Biliary Intraepithelial Neoplasia Research Articles

Clinical implications of pediatric biliary intraepithelial neoplasia diagnosed from a choledochal cyst specimen

BackgroundBiliary intraepithelial neoplasia (BilIN), a noninvasive precursor of cholangiocarcinoma, can manifest malignant transformation. Since cholangiocarcinoma (CCA) may progress due to chronic inflammation in the bile ducts and gallbladder, choledochal cysts are considered a precursor to CCA. However, BilIN has rarely been reported in children, to date.MethodsWe reviewed medical records of patients (< 18 years of age, n = 329) who underwent choledochal cyst excision at Asan Medical Center from 2008 to 2022. BilIN was diagnosed in 15 patients. Subsequent analyses were performed of the demographics, surgical procedures, clinical course, and outcomes in these patients. Subgroup analysis and multivariate logistic regression test were performed to identify factors influencing BilIN occurrence.ResultsThe mean age of the patients included in our study was 40.1 ± 47.6 months. In 15 patients, BilIN of various grades was diagnosed. Todani type I was prevalent in 80% of the patients. The median age at surgery was 17 months. During a mean follow-up of 63.3 ± 94.0 months, no adverse events such as stone formation in the remnant intrapancreatic common bile duct and intrahepatic duct or cholangiocarcinoma were observed, indicating a favorable outcome until now.ConclusionsThe potential progression of choledochal cysts to BilIN in children was demonstrated. These results could underscore the importance of early and comprehensive excision of choledochal cysts, including resection margins for associated lesions and more thorough postoperative surveillance in patients with or at risk of BilIN.

Early detection of occupational cholangiocarcinoma in a high-risk patient under intensive surveillance: a case study

BackgroundOccupational cholangiocarcinoma is associated with exposure to organic solvents, such as dichloromethane (DCM) and 1,2-dichloropropane (DCP). This report describes a case of occupational cholangiocarcinoma detected through regularly imaging following the discovery of elevated serum γ-glutamyl trans peptidase (γ-GTP) levels revealed during regular checkup.Case presentationA 43-year-old man who had been working in a printing company with 15 years of exposure to organic solvents presented to our hospital owing to abnormalities found during a routine checkup. Ultrasound (US) imaging revealed thickening of the gallbladder wall accompanied by gallstones, although in the blood tests, γ-GTP levels were within normal range. Given the high risk of cholangiocarcinoma development, the patient underwent regular monitoring with abdominal US and blood tests at a local doctor's office. At the age of 48, his serum γ-GTP level mildly elevated for the first time, prompting the initiation of semi-annual magnetic resonance cholangiopancreatography (MRCP). By the age of 50 years, dilation in B8 was detected, and one and a half years later, a tumor on the central side of the B8 dilation appeared. The patient was diagnosed with intrahepatic cholangiocarcinoma, which was treated with anterior sectionectomy. Pathological examination revealed an adenocarcinoma with a papillary glandular ductal structure at the root of the B8. In addition, biliary intraepithelial neoplasia (BilIN) and dysplasia have been identified around the tumor and periphery bile ducts and in noncancerous bile ducts. Postoperatively, the patient received 6 months of adjuvant chemotherapy with S-1monotherapy. Eight months after surgery, the patient remained under observation with no signs of recurrence.ConclusionsWe report a case of occupational cholangiocarcinoma detected during a prolonged period of regular follow-up after exposure to DCM and DCP. Given the delayed carcinogenesis process, occupational cholangiocarcinomas manifest long after exposure to organic solvents, therefore, ongoing screening is extremely important. Vigilance is essential to avoid underdiagnosis, particularly for individuals who are at an increased risk of developing this form of cancer. Continuous monitoring is key to the early detection and effective management of occupational cholangiocarcinoma.

Plastoquinone-Derivative SkQ1 Improved the Biliary Intraepithelial Neoplasia during Liver Fluke Infection.

