Intraductal papillary neoplasms of the bile duct (IPNB) shows favorable prognosis and is regarded as a biliary counterpart of intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Although activating point mutations of GNAS at codon 201 have been detected in approximately two thirds of IPMNs of the pancreas, there have been few studies on GNAS mutations in IPNBs. This study investigates the status of GNAS and KRAS mutations and their association with clinicopathological factors in IPNBs. We examined the status of GNAS mutation at codon 201 and KRAS mutation at codon 12&13, degree of mucin production and immunohistochemical expressions of MUC mucin core proteins in 29 patients (M/F = 15/14) with IPNB in intrahepatic and perihilar bile ducts (perihilar IPNB) and 6 patients (M/F = 5/1) with IPNB in distal bile ducts (distal IPNB). GNAS mutations and KRAS mutations were detected in 50% and 46.2% of IPNBs, respectively. There was no significant correlation between the status of GNAS mutation and clinicopathological factors in IPNBs, whereas, the status of KRAS mutation was significantly inversely correlated with the degree of MUC2 expression in IPNBs (p<0.05). All IPNBs with GNAS mutation only showed high-mucin production. Degree of mucin production was significantly higher in perihilar IPNBs than distal IPNBs (p<0.05). MUC2 and MUC5AC expression was significantly higher in IPNBs with high-mucin production than those with low-mucin production (p<0.01 and p<0.05, respectively). In conclusions, this study firstly disclosed frequent GNAS mutations in IPNBs, similarly to IPMNs. This may suggest a common histopathogenesis of IPNBs and IPMNs. The status of KRAS mutations was inversely correlated to MUC2 expression and this may suggest heterogeneous properties of IPNBs. IPNBs with high-mucin production are characterized by perihilar location and high expression of MUC2 and MUC5AC, irrespective of the status of GNAS and KRAS mutations.
Citation: Sasaki M, Matsubara T, Nitta T, Sato Y, Nakanuma Y (2013) GNAS and KRAS Mutations are Common in Intraductal Papillary Neoplasms of the Bile Duct. PLoS ONE 8(12): e81706. https://doi.org/10.1371/journal.pone.0081706
Editor: Andreas-Claudius Hoffmann, West German Cancer Center, Germany
Received: August 21, 2013; Accepted: October 25, 2013; Published: December 2, 2013
Copyright: © 2013 Sasaki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported in part by a Grant-in Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports and Science and Technology of Japan (22390067). There is nothing to disclose regarding this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Intraductal papillary neoplasm of the bile duct (IPNB) is characterized by dilated intrahepatic bile ducts filled with noninvasive papillary or villous biliary neoplasm covering delicate fibrovascular stalks . IPNB includes the previous categories of biliary papilloma and papillomatosis, and the term IPNB has been adopted in WHO classification 2010 . IPNB is regarded as a biliary counterpart of intraductal papillary mucinous neoplasm of the pancreas (IPMN) and shows favorable prognosis –. In addition, IPNBs are not infrequently associated with invasive carcinoma (IPNB with an associated with invasive carcinoma) . Some IPNBs show excessive mucin secretion and are described using the terms “mucin-producing bile duct tumor”, “mucin-hypersecreting bile duct tumor”, or “IPNB with macroscopically mucin-producing biliary tumor”–. This subset of IPNB is associated with hepatolithiasis  and has a tendency to show intestinal differentiation , , .
