Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice.

Background: Secretin stimulates bicarbonate secretion in large ducts by interaction with receptors by a cAMP-dependent pathway [1]. In BDL rats, the growth of large (but not small) cholangiocytes is activated by the cAMP/ERK1/2/Elk-1 pathway; and is associated with increased secretin receptor (SR) expression [2]. The aim of our study was to define the role of secretin in experimental models of cholestatic mice. Methods: We used (SEC+/+) (wild-type, WT) and SEC-/- male mice that underwent sham surgery or BDL for 7 days treated with saline or secretin. Then, we used: (i) liver sections to evaluate secretin protein expression, cholangiocyte growth, apoptosis, the presence of VEGF-A/C and NGF; (ii) purified and immortalized small and large BDL cholangiocytes to measure PCNA protein expression and the same previous growth factors. Results: In vivo, we demonstrated that: (i) secretin is expressed in normal liver by large bile ducts; and (ii) secretin expression increases in large bile ducts following BDL. Isolated large cholangiocytes from BDL WT mice express higher levels of the mRNA for secretin and secrete greater amounts of this hormone compared to normal large cholangiocytes. Knockout of the secretin gene reduced BDL-induced increase in large IBDM compared to WT BDL mice, an event that was associated with enhanced apoptosis of large cholangiocytes and decreased expression of VEGF-A/C and NGF. Concomitant with reduced large IBDM there was increased small IBDM in SEC-/- KO BDL mice compared to WT BDL mice. After prolonged treatment, secretin increased the biliary expression of PCNA, VEGF-A/C and NGF. Conclusions: The study introduces the novel concept that cholangiocytes express and secrete secretin and that manipulation of this gene may be an important target for the management of biliary disorders.

Adenosine triphosphate (ATP) is released from cholangiocytes into bile and is a potent secretogogue by increasing intracellular Ca²(+) and stimulating fluid and electrolyte secretion via binding purinergic (P2) receptors on the apical membrane. Although morphological differences exist between small and large cholangiocytes (lining small and large bile ducts, respectively), the role of P2 signaling has not been previously evaluated along the intrahepatic biliary epithelium. The aim of these studies therefore was to characterize ATP release and P2-signaling pathways in small (MSC) and large (MLC) mouse cholangiocytes. The findings reveal that both MSCs and MLCs express P2 receptors, including P2X4 and P2Y2. Exposure to extracellular nucleotides (ATP, uridine triphosphate, or 2',3'-O-[4-benzoyl-benzoyl]-ATP) caused a rapid increase in intracellular Ca²(+) concentration and in transepithelial secretion (I(sc)) in both cell types, which was inhibited by the Cl(-) channel blockers 5-nitro-2-(-3-phenylpropylamino)-benzoic acid (NPPB) or niflumic acid. In response to mechanical stimulation (flow/shear or cell swelling secondary to hypotonic exposure), both MSCs and MLCs exhibited a significant increase in the rate of exocytosis, which was paralleled by an increase in ATP release. Mechanosensitive ATP release was two-fold greater in MSCs compared to MLCs. ATP release was significantly inhibited by disruption of vesicular trafficking by monensin in both cell types. These findings suggest the existence of a P2 signaling axis along intrahepatic biliary ducts with the "upstream" MSCs releasing ATP, which can serve as a paracrine signaling molecule to "downstream" MLCs stimulating Ca²(+)-dependent secretion. Additionally, in MSCs, which do not express the cystic fibrosis transmembrane conductance regulator, Ca²(+)-activated Cl(-) efflux in response to extracellular nucleotides represents the first secretory pathway clearly identified in these cholangiocytes derived from the small intrahepatic ducts.