Carcinogenic food-borne liver fluke infections are a serious epidemiological threat worldwide. The major complications of Opisthorchis felineus infection are chronic inflammation and biliary intraepithelial neoplasia. Although evidence has accumulated that increased reactive oxygen species production is observed in liver fluke infection, a direct relationship between the oxidative stress and biliary intraepithelial neoplasia has not been shown. Quinones and SkQ1, a derivative of plastoquinone, have been demonstrated to be cytoprotective in numerous liver injuries due to their potent antioxidant properties. This study is aimed to assess the level of biliary intraepithelial neoplasia in O. felineus-infected hamsters after treatment with mitochondria-targeted SkQ1. SkQ1 significantly reduced the biliary intraepithelial neoplasia, which was accompanied by a decrease in lipid and DNA oxidation byproducts, mRNA expression and level of proteins associated with inflammation (TNF-α, CD68) and fibrogenesis (CK7, αSMA), and was also associated with an activation of the Keap1-Nrf2 pathway. Thus, a direct relationship was found between oxidative stress and the severity of biliary intraepithelial neoplasia in O. felineus-infected hamsters. The hepatoprotective effect of plastoquinone-derivative SkQ1 was established; therefore, this compound is a promising agent in complex therapy in the treatment of opisthorchiasis.

Incidental precancerous and cancerous neoplasms in routine cholecystectomy specimens – A fascinating series of cases in a tertiary care center

Laparoscopic cholecystectomy is one of the most commonly performed surgeries for benign gall bladder pathologies. Pre-cancerous and malignant gall bladder neoplasms often have overlapping non-specific clinical manifestation in their early stages. Radiological findings are also non-specific in the early stages with the presence of cholelithiasis in a significant proportion of cases. Cholelithiasis is said to be one of the important comorbid risk factors for gall bladder carcinoma (GBC) as a source of constant irritation to biliary mucosa. In such cases, cholecystectomy done for benign indications may lead to a histopathological diagnosis of incidental gall bladder carcinomas. Pancreaticobiliary type of adenocarcinoma is the most common subtype of GBC with two described cases in this series. Invasive foci need to be ruled out in. All cases of high-grade biliary intraepithelial neoplasia as were seen in the third case. Intracholecystic papillary neoplasm is a recently described entity with one reported case in this series that was associated with invasive adenocarcinoma. Signet ring adenocarcinoma is a rarely encountered adenocarcinoma in gall bladder with worse outcome. One such case has been described here where the patient has succumbed to death within 3 months of surgery and during the course of adjuvant therapy.

Intraductal papillary neoplasm of the bile duct: The new frontier of biliary pathology.

Intraductal papillary neoplasms of the bile duct (IPNBs) represent a rare variant of biliary tumors characterized by a papillary growth within the bile duct lumen. Since their first description in 2001, several classifications have been proposed, mainly based on histopathological, radiological and clinical features, although no specific guidelines addressing their management have been developed. Bile duct neoplasms generally develop through a multistep process, involving different precursor pathways, ranging from the initial lesion, detectable only microscopically, i.e. biliary intraepithelial neoplasia, to the distinctive grades of IPNB until the final stage represented by invasive cholangiocarcinoma. Complex and advanced investigations, mainly relying on magnetic resonance imaging (MRI) and cholangioscopy, are required to reach a correct diagnosis and to define an adequate bile duct mapping, which supports proper treatment. The recently introduced subclassifications of types 1 and 2 highlight the histopathological and clinical aspects of IPNB, as well as their natural evolution with a particular focus on prognosis and survival. Aggressive surgical resection, including hepatectomy, pancreaticoduodenectomy or both, represents the treatment of choice, yielding optimal results in terms of survival, although several endoscopic approaches have been described. IPNBs are newly recognized preinvasive neoplasms of the bile duct with high malignant potential. The novel subclassification of types 1 and 2 defines the histological and clinical aspects, prognosis and survival. Diagnosis is mainly based on MRI and cholangioscopy. Surgical resection represents the mainstay of treatment, although endoscopic resection is currently applied to nonsurgically fit patients. New frontiers in genetic research have identified the processes underlying the carcinogenesis of IPNB, to identify targeted therapies.