Guanine nucleotide-binding protein, α-stimulating activity polypeptide (GNAS) encodes the α-subunit of the stimulatory G-protein (Gαs), which mediates the regulation of adenylate cyclase activity through G-protein-coupled receptors. Activating mutations of GNAS at codon 201 have been detected in approximately two thirds of IPMNs of the pancreas , . In addition, frequent GNAS mutations were reported in pituitary adenomas, colorectal villous adenomas and pyloric gland adenomas , . In contrast to frequent mutation of GNAS in IPMNs, there have been few GNAS mutations in usual invasive ductal carcinoma of the pancreas (PDAC) , . There have been only a few studies on GNAS mutation in cholangiocarcinomas and IPNBs, to our knowledge –. GNAS mutations were reportedly uncommon in IPNBs in a previous study . GNAS mutation was not detected in cholangiocarcinomas and biliary intraepithelial neoplasia (BilIN) in our previous study . v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation is an early event within PanINs and occurs in up to 90% of early PanINs and in 95% pancreatic adenocarcinomas (PDACs), . KRAS mutation was detected in about a third of cholangiocarcinomas and BilINs .
Given frequent GNAS mutations in IPMNs, which are similar to IPNBs, we examined the status of GNAS and KRAS mutations. Furthermore, we analyzed their association with clinicopathological factors including the degree of mucin production and the expression of MUC mucin core proteins (MUC1, MUC2, MUC5AC, MUC5B and MUC6).
Materials and Methods
Classification of the biliary tree and IPNBs
The biliary tree is divided into intrahepatic, perihilar (the right, left and common hepatic ducts) and distal bile duct (extrahepatic bile ducts distal to the insertion of the cystic duct) . The intrahepatic bile duct is classified as described previously . IPNB were classified into perihilar and distal IPNB based on the location of tumor according to the TNM classification of malignant tumors .
Preparation of human IPNB tissue specimens
We examined 29 patients with perihilar IPNB (12 non-invasive, 3 microinvasive and 14 invasive, M/F = 15/14) and 6 distal IPNB (all invasive M/F = 5/1). We defined the IPNB with microinvasion as IPNB with small foci of invasion in which the deepest invasion is limited to the mucosa in this study. The clinical and pathological features were summarized in Table. 1. Mucinous cystic neoplasms with ovarian-like stroma were excluded. Participants provided their written informed consent to participate in this study. The Ethics Committee of Kanazawa University approved this study and consent procedure. When the written consent was not obtained because of old samples, such samples were handled anonymously. The respective Ethics Committee also approved this. All of these specimens were obtained from the liver disease file of our department and affiliated hospitals. These specimens were fixed in 10% buffered formalin and embedded in paraffin, and more than 20 serial 3 µm thin sections were cut from each paraffin block. Some sections were stained for hematoxylin and eosin (HE) stain and the remaining were used for the following DNA extraction, mucin stain and immunostaining.
Extraction of DNA samples and GNAS and KRAS mutation analysis
IPNBs and background livers were visualized and scraped off from 2 to 3 serial whole sections (3 µm) and DNA was isolated using the QIAMP DNA kit (QIAGEN). Approximately 10,000 cells were harvested from each lesion with and estimated tumour cellularity of >80%. Isolated DNA was then subjected to PCR amplification of the region of the GNAS gene coding codon 201 and KRAS gene containing codons 12 and 13. The forward and reverse primers for the GNAS codon 201 were 5′-ACTGTTTCGGTTGGCTTTGGTGA-3′ and 5′-AGGGACTGGGGTGAATGTC -AAGA-3′ and the primers for KRAS gene containing codons 12 and 13 were 5′-AGGCCTGCTGAAAATGACTG-3′ and 5′-ATCAAAGAATGGTCCTGCAC -3′, respectively. Amplifications were done by initial denaturation at 94°C for 3 min followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min, followed by 10 min final extension at 72°C using TaqDNA polymerase (Takara EX Taq; Takara Bio). These PCR products were then purified using the QIAGEN PCR purification kit (QIAGEN) and sequenced by the Big Dye cyclic sequencing kit and ABI 310 sequencer (Applied Biosystems, Forster City, CA).
Mucin stain and semiquantitative evaluation
Each section was processed for double mucin stain with periodic acid Schiff stain after diastase-digestion and alcian blue (pH 2.5) (d-PAS/AB). Amount of mucin production was evaluated sumiquantitatively (score 0–3), being based on d-PAS/alcian blue positive mucin on the cell surface of tumor cells. Figure 1 demonstrates degrees of surface and intracellular mucin productions and showed examples.