Bile ducts are heterogenous in structure and function, and primary sclerosing cholangitis (PSC) damages specific bile ducts leading to ductular reaction (DR), mast cell (MC) infiltration, increased histamine release, inflammation, and fibrosis. Bile duct ligation (BDL) induces large duct damage via cyclic adenosine monophosphate (cAMP)/extracellular signal‐related protein kinase (ERK) signaling, and large cholangiocytes express H2 histamine receptor (H2HR). We evaluated how MCs interact with large cholangiocytes during cholestasis. Male wild‐type (WT) and MC‐deficient (KitW‐sh) mice 10–12 weeks of age were subjected to BDL for 7 days. Select KitW‐sh mice were injected with MCs pretreated with control or H2HR antagonist (ranitidine, 25 μm, 48 h) via tail vein injection. In vitro, MC migration toward small mouse cholangiocytes (SMCCs) and large mouse cholangiocytes (LMCCs) treated with lipopolysaccharide or histamine (±ranitidine) was measured. LMCCs were stimulated with MC supernatants pretreated with control, α‐methyl‐dl‐histidine (to block histamine release), or ranitidine. Liver damage, large duct DR/senescence, inflammation, fibrosis, and cAMP/ERK immunoreactivity increased in BDL WT and KitW‐sh+MC mice but decreased in BDL KitW‐sh and KitW‐sh+MC‐H2HR mice. In vitro, MCs migrate toward damaged LMCCs (but not SMCCs) blocked by inhibition of H2HR. Loss of MC histamine or MC‐H2HR decreases LMCC proliferation, senescence, H2HR, and cAMP/ERK levels. Human PSC livers have increased MC number found near DR, senescent ducts, and H2HR‐positive ducts. Conclusion: Infiltrating MCs preferentially interact with large ducts via H2HR signaling promoting biliary and liver damage. Mediation of MCs may be a therapeutic strategy for PSC.

During bile duct ligation (BDL), the growth of large cholangiocytes is regulated by the cyclic adenosine monophosphate (cAMP)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and is closely associated with increased secretin receptor (SR) expression. Although it has been suggested that SR modulates cholangiocyte growth, direct evidence for secretin-dependent proliferation is lacking. SR wild-type (WT) (SR(+/+)) or SR knockout (SR(-/-)) mice underwent sham surgery or BDL for 3 or 7 days. We evaluated SR expression, cholangiocyte proliferation, and apoptosis in liver sections and proliferating cell nuclear antigen (PCNA) protein expression and ERK1/2 phosphorylation in purified large cholangiocytes from WT and SR(-/-) BDL mice. Normal WT mice were treated with secretin (2.5 nmoles/kg/day by way of osmotic minipumps for 1 week), and biliary mass was evaluated. Small and large cholangiocytes were used to evaluate the in vitro effect of secretin (100 nM) on proliferation, protein kinase A (PKA) activity, and ERK1/2 phosphorylation. SR expression was also stably knocked down by short hairpin RNA, and basal and secretin-stimulated cAMP levels (a functional index of biliary growth) and proliferation were determined. SR was expressed by large cholangiocytes. Knockout of SR significantly decreased large cholangiocyte growth induced by BDL, which was associated with enhanced apoptosis. PCNA expression and ERK1/2 phosphorylation were decreased in large cholangiocytes from SR(-/-) BDL compared with WT BDL mice. In vivo administration of secretin to normal WT mice increased ductal mass. In vitro, secretin increased proliferation, PKA activity, and ERK1/2 phosphorylation of large cholangiocytes that was blocked by PKA and mitogen-activated protein kinase kinase inhibitors. Stable knockdown of SR expression reduced basal cholangiocyte proliferation. SR is an important trophic regulator sustaining biliary growth. The current study provides strong support for the potential use of secretin as a therapy for ductopenic liver diseases.

Biliary tree stem cells (BTSCs) are multipotent stem cells located in peribiliary glands (PBGs) of extrahepatic and large intrahepatic bile ducts (1). Primary sclerosing cholangitis (PSC) is characterised by fibro-stenosing strictures involving extrahepatic and/or large intrahepatic bile ducts. Mechanisms leading to bile duct injury are poorly understood (2). Our aims are to study the role of BTSC in the pathogenesis of biliary fibrosis in PSC. Specimens containing extrahepatic or large intrahepatic bile ducts were obtained from normal liver (n=6), liver explants from patients with PSC (n=11), and primary biliary cirrhosis (n=6). Specimens were processed for histology, immunohistochemistry and immunofluorescence. In PSC samples, progressive hyperplasia and mucinous metaplasia of PBGs were observed in large ducts with fibrosis, but not in inflamed ducts without fibrosis. PBG hyperplasia was associated with progressive biliary fibrosis and the occurrence of dysplastic lesions. Hyperplasia of PBGs was determined by the expansion of biliary tree stem cells, which sprouted towards the surface epithelium. In PSC, PBGs and myofibroblasts displayed enhanced expression of Hedgehog pathway components. PBGs in ducts with onion skin-like fibrosis expressed epithelial-to-mesenchymal transition traits associated with components of Hedgehog pathway, markers of senescence and autophagy. The biliary tree stem cell compartment is activated in PSC, its activation contributes to biliary fibrosis, and is sustained by the Hedgehog pathway. Our findings suggest a key role for peribiliary glands in the progression of bile duct lesions in PSC and could explain the associated high risk of cholangiocarcinoma. This work was supported by grants from MIUR FIRB 2010 and MIUR PRIN-2009.