Intrahepatic cholangiocarcinoma with extensive intraductal extension of high-grade biliary intraepithelial neoplasia: a case report

BackgroundIntrahepatic cholangiocarcinoma (ICC) is frequently associated with precursor lesions, and biliary intraepithelial neoplasia (BilIN) may play a significant role in the development of ICC. However, the exact sequence and progression of these lesions remain to be elucidated. We report a rare case of ICC that exhibited extensive longitudinal intraductal extension of high-grade BilIN in the posterior bile ducts and involved the hepatic hilum and the peripheral hepatic parenchyma.Case presentationA 70-year-old female presented with anorexia. Computed tomography (CT) revealed a 15 mm enhancing intrahepatic tumor extending to the right intrahepatic secondary confluence. This was associated with a 7 mm diameter cystic dilatation of the segment 6 bile duct (B6). Endoscopic retrograde cholangiopancreatography (ERCP) revealed stenosis at the bifurcation of the posterior bile duct branch. Bile cytology confirmed the diagnosis of adenocarcinoma cells. Therefore, the patient was diagnosed with an ICC involving the right glissonean pedicle and underwent a right hepatectomy and lymph node dissection. Histologic examination revealed the tumor consisted of moderately differentiated adenocarcinoma. In connection with this lesion, diffuse intraductal atypical epithelial cells, which were diagnosed as high-grade BilIN, was observed not only in the dilated B6 but in the entire posterior bile ducts, which measured approximately 120 mm in diameter. Furthermore, two distinct foci of adenocarcinomas were identified in the peripheral hepatic parenchyma. A lymph node metastasis was also present. The pathological diagnosis was ICC pT4N1M0 stage IVA. The patient underwent adjuvant chemotherapy and has shown no recurrence 5 years after surgery. Imaging modalities were unable to accurately assess the extent of the intraductal neoplastic lesions due to their low papillary or sessile intraductal tubular growth. No risk factors for BilIN development, which has the potential to predispose to cholangiocarcinoma, were identified in the present case.ConclusionsWe present a case of ICC involving the right hepatic hilum, accompanied by extensive longitudinal extensions of high-grade BilIN and multifocal microscopic invasions in peripheral hepatic parenchyma. Notably, the intraductal lesions involved the entire posterior intrahepatic bile ducts. The presence of biliary neoplasia with extensive intraductal extension, in conjunction with ICC, should be considered as a variant of BilIN.

INTRAHEPATIC BILIARY PROLIFERATIONS: HISTOPATHOLOGY AND POTENTIAL IMMUNOHISTOCHEMICAL MARKERS.

•Intrahepatic biliary proliferations represent a spectrum varying from reactive to malignant entities. •Clinical and imaging patterns may be similar, requiring histopathological and immunohistochemistry for precise diagnosis. Intrahepatic biliary proliferations represent a spectrum from reactive (ductular reaction, some with atypical architecture), hamartomatous (von Meyenburg complex), benign (bile duct adenoma) and precursor/borderline entities (biliary intraepithelial neoplasia, intraductal papillary neoplasm of the bile duct) to fully malignant (cholangiocarcinoma) neoplasms. Clinical pictures and even imaging patterns may be similar, requiring refined studies aiming at histopathological and immunohistochemistry for more precise diagnosis, essential for correct patient management. This article discusses updated concepts and definitions of most relevant entities aiming more specifically at the differential diagnosis in practice, focusing on morphology and immunohistochemistry, with a discussion of potential markers to help distinguishing between benign and malignant lesions.

Significance of morphological studies in the diagnostics of bilial intraepithelial neoplasia in nonparasitic liver cysts

Biliary intraepithelial neoplasia (BilIN) according to modern data is considered as an obligate precancer of the bile ducts. As BILIN progresses, cholangiocarcinoma often develops, characterized by a low five-year survival rate. The aim of this work is to describe the morphological characteristics of BilIN in the epithelium of nonparasitic liver cysts. The material is described after primary and repeated (15) surgical interventions in 99 patients. The morphological manifestations of intraepithelial neoplasia in non-parasitic dysontogenetic liver cysts were studied, their role in the development of liver cholangiocarcinoma was established.

