma (D, x200). Focal high-grade intraepithelial neoplasia (BilIN-3) of the cystic duct was detected (E, x200), focally resembling gastric-type intraductal papillary neoplasm (IPN) with direct transition into the pyloric gland adenoma (F, x200). thus confirmed histopathologically, and the tumour was finally staged at pT1, pNX, pMX, G1, L0, V0, R0. Metastases had been excluded by CT scan.
CGH analysis was performed from the pyloric gland adenoma as described previously [17] and revealed chromosomal gains at 7p, 7q11q21, 15q, 16p, 20, losses at 6p23pter, 6q, 18, and amplifications at 1q and 6p21p22 in the pyloric gland adenoma ( Figure 5). Sequencing analysis of KRAS exon 1 and 2 showed a point mutation at exon 1, codon 12 (c.35G>T; p.G12V) and wildtype sequence at exon 2 in the pyloric gland adenoma and the same mutation in the adjacent BilIN-3. The patient recovered well and was discharged 12 days after surgery. He presently shows no signs of tumour relapse 12 months after tumour resection.
CD10 are generally negative, but may indicate transition from gastric to intestinal differentiation [2,3,8,12]. Additionally, Ki67 expression and p53 mutations can be used to detect malignant transformation [3]. The presence of adenocarcinoma may be difficult to recognize since the cytology of adenocarcinoma developing in pyloric gland adenoma is known to show rather mild abnormalities without the classical signs of intraepithelial neoplasia/dysplasia [3]. However, transition from round or oval nuclei to elongated or pleomorphic nuclei with loss of polarity in conjunction with so-called lateral expansion or branching of glands indicates the presence of adenocarcinoma, as found focally also in the present case ( Figure 3) [3]. In another study describing 3 pyloric gland adenocarcinomas of the extrahepatic bile ducts the authors observed pyloric gland metaplasia adjacent to the adenocarcinoma in 2 of 3 cases and suggested them as probable precursor lesion [16]. These tumours did not arise from pyloric gland adenomas, but displayed a similar immunophenotype with positive expression of MUC5AC and MUC6 and negativity for MUC2 and CDX2 [16]. Squamous morules (or spindle cell metaplasias) are reported to occur in 23% of cases [9], but were not observed in the present case.
Histopathological differential diagnoses of polypous intraductal bile duct lesions include adenomas of the gallbladder and extrahepatic bile ducts. They can be divided into a tubular, papillary, and tubulopapillary type based on their growth pattern, and cytologically into a pyloric-gland, intestinal, foveolar, and biliary type [1]. IPN of gastric, pancreatobiliary, intestinal or oncocytic phenotype, and mucinous cystic neoplasms should also be considered [1]. Furthermore, concomitant intraductal papillary mucinous neoplasms (IPMN) [5,13] have been observed in cases of pancreatic pyloric gland adenomas. In the pancreas, gastric-type IPMN are usually located as small cystic lesions in branching ducts, harboring only mild/low-grade atypia and immunohistochemically expressing MUC5AC, but not MUC1, MUC2 or CDX2. They are associated with a rather favorable clinical prognosis compared to the other subtypes of IPMN [19]; adenocarcinoma occurs in 10-15% [19]. Invasive adenocarcinoma may also develop in IPN of the bile duct [1]. BilIN of the gallbladder and extrahepatic bile ducts are associated with lithiasis in up to 3%, familial adenomatous polyposis coli, sclerosing cholangitis, and pancreatobiliary reflux [1]. Additionally, BilIN-3 usually arises in a association with pyloric and intestinal metaplasia, as observed in the present case, with an abrupt transition between normal and atypical columnar cells [1]. As reported, BilIN immunohistochemically also expresses p53 [1].
KRAS codon 12 mutations have been previously reported in two cases of pyloric gland adenoma of the main pancreatic duct, and probably support the neoplastic nature of this tumour [4,5]. KRAS (13-100%) and TP53 (50%) mutations have been described for IPMN of the pancreas before [19]. Also in the present case, the pyloric gland adenoma and the BilIN-3 harbored a KRAS codon 12 mutation, indicating a possible metaplasia-dysplasia-carcinoma sequence with a common tumourigenesis.

Conclusions
In conclusion, a pyloric gland adenoma evolving in the cystic duct is very rare, but may sometimes be overlooked and therefore should be considered as a differential diagnosis for obstructive bile duct tumours. An association with other premalignant bile duct lesions such as BilIN may be observed. ERCP-guided biopsy with histopathological examination is necessary to establish the diagnosis. Due to the high risk of evolving adenocarcinoma, surgical resection should be performed whenever possible.