The objective of this article is to summarize the findings related to the notion that cholangiocytes, within small and large intrahepatic ducts, are heterogeneous regarding (1) morphology; (2) secretion in response to hormones and peptides and to bile acids; and (3) proliferation in response to injury or toxins, including bile duct ligation (BDL), acute carbon tetrachloride (CCl 4 ) administration, chronic feeding of bile salts (i.e., taurocholate [TC] or taurolithocholate [TLC]) or alpha-naphthylisothiocyanate (ANIT). After an overview of the morphology of the biliary epithelium, we provide a summary of cholangiocyte function, the in vivo models, and the in vitro experimental tools (i.e., small and large cholangiocytes or small and large intrahepatic bile duct units [IBDU]), which allowed us to demonstrate cholangiocyte heterogeneity. After a discussion on the receptors, transporters, and channels that are heterogeneously expressed by cholangiocytes, we discuss the different-sized ducts that differentially respond to injury and toxins. Finally, we review the human diseases that selectively target specific-sized ducts.

Gene Expression Profiles During Hepatic Stellate Cell Activation in Culture and In Vivo

Epithelial-mesenchymal transition (EMT) of biliary epithelial cells (BECs) plays major roles in many cholangiopathies. This study evaluated whether EMT of BECs has a role in hepatolithiasis-induced biliary fibrosis and types of BECs that are involved. The expression of EMT-related proteins and epidermal growth factor receptor was evaluated by immunohistochemistry of liver tissues from 102 patients with hepatolithiasis, 32 patients with post-hepatitis cirrhosis, and 48 normal livers. Antibodies against E-cadherin, β-catenin, and cytokeratin were used to identify epithelial cells and antibodies against vimentin, S100A4, podoplanin, and α-smooth muscle actin (α-SMA) were used to identify mesenchymal cells. The relationship between clinical and histological parameters and immunohistochemistry findings in BECs, and the surrounding stroma were evaluated. Loss of E-cadherin and acquisition of S100A4 and vimentin were observed in BECs. In all BECs, cytokeratin and β-catenin expression were unchanged, while podoplanin and α-SMA were not expressed. Although hepatic fibrosis was more severe in post-hepatitis cirrhosis, EMT of BECs was more widespread in hepatolithiasis. In hepatolithiasis, EMT-related proteins were more highly expressed in small bile ducts than in medium or large bile ducts. Their expression was associated with the severity of biliary fibrosis and the expressions of epidermal growth factor receptor. Expression of α-SMA in fibroblasts from the portal space was closely linked to pathological changes in small bile ducts and EMT-related protein expressions in BECs. Proliferating cholangiocytes that form small bile ducts may contribute to cholangiopathies in hepatolithiasis through an EMT-like phenomenon or through interactions with stromal myofibroblasts.

Cholangiocarcinomas (CCs) are primary hepatobiliary carcinomas of increasing importance and with major biological and therapeutic challenges. CCs may occur in any segment along the biliary tract and are presently also classified into two major categories according to their anatomic location. CCs arising from small bile duct and ductules to segmental large bile ducts are designated as intrahepatic or peripheral CC (ICC). Tumors originating from large bile ducts at the hilum (right or left hepatic bile duct or at their junction) or along the extrahepatic biliary tree are designated as extrahepatic bile duct carcinomas (BDC) or extrahepatic CC. Features of cholangiocyte differentiation characterize them; traditionally, they are thought to derive from the malignant transformation of bile duct epithelial cells and histologically classified as adenocarcinoma and rare variants. Recent data emphasized the significant degree of CCs’ heterogeneity in terms of their epidemiology and risk factors, pathological and molecular features, pathogenesis, and clinical behaviors and treatment and underlined the role of hepatic stem/progenitor cells as cell origin of a proportion of CC and their possible overlap with the major primary malignant tumor of the liver, namely hepatocellular carcinoma (HCC); precursor lesions and early lesions have been characterized underlining the existence of multistep carcinogenesis process. Overall, these data result in proposal of new histological or molecular classifications that could soon replace current anatomic-based classification and have major impact on establishment of prognosis and on development of novel target treatment approaches.