Intraductal neoplasms of the pancreatobiliary tract: navigating the alphabet.

Cancers of the pancreatobiliary tract are diseases with unfavourable prognoses. In the last couple of decades, two types of lesions have been described as precursors that precede pancreatobiliary cancers. These include incidental microscopic (flat) lesions known as pancreatic intra-epithelial neoplasia and biliary intra-epithelial neoplasia, and grossly visible, mass-forming lesions (tumoral intra-epithelial neoplasia) including intraductal papillary mucinous neoplasms, intraductal oncocytic papillary neoplasms, intraductal tubulopapillary neoplasms, intraductal papillary neoplasms of the bile duct and intracholecystic papillary neoplasms. Early detection and adequate treatment of these precursor lesions, especially the second group, have the potential to prevent pancreatobiliary cancer or at least improve its prognosis. In this review, we discuss their histopathology and recent updates on molecular profiling of these intraductal neoplasms of the pancreatobiliary tract.

The existence of biliary intraepithelial neoplasia at resection margin is not a risk factor for recurrence of biliary tract cancer

Abstract Background Invasive carcinoma or dysplasia (biliary intraepithelial neoplasia [BilIN]) is often found in the surgical stumps of biliary tract cancer. We investigated whether the presence of BilIN at the surgical margin is a risk factor for recurrence. Materials and Methods Seventy-five consecutive patients who underwent curative resection for biliary tract cancer were included in this study. We retrospectively evaluated the surgical margin status (negative, BilIN, or positive) and analyzed the postoperative disease recurrence rate and recurrence pattern by the status. Results The disease-free survival rate was significantly worse in the positive margin group than in the negative margin group (11 vs 18 months, P = 0.027). There were no statistical differences between the BilIN-positive group and the other two groups. In the multivariate analysis, infiltrative growth c (hazard ratio [HR], 3.348; 95% confidence interval [CI], 1.368–8.195; P = 0.008) and pathological exhumed margin (HR, 2.670; 95% CI, 1.097–6.495; P = 0.030) were independent recurrence factors. The margin status of BilIN was not associated with recurrence (HR, 0.867; 95% CI, 0.491–2.946; P = 0.687). Conclusions The BilIN component at the surgical margin was not associated with short-term recurrence, but the pathological exhumed margin positive and infiltrative growth c were. Infiltrative growth c may be a potential predictive factor for recurrence.

Simultaneous activation of Kras-Akt and Notch pathways induces extrahepatic biliary cancer via the mTORC1 pathway.

Biliary tract cancer (BTC) has poor prognosis. The Notch receptor is aberrantly expressed in extrahepatic cholangiocarcinoma (eCCA). However, the role of Notch signaling in the initiation and progression of eCCA and gallbladder (GB) cancer remains unknown. Therefore, we investigated the functional role of Notch signaling during tumorigenesis of the extrahepatic bile duct (EHBD) and GB. Activation of Notch signaling and oncogenic Kras resulted in the development of biliary intraepithelial neoplasia (BilINs) in the EHBD and GB, which were premalignant lesions that progressed to adenocarcinoma in mice. The expression of genes involved in the mTORC1 pathway was increased in biliary spheroids from Hnf1b-CreERT2; KrasLSL-G12D ; Rosa26LSL-NotchIC mice and inhibition of the mTORC1 pathway suppressed spheroid growth. Additionally, simultaneous activation of the PI3K-AKT and Notch pathways in EHBD and GB induced biliary cancer development in mice. Consistent with this, we observed a significant correlation between activated NOTCH1 and phosphorylated Ribosomal Protein S6 (p-S6) expression in human eCCA. Furthermore, inhibition of the mTORC1 pathway suppressed the growth of Notch-activated human biliary cancer cells in vitro and in vivo. Mechanistically, the Kras/Notch-Myc axis activated mTORC1 through TSC2 phosphorylation in mutant biliary spheroids. These data indicate that inhibition of the mTORC1 pathway could be an effective treatment strategy for Notch-activated human eCCA. © 2023 The Pathological Society of Great Britain and Ireland.