Immunostaining and semiquantitative evaluation
The expression of MUC1, MUC2, MUC5AC, MUC5B and MUC6 mucin was immunohistochemically assessed using a standard method as described previously . Primary antibodies used in this study were shown in Table 2. A similar dilution of the control mouse IgG (Dako) was applied as negative control. The expression of MUC mucin core proteins (MUC1, MUC2, MUC5AC, MUC5B and MUC6) was evaluated according to the percentage of positive cells in each lesion: Score 0, less than 1%; score 1, 1–30%; score 2, 30–70%; score 3, more than 70%.
The Wilcoxon rank sum test and Kruscal-Wallis test with Dunn posttest was used in statistical analysis for the difference between 2 groups and among 3 or more groups, respectively. A p value <0.05 was considered significant. Correlation between 2 groups was assessed using Spearman's correlation test. A p value <0.05 was considered significant.
Sequencing analysis was successfully performed in 30 gene samples extracted from the cases of IPNB. GNAS mutation was detected in 15 cases (50%) of 30 IPNBs, including 12 perihilar IPNBs (50%) and 3 distal IPNBs (50%)(Table 3). All GNAS mutation was at codon 201; cDNA 602G>A and cDNA 601C>A mutants were detected in 14 and 2 cases, respectively (Table 3, Figure 2A). One case harbored both cDNA 602G>A and cDNA 601C>A mutations. There was no significant difference between GNAS mutation and the degree of mucin secretion and other clinicopathological factors (Table 4).
A) Representative sequencing trace of an IPNB without GNAS mutation (wild type, WT) and IPNBs with GNAS mutations. An arrow indicates miss-sense mutation. B) Representative sequencing trace of an IPNB without KRAS mutation (wild type, WT) and IPNBs with KRAS mutations. An arrow indicates miss-sense mutation.
Sequencing analysis was successfully performed in 26 gene samples extracted from the cases of IPNB. Sequencing analysis for KRAS was failed in 4 gene samples for unknown reason. KRAS mutation was detected in 12 cases (46.2%) including 10 perihilar IPNB (45.5%) and 2 distal IPNBs (50%)(Table 3). Ten cases with KRAS mutation showed GGT to GAT at codon 12 and 2 cases harbored both GGT to GAT and GGT to GCT at codon 12 (Figure 2B). KRAS mutation was significantly inversely correlated with MUC2 expression (Table 4). There was no significant correlation between GNAS and KRAS mutations.
Mucin production in IPNBs
All IPNBs showed mucin production to various degrees. Twenty and 15 patients were divided into high- and low- mucin production, respectively. Representative histology of IPNBs with high- and low-mucin production was shown in Figure 3A and 3B. The degree of mucin production was significantly higher in perihilar IPNBs than distal IPNBs (p<0.05). (Figure 3C).
A) An example of IPNB with high- mucin production. IPNB with high- mucin production is composed of tall columnar tumor cells showing abundant mucin production in double mucin stain with periodic acid Schiff stain after diastase-digestion and alcian blue (pH2.5) (d-PAS/AB) (scores; surface 3, cytoplasmic 3). The tumor cells show extensive immunoreactivity for MUC2 and MUC5AC. B) An example of IPNBs with low-mucin production. IPNB with low-mucin production is composed of cuboidal tumor cells showing less mucin production (scores; surface 0, cytoplasmic 1). The tumor cells show no immunoreactivity for MUC2 and focal immunoreactivity for MUC5AC. Hematoxylin and eosin, d-PAS/AB and the immunostaining for MUC2 and MUC5AC and hematoxylin. x200. C) Semiquantitative evaluation of the degree of mucin production in perihilar IPNBs with and without invasion and distal IPNBs (all with invasion). White column, score 1; half-tone column, score 2; black column, score 3. *, p<0.05. Non-I, without invasion; inv, with invasion.