Parasitic infection of the biliary tree is caused by liver flukes, namely Clonorchis sinensis and Opisthorchis viverrini. These flukes reside in the peripheral small bile ducts of the liver and produce chronic inflammation of the bile duct, bile duct dilatation, mechanical obstruction, and bile duct wall thickening. On imaging, peripheral small intrahepatic bile ducts are dilated, but the large bile ducts and extrahepatic bile ducts are not dilated or slightly dilated. There is no visible caused of obstruction. Sometimes, in heavy infection, adult flukes are demonstrated on sonography, CT or MR cholangiography as small intraluminal lesions. The flukes in the gallbladder may appear as floating, small objects on sonography. Chronic infection may result in cholangiocarcinoma of the liver parenchyma or along the bile ducts. Human infection of Fasciola hepatica, a cattle flukes, may occur inadvertently, and the flukes migrate in the liver (hepatic phase) and reside the bile ducts (biliary phase). Image findings in the hepatic phase present with multiple, small, clustered, necrotic cavities or abscesses in the peripheral parts of the liver, showing "tunnels and caves" sign, reflecting parasite migration in the liver parenchyma. In the biliary phase, the flukes are demonstrated in the intra- and extrahepatic bile ducts and the gallbladder as small intraluminal flat objects, sometimes moving spontaneously. Bile ducts are dilated.

Recently, there have been sporadic reports of lithotomy using endoscopic sphincterotomy combined with large balloon dilation (EPLBD) against large or multiple bile duct stones. However, there are not many reports so far concerning this procedure. Therefore, we decided to discuss the results of EPLBD against large or multiple bile duct stones. Stone retrieval using EPLBD was performed with 59 patients of choledocholithiasis, A) with 13 mm or more in shortest dimension, or B) multiple (≥3) bile duct stones, with the smallest more than 10 mm in shortest dimension. The papilla treated with endoscopic sphincterotomy (EST) was dilated using a 12-20 mm balloon suitable for the biliary ductal size. The success rate for the first lithotomy for choledocholithiasis was 83.1% (49/59). The final lithotomy rate was 100% (59/59). The time required for lithotomy was 43.7 (12-125) minutes and the number of treatment was 1.3 (1-4) on average. Lithotripsy was needed in 13.6% (8/59). The incidence of coincidental events associated with the procedure was 6.8% (4/59). No pancreatitis was noted. An endoscopic treatment using EST plus large balloon dilation against large or multiple bile duct stones was suggested to be safe and effective.

Intrahepatic cholangiocarcinoma (iCCA) exhibits different blood imaging features and prognosis depending on histology. To clarity histopathological growth patterns (HGPs) and vascularization processes of iCCA, we collected 145 surgical specimens and histologically classified them into large bile duct (LBD) (20 cases), small bile duct (SBD) (54), cholangiolocarcinoma (CLC) (35), combined SBD–CLC (cSBD–CLC) (26), and ductal plate malformation (DPM) (10) (sub)types. According to the invasive pattern at the interface between tumor and adjacent background liver, HGPs were classified into desmoplastic, pushing, and replacing HGPs. Desmoplastic HGP predominated in LBD type (55.5%), while replacing HGP was common in CLC (82.9%) and cSBD–CLC (84.6%) subtypes. Desmoplastic HGP reflected angiogenesis, while replacing HGP showed vessel co-option in addition to angiogenesis. By evaluating microvessel density (MVD) using vascular markers, ELTD1 identified vessel co-option and angiogenesis, and ELTD1-positive MVD at invasive margin in replacing HGP was significantly higher than those in desmoplastic and pushing HGPs. REDD1, an angiogenesis-related marker, demonstrated preferably higher MVD in the tumor center than in other areas. iCCA (sub)types and HGPs were closely related to vessel co-option and immune-related factors (lymphatic vessels, lymphocytes, and neutrophils). In conclusion, HGPs and vascular mechanisms characterize iCCA (sub)types and vessel co-option linked to the immune microenvironment.