Understanding the Clinical Significance of MUC5AC in Biliary Tract Cancers.

Biliary tract cancers (BTC) arise from biliary epithelium and include cholangiocarcinomas or CCA (including intrahepatic (ICC) and extrahepatic (ECC)) and gallbladder cancers (GBC). They often have poor outcomes owing to limited treatment options, advanced presentations, frequent recurrence, and poor response to available systemic therapy. Mucin 5AC (MUC5AC) is rarely expressed in normal biliary epithelium, but can be upregulated in tissues of benign biliary disease, premalignant conditions (e.g., biliary intraepithelial neoplasia), and BTCs. This mucin's numerous glycoforms can be divided into less-glycosylated immature and heavily-glycosylated mature forms. Reported MUC5AC tissue expression in BTC varies widely, with some associations based on cancer location (e.g., perihilar vs. peripheral ICC). Study methods were variable regarding cancer subtypes, expression positivity thresholds, and MUC5AC glycoforms. MUC5AC can be detected in serum of BTC patients at high concentrations. The hesitancy in developing MUC5AC into a clinically useful biomarker in BTC management is due to variable evidence on the diagnostic and prognostic value. Concrete conclusions on tissue MUC5AC are difficult, but serum detection might be relevant for diagnosis and is associated with poor prognosis. Future studies are needed to further the understanding of the potential clinical value of MUC5AC in BTC, especially regarding predictive and therapeutic value.

Biliary Intraepithelial Neoplasia (BilIN) Diagnosed From Choledochal Cyst in 7-Year-Old Girl With Underlying Anomalous Pancreaticobiliary Ductal Union (APBDU)

Biliary Intraepithelial Neoplasia (BilIN) Diagnosed From Choledochal Cyst in 7-Year-Old Girl With Underlying Anomalous Pancreaticobiliary Ductal Union (APBDU)

Gallbladder adenocarcinomas undergo subclonal diversification and selection from precancerous lesions to metastatic tumors.

We aimed to elucidate the evolutionary trajectories of gallbladder adenocarcinoma (GBAC) using multi-regional and longitudinal tumor samples. Using whole-exome sequencing data, we constructed phylogenetic trees in each patient and analyzed mutational signatures. A total of 11 patients including 2 rapid autopsy cases were enrolled. The most frequently altered gene in primary tumors was ERBB2 and TP53 (54.5%), followed by FBXW7 (27.3%). Most mutations in frequently altered genes in primary tumors were detectable in concurrent precancerous lesions (biliary intraepithelial neoplasia, BilIN), but a substantial proportion was subclonal. Subclonal diversity was common in BilIN (n=4). However, among subclones in BilIN, a certain subclone commonly shrank in concurrent primary tumors. In addition, selected subclones underwent linear and branching evolution, maintaining subclonal diversity. Combined analysis with metastatic tumors (n=11) identified branching evolution in 9 patients (81.8%). Of these, 8 patients (88.9%) had a total of 11 subclones expanded at least 7-fold during metastasis. These subclones harbored putative metastasis-driving mutations in cancer-related genes such as SMAD4, ROBO1, and DICER1. In mutational signature analysis, 6 mutational signatures were identified: 1, 3, 7, 13, 22, and 24 (cosine similarity >0.9). Signatures 1 (age) and 13 (APOBEC) decreased during metastasis while signatures 22 (aristolochic acid) and 24 (aflatoxin) were relatively highlighted. Subclonal diversity arose early in precancerous lesions and clonal selection was a common event during malignant transformation in GBAC. However, selected cancer clones continued to evolve and thus maintained subclonal diversity in metastatic tumors.

A Case of Intracholecystic Papillary Neoplasm of the Gallbladder with a Brief Review of the Literature

Gallbladder polyps are frequently found on abdominal ultrasonography. Most gallbladder polyps are benign. However, the risk of malignancy increases when the polyp size exceeds 10 mm. The fifth edition of the World Health Organization Classification of tumors proposed three types of preinvasive neoplasms of the gallbladder: adenoma, intracholecystic papillary neoplasm (ICPN), and biliary intraepithelial neoplasia (BilIN). Herein, we report a case of a growing gallbladder polyp that was diagnosed with ICPN after cholecystectomy, along with a brief literature review.

Development of extrahepatic bile ducts and mechanisms of tumorigenesis: Lessons from mouse models.

The biliary system is a highly branched tubular network consisting of intrahepatic bile ducts (IHBDs) and extrahepatic bile ducts (EHBDs). IHBDs are derived from hepatic progenitor cells, while EHBDs originate directly from the endoderm through a separate branching morphogenetic process. Traits that are important for cancer are often found to overlap in developmental and other processes. Therefore, it has been suggested that intrahepatic cholangiocarcinomas (iCCAs) and extrahepatic cholangiocarcinomas (eCCAs) have different developmental mechanisms. While much evidence is being gathered on the mechanism of iCCAs, the evidence for eCCA is still very limited. The main reason for this is that there are very few appropriate animal models for eCCA. We can gain important insights from these animal models, particularly genetically engineered mouse models (GEMMs). GEMMs are immunocompetent and mimic human CCA subtypes with a specific mutational pattern, allowing the development of precancerous lesions, that is, biliary intraepithelial neoplasia (BilIN) and intraductal papillary neoplasm of the bile duct (IPNB). This review provides a summary of the pathogenesis and mechanisms of eCCA that can be revealed by GEMMs. Furthermore, we discuss several clinical questions, such as whether BilIN and IPNB really become malignant, whether the peribiliary gland is the origin of eCCAs, and others.

S1869 A Case of Biliary Intraepithelial Neoplasia of the Extrahepatic Bile Duct

Introduction: Biliary intraepithelial neoplasia (BilIN) is a relatively new diagnosis that refers to flat or micropapillary precursor lesions of the bile duct which can develop into adenocarcinomas. Herein, we present a case of an extrahepatic biliary intraepithelial neoplasia that was found during endoscopic retrograde cholangiopancreatography (ERCP). Case Description/Methods: 54-year-old male with a history of hypertension and tobacco abuse presented with sharp, right upper quadrant abdominal pain with intermittent nausea and vomiting of one month duration. On admission, he was hemodynamically stable, with normal labs including liver function tests. MRI/MRCP revealed intra- and extra-hepatic duct dilation up to 12 mm with no intraductal filling defect or stricture and a normal gallbladder. (Figure A).The patient underwent an EUS which revealed common bile duct dilation up to 12 mm with a hypoechoic lesion in the distal bile duct, just proximal to the ampulla (Figure B). Given the EUS findings, an ERCP was performed. Cholangiogram revealed diffuse dilation of the biliary tree with no obvious stricture or filling defect (Figure C). The biliary tree was swept, and nothing was found. Due to the suspicious lesion seen on the EUS we performed distal common bile duct brushing and biopsies. Histopathology revealed biliary intraepithelial neoplasia, grade 2 (Figure D). His pain was controlled and was discharged with plans for pancreaticoduodenectomy (Whipple’s) procedure. Discussion: BilIN refers to flat or micropapillary precursor lesions of the bile duct which can develop into adenocarcinomas. They are commonly found in liver samples of chronic biliary and liver diseases such as hepatolithiasis, primary sclerosing cholangitis, choledochal cyst, chronic hepatitis C, and alcoholic cirrhosis. However, they are rarely found in clinical practice since they are not easily accessible, nor do they usually cause bile duct obstruction. The natural course of BillN is not well understood and in 2017, diagnostic criteria were created for lesions found in the intrahepatic duct based on their cellular and structural atypia. Lesions in the extrahepatic duct do occur, however, very rarely. This case is unusual in that the BilIN caused symptoms suggestive of biliary obstruction with a dilated bile duct and because the BillN was found in the extrahepatic duct. The case highlights the importance of considering BilIN in biliary obstruction and perform biopsies to identify the lesions prior to development of malignancy.Figure 1.: (A) MRI T2 flare reveals intra- and extra-hepatic duct dilation up to 12 mm with no intraductal filling defect or stricture. (B) EUS reveals hypoechoic lesion in the distal bile duct, just proximal to the ampulla. (C) ERCP cholangiogram reveals diffuse dilation of the biliary tree with no obvious stricture or filling defect. (D) Hematoxylin and eosin stain reveals dysplastic biliary epithelium (flat architecture; enlarged, hyperchromatic nuclei with nucleoli and some pseudostratification, rare mitotic figures) consistent with BilIN grade 2.

Abstract 6083: Evolutionary trajectories during carcinogenesis and metastasis in gallbladder adenocarcinoma

Abstract Background: There is no study so far that analyzed the patterns of clonal evolution from the initiation of carcinogenesis to distant metastasis in patients with gallbladder adenocarcinoma (GBAC). This study aimed to elucidate the evolutionary trajectories of GBAC using multi-regional and longitudinal tumor samples. Methods: Whole-exome sequencing was conducted on tumors and matched normal samples with the coverage of 300x and 200x, respectively. Using PyClone, CITUP, MapScape, and TimeScape, phylogenetic trees were visualized in each patient considering each tumor’s location and timing of acquisition. Mutational signatures were analyzed using Mutalisk. Results: Between 2013 and 2018, a total of 11 patients (male, 5) including 2 rapid autopsy cases were enrolled. The median age was 70 (range, 59-75) years. The dataset consisted of 11 normal samples, 4 biliary intraepithelial neoplasia (BilIN), 11 primary tumors, and 30 metastatic tumors. The most frequently altered gene was ERBB2 (54.5%), followed by TP53 (45.5%) and FBXW7 (27.3%). Of 6 patients with analyzable tumor ploidy, 2 patients (33.3%) had whole genome doubling (WGD) in both primary and metastatic tumors, and 1 patient (16.7%) had WGD not in the primary tumor but in the liver metastasis. In the BilIN analysis (n=4), most mutations in frequently altered genes in GBAC were detectable from the BilIN stage, but some of them were subclonal. In these 4 patients, the fittest subclone in BilIN underwent linear and branching evolution by acquiring additional subclonal mutations and thus expanded in the primary tumor, suggesting a selective sweep phenomenon. In combined analysis with metastatic tumors (n=11), branching and linear evolution was identified in 9 (81.8%) and 2 (18.2%) patients, respectively. Of the 9 patients with branching evolution, eight (88.9%) had a total of 11 subclones expanded at least 7-fold in regional or distant metastasis. These subclones harbored putative metastasis-driving mutations in tumor suppressor genes such as SMAD4, ROBO1, and DICER1. Metastases were polyclonal in all patients. However, metastatic lesions in the same or adjacent organs showed similar clonal compositions and there was evidence of metastasis-to-metastasis spread. In mutational signature analysis, we identified 6 mutational signatures: signatures 1 (age), 3 (DNA double-strand break-repair), 7 (ultraviolet), 13 (APOBEC), 22 (aristolochic acid), and 24 (aflatoxin) (cosine similarity values ≥ 0.9). Among them, signatures 1 and 13 were dominant at the carcinogenesis stage while signatures 22 and 24 were dominant at the metastasis stage. Conclusions: We have constructed evolutionary trajectories of individual patients, highlighting the role of each clone and the dynamics among clones during carcinogenesis and metastasis. This novel approach may help us move forward to precision medicine that enables early detection of carcinogenesis and metastasis. Citation Format: Minsu Kang, Hee Young Na, Soomin Ahn, Ji-Won Kim, Sejoon Lee, Soyeon Ahn, Ju Hyun Lee, Jeonghwan Youk, Haesook T. Kim, Kui-Jin Kim, Koung Jin Suh, Jun Suh Lee, Se Hyun Kim, Jin Won Kim, Yu Jung Kim, Keun-Wook Lee, Yoo-Seok Yoon, Jee Hyun Kim, Jin-Haeng Chung, Ho-Seong Han, Jong Seok Lee. Evolutionary trajectories during carcinogenesis and metastasis in gallbladder adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6083.

Cancerization of ducts in hilar cholangiocarcinoma.

Invasive cancers that arise from ductal structures can infiltrate and colonize pre-existing ducts in a process referred to as cancerization of ducts (COD). COD in cholangiocarcinoma is an under-studied process whose clinical significance remains poorly understood. Even though both cancerized ducts and biliary intraepithelial neoplasias (BilINs) show dysplastic changes, hallmarks of COD are (i) an abrupt transition from the normal/reactive epithelium to severe dysplasia and (ii) close proximity to invasive carcinoma with similar cytologic features. We investigated 113 cases of surgically resected hilar cholangiocarcinoma and identified COD in 37 cases (33%). Using immunohistochemistry, we found that COD and adjacent invasive carcinoma had a concordant pattern of p53 and SMAD4 staining in 95% (21/22) and 100% (21/21) of cases, respectively. In contrast, BilINs and cancerized ducts showed significantly lower levels of concordance in p53 and SMAD4 staining at 44% (8/18) and 47% (8/17) of cases, respectively (P = 0.0007 and 0.0001, respectively). By univariate analysis, positive lymph node metastasis (P = 0.027), positive final bile duct margin (P = 0.021), and the presence of COD (P = 0.020) were associated with decreased overall survival. We further performed multivariate analysis to demonstrate that positive lymph node metastasis (P = 0.031), positive final bile duct margin (P = 0.035), and COD (P = 0.0051) were correlated with decreased overall survival. Together, our study highlights that COD is a clinically significant process in hilar cholangiocarcinoma that can be identified using morphological criteria in conjunction with p53 and SMAD4 immunolabeling.

Global changes in gene expression related to Opisthorchis felineus liver fluke infection reveal temporal heterogeneity of a mammalian host response

The food-borne trematode Opisthorchis felineus colonizes bile ducts of the liver of fish-eating mammals including humans. Among chronically infected individuals, this opisthorchiasis involves hepatobiliary problems, including chronic inflammation, periductal fibrosis, biliary intraepithelial neoplasia, and even cholangiocarcinoma. Despite numerous studies at the pathomorphological level, the systemic response and cellular pathogenesis of these disorders are not well studied.To conduct in-depth research and to gain insights into the mechanism by which O. felineus infection causes precancerous liver lesions, we (i) applied a next-generation-sequencing–based technology (high-throughput mRNA sequencing) to identify differentially expressed genes in the liver of golden hamsters infected with O. felineus at 1 and 3 months postinfection and (ii) verified the most pronounced changes in gene expression by western blotting and immunohistochemistry.A total of 2151 genes were found to be differentially expressed between uninfected and infected hamsters (“infection” factor), whereas 371 genes were differentially expressed when we analyzed “time × infection” interaction. Cluster analysis revealed that sets of activated genes of cellular pathways were different between acute (1 month postinfection) and chronic (3 months postinfection) opisthorchiasis. This enriched KEGG pathways were “Cell adhesion molecules”, “Hippo signaling”, “ECM-receptor interaction”, “Cell cycle”, “TGF-beta”, and “P53 signaling”. Moreover, epithelial–mesenchymal transition was the most enriched (q-value = 2.2E-07) MSigDB hallmark in the set of differentially expressed genes of all O. felineus–infected animals. Transcriptomic data were supported by the results of western blotting and immunohistochemistry revealing the upregulation of vimentin, N-cadherin, and α-smooth muscle actin postinfection.Our data expand knowledge about global changes in gene expression in the O. felineus–infected host liver and contribute to understanding the biliary neoplasia associated with the liver fluke infection.