Obstructive jaundice has rarely been reported in liver amyloidosis. We report here an autopsy of a 55-year-old woman with multiple myeloma and obstructive jaundice due to the deposition of amyloid-like substances in the walls and lumina of the extrahepatic bile duct and intrahepatic large bile ducts, resulting in obstruction and narrowing of the bile ducts. The amyloid-like deposits were negative with Congo red stain, and were negative immunohistochemically for IgG, IgA, IgM, kappa chain, beta-2-microglobulin, and amyloid A and P components. However, they were positive for lambda chain, and ultrastructurally composed of non-branching filaments with a diameter of 7-10 nm. This is the first case of obstructive jaundice due to histologically confirmed amyloid-like deposits in the biliary system.

Primary sclerosing cholangitis (PSC) is a chronic progressive inflammatory disease of the bile ducts that is surrounded by much uncertainty. First of all, it is a rare disease, with reported prevalence between 0 and 31.7 per 100,000 individuals,1Boonstra K. Beuers U. Ponsioen C.Y. Epidemiology of primary sclerosing cholangitis and primary biliary cirrhosis: a systematic review.J Hepatol. 2012; 56: 1181-1188Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar,2Barner-Rasmussen N. Pukkala E. Jussila A. et al.Epidemiology, risk of malignancy and patient survival in primary sclerosing cholangitis: a population-based study in Finland.Scand J Gastroenterol. 2020; 55: 74-81Crossref PubMed Scopus (31) Google Scholar and establishing a diagnosis can be difficult. The primary pathology is hidden deep in the liver, and there is no specific and easy noninvasive diagnostic test for PSC. Histologic features of fibrosing cholangiopathy are present in <20% of liver biopsy specimens obtained from patients with PSC and can also be seen in cases of secondary sclerosing cholangitis. Current US and European guidelines do not recommend liver biopsy for making a diagnosis of PSC except for suspected cases of small duct PSC and pediatric cases.3Lindor K.D. Kowdley K.V. Harrison M.E. ACG clinical guideline: primary sclerosing cholangitis.Am J Gastroenterol. 2015; 110: 646-659Crossref PubMed Scopus (358) Google Scholar,4European Association for the Study of the LiverEASL clinical practice guidelines: management of cholestatic liver diseases.J Hepatol. 2009; 51: 237-267Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar In addition, biliary tract abnormalities can be found on magnetic resonance cholangiopancreatography (MRCP) or endoscopic retrograde cholangiography (ERC) in a wide array of conditions (see Supplementary Table 1). Furthermore, the course of PSC can be variable. Liver transplant–free survival ranges between 14.5 and 21.3 years in large cohorts.5Weismüller T.J. Trivedi P.J. Bergquist A. et al.Patient age, sex, and inflammatory bowel disease phenotype associate with course of primary sclerosing cholangitis.Gastroenterology. 2017; 152: 1975-1984Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar,6Boonstra K. Weersma R.K. van Erpecum K.J. et al.Population-based epidemiology, malignancy risk, and outcome of primary sclerosing cholangitis.Hepatology. 2013; 58: 2045-2055Crossref PubMed Scopus (460) Google Scholar An important risk factor affecting survival is the development of cholangiocarcinoma (CCA). This has a cumulative incidence of approximately 20% at 20 years after diagnosis, but one third of CCA cases become manifest within 1 year after the diagnosis of PSC.5Weismüller T.J. Trivedi P.J. Bergquist A. et al.Patient age, sex, and inflammatory bowel disease phenotype associate with course of primary sclerosing cholangitis.Gastroenterology. 2017; 152: 1975-1984Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar,6Boonstra K. Weersma R.K. van Erpecum K.J. et al.Population-based epidemiology, malignancy risk, and outcome of primary sclerosing cholangitis.Hepatology. 2013; 58: 2045-2055Crossref PubMed Scopus (460) Google Scholar Last but not least, the cause of PSC is still enigmatic, and consequently, there is no therapy that has been proven to modify disease progression. In recent years, there has been an upsurge of pilot studies and phase 2 clinical trials. Phase 2 and 3 trials are hampered by the lack of established surrogate endpoints for clinical outcome, the heterogeneity of the disease course, and the lack of clear definitions for many key clinical disease characteristics.7Ponsioen C.Y. Chapman R.W. Chazouillères O. et al.Surrogate endpoints for clinical trials in primary sclerosing cholangitis: review and results from an International PSC Study Group consensus process.Hepatology. 2016; 63: 1357-1367Crossref PubMed Scopus (124) Google Scholar While the available national and international clinical practice guidelines predominantly focus on clinical management, the International PSC Study Group (IPSCSG) has recognized that there is a vital need for clinical trials to use uniform definitions for the diagnosis of PSC and for assessing disease severity and outcomes.3Lindor K.D. Kowdley K.V. Harrison M.E. ACG clinical guideline: primary sclerosing cholangitis.Am J Gastroenterol. 2015; 110: 646-659Crossref PubMed Scopus (358) Google Scholar,4European Association for the Study of the LiverEASL clinical practice guidelines: management of cholestatic liver diseases.J Hepatol. 2009; 51: 237-267Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar,8Chapman M.H. Thorburn D. Hirschfield G.M. et al.British Society of Gastroenterology and UK-PSC guidelines for the diagnosis and management of primary sclerosing cholangitis.Gut. 2019; 68: 1356-1378Crossref PubMed Scopus (135) Google Scholar Therefore, the IPSCSG has commissioned a consensus process among its experts to issue a set of definitions to aid clinical trialists designing much needed clinical trials. To this end, an extensive consensus process was initiated, applying a hybrid between a Delphi process and a nominal group process, as was previously used by the IPSCSG and the European Crohn's and Colitis Organization.7Ponsioen C.Y. Chapman R.W. Chazouillères O. et al.Surrogate endpoints for clinical trials in primary sclerosing cholangitis: review and results from an International PSC Study Group consensus process.Hepatology. 2016; 63: 1357-1367Crossref PubMed Scopus (124) Google Scholar,9Dignass A. Eliakim R. Magro F. et al.Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 1: definitions and diagnosis.J Crohns Colitis. 2012; 6: 965-990Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar Where necessary, an additional survey was conducted among all IPSCSG members (see the Supplementary Methods for details). Results are presented as a comprehensive set of consensus statements (CSs) and diagnostic criteria for PSC. Although patients with PSC are increasingly diagnosed at earlier stages of disease based on asymptomatic serum liver enzyme abnormalities, at least 45% of patients PSC present with symptoms on either a constant or intermittent basis, which significantly reduces quality of life.10Broome U. Olsson R. Lööf L. et al.Natural history and prognostic factors in 305 Swedish patients with primary sclerosing cholangitis.Gut. 1996; 38: 610-615Crossref PubMed Google Scholar Furthermore, more than 22% of asymptomatic patients may develop symptoms within 5 years. The most common clinical symptoms associated with PSC are fatigue; pruritus; right upper quadrant (RUQ) abdominal pain; and emotional distress, which may include anxiety and depression symptoms. Pruritus and abdominal pain can fluctuate dramatically, and this may be suddenly worsened because of the development of a biliary obstruction or acute bacterial cholangitis. Therefore, clinical evaluation with laboratory testing and imaging may be necessary in the event of the sudden appearance or worsening of symptoms. Emotional distress for many patients is attributable to chronic anxiety about the uncertain etiology of the disease, the lack of effective therapy, and the risk of malignancy, among others.11Ranieri V. Kennedy E. Walmsley M. et al.The Primary Sclerosing Cholangitis (PSC) Wellbeing study: understanding psychological distress in those living with PSC and those who support them.PLoS One. 2020; 15e0234624Crossref PubMed Scopus (10) Google Scholar The incorporation of clearly defined patient-reported symptoms into the design of clinical trials is both desirable from a patient-centered perspective and highly encouraged by regulatory agencies12Ponsioen C.Y. Lindor K.D. Mehta R. et al.Design and endpoints for clinical trials in primary sclerosing cholangitis.Hepatology. 2018; 68: 1174-1188Crossref PubMed Scopus (43) Google Scholar (Box 1, CS 1.1). Recently, 2 such tools have emerged. One patient-reported outcome (PRO) dedicated to PSC showed excellent reliability and was able to discern patients with cirrhosis and those with a history of depression.13Younossi Z.M. Afendy A. Stepanova M. et al.Development and validation of a primary sclerosing cholangitis-specific patient-reported outcomes instrument: the PSC PRO.Hepatology. 2018; 68: 155-165Crossref PubMed Scopus (21) Google Scholar A more recently developed tool to assess patient-reported symptoms, the Simple Cholestatic Complaints Score, was developed through digital surveys of patients and showed good criterion and construct validity and a very high test-retest reproducibility.14Munster K.N. Dijkgraaf M.G.W. Gennep S. et al.The Simple Cholestatic Complaints Score is a valid and quick patient-reported outcome measure in primary sclerosing cholangitis.Liver Int. 2020; 40: 2758-2766Crossref PubMed Scopus (0) Google Scholar Further validation of these tools in larger and more diverse cohorts is needed.Box 1Consensus StatementsTabled 11.Clinical symptoms1.1.Assessment of symptoms in patients with PSC for clinical trial purposes should be performed through disease-specific and validated quantitative tools.1.2.Assessment of symptoms in patients with PSC should take into consideration and be distinguished from symptoms attributable to active inflammatory bowel disease and other coexistent conditions.2.Laboratory markers2.1.The presence of significant elevations of serum aminotransferase activity (>5 × ULN) and/or IgG (>2 × ULN) can be indicative of features of coexisting autoimmune hepatitis and should trigger consideration of liver biopsy for diagnostic and stratification purposes when considering enrolling a subject in a clinical trial.2.2.Serum IgG4 concentrations can be elevated in patients with PSC, in the absence of classical features of IgG4-related disease, but IgG4 elevations are not validated markers of disease diagnosis, severity, prognosis, or treatment.3.Imaging3.1.The pattern of PSC can be defined according to the appearances of cholangiography using high-quality magnetic resonance cholangiography (MRC) (at least 1.5 T):3.1.1.Small duct when a recent cholangiogram (not older than 1 year) is normal3.1.2.Intrahepatic when there are intrahepatic changes but the common ducts and first-order ducts are normal3.1.3.Extrahepatic disease when the common ducts and/or the first-order ducts are involved4.Histology4.1.Histologic findings in a liver biopsy sample from patient suspected to have PSC can be defined (classified) as follows:4.1.1.Typical of PSC: features of fibrosing cholangiopathy present (periductal fibrosis, fibro-obliterative duct lesions)4.1.2.Compatible with PSC: features of chronic biliary disease present (see text for further details) without typical bile duct lesions4.1.3.Atypical for PSC: features atypical for PSC present. Examples include•unusually prominent inflammatory activity suggesting the possibility of PSC-AIH overlap syndrome•features suggesting an alternative diagnosis, such as fatty liver disease, or IgG4-associated cholangitis.4.2.A liver biopsy specimen showing typical or compatible histologic features is required to establish a diagnosis of small duct PSC.4.3.In a person with PSC, a liver biopsy specimen showing at least moderate interface hepatitis in addition to features compatible with or diagnostic of PSC is required to establish a diagnosis of PSC with features of AIH or PSC-AIH overlap syndrome.4.4.The histologic features that indicate disease progression (staging) in PSC include fibrosis, bile duct loss, and evidence of chronic cholestasis (demonstrated by deposition of copper-associated protein in periportal hepatocytes).5.Concurrent IBD5.1.The definition and description of IBD in PSC should follow the European Crohn's and Colitis Organization (ECCO) guidelines and Montreal classification. Any patient with established PSC should undergo a full ileocolonoscopy with histology before concurrent IBD may be excluded.5.2.When assessing the existence of backwash ileitis, it is recommended that the ECCO definition be followed. This defines backwash ileitis as continuous extension of macroscopic or histologic inflammation from the cecum into the most distal ileum.6.Staging6.1.Disease staging of PSC is defined as the histologic progression from initial disease onset to cirrhosis.6.2.A clinically significant change in disease stage is defined by a 1-point change in the Ludwig or Nakanuma system or a 2-point change in the Ishak system.6.3.Histologic cirrhosis is a clinically significant endpoint.6.4.Cirrhosis (stage 5–6 by Ishak) in PSC can be defined by an LS of >14.4 kPa measured by VCTE.6.5.Cirrhosis (stage 5–6 or greater by Ishak) in PSC can be defined by an ELF score of >9.8 or FibroTest of >7.3.7.Clinical endpoints7.1.Liver transplant:Liver transplant is an appropriate endpoint in PSC clinical trials. However, divergent indications for transplant listing (end-stage disease, dysplasia, CCA, symptoms, intractable cholangitis) and method of transplantation (deceased vs living donation) should not be grouped into a single endpoint because of the wide heterogeneity of clinical practice.7.2.Liver-related death:Liver-related death in the setting of PSC is defined as death from clinically significant portal hypertension, synthetic liver dysfunction, or specific complications from biliary disease such as hepatobiliary infections.8.Cancer development8.1.Cholangiocarcinoma:8.1.1.CCA in PSC requires pathologic confirmation and is classified according to CCA in general.8.1.2.Dysplastic changes considered precursors to CCA in PSC are defined by standard criteria.8.2.Colorectal carcinoma:8.2.1.CRC in the setting of PSC is defined according to standard criteria.9.Pediatric PSC9.1.Pediatric PSC is not a specific disease phenotype. PSC in children should be described in terms of small vs large duct involvement, whether features of overlap with AIH are present or absent, and whether IBD is present or absent, as in adult patients.9.2.Terms such as juvenile sclerosing cholangitis and autoimmune sclerosing cholangitis should be avoided.9.3.When gGT is substituted for ALP, the consensus definitions in this document are applicable to children.10.Recurrent PSC10.1.The diagnosis of rPSC is based on the presence of compatible radiologic changes (using high-quality MR) with/or without histologic findings, along with the exclusion of other causes of similar findings. Open table in a new tab Tabled 11.Clinical symptoms1.1.Assessment of symptoms in patients with PSC for clinical trial purposes should be performed through disease-specific and validated quantitative tools.1.2.Assessment of symptoms in patients with PSC should take into consideration and be distinguished from symptoms attributable to active inflammatory bowel disease and other coexistent conditions.2.Laboratory markers2.1.The presence of significant elevations of serum aminotransferase activity (>5 × ULN) and/or IgG (>2 × ULN) can be indicative of features of coexisting autoimmune hepatitis and should trigger consideration of liver biopsy for diagnostic and stratification purposes when considering enrolling a subject in a clinical trial.2.2.Serum IgG4 concentrations can be elevated in patients with PSC, in the absence of classical features of IgG4-related disease, but IgG4 elevations are not validated markers of disease diagnosis, severity, prognosis, or treatment.3.Imaging3.1.The pattern of PSC can be defined according to the appearances of cholangiography using high-quality magnetic resonance cholangiography (MRC) (at least 1.5 T):3.1.1.Small duct when a recent cholangiogram (not older than 1 year) is normal3.1.2.Intrahepatic when there are intrahepatic changes but the common ducts and first-order ducts are normal3.1.3.Extrahepatic disease when the common ducts and/or the first-order ducts are involved4.Histology4.1.Histologic findings in a liver biopsy sample from patient suspected to have PSC can be defined (classified) as follows:4.1.1.Typical of PSC: features of fibrosing cholangiopathy present (periductal fibrosis, fibro-obliterative duct lesions)4.1.2.Compatible with PSC: features of chronic biliary disease present (see text for further details) without typical bile duct lesions4.1.3.Atypical for PSC: features atypical for PSC present. Examples include•unusually prominent inflammatory activity suggesting the possibility of PSC-AIH overlap syndrome•features suggesting an alternative diagnosis, such as fatty liver disease, or IgG4-associated cholangitis.4.2.A liver biopsy specimen showing typical or compatible histologic features is required to establish a diagnosis of small duct PSC.4.3.In a person with PSC, a liver biopsy specimen showing at least moderate interface hepatitis in addition to features compatible with or diagnostic of PSC is required to establish a diagnosis of PSC with features of AIH or PSC-AIH overlap syndrome.4.4.The histologic features that indicate disease progression (staging) in PSC include fibrosis, bile duct loss, and evidence of chronic cholestasis (demonstrated by deposition of copper-associated protein in periportal hepatocytes).5.Concurrent IBD5.1.The definition and description of IBD in PSC should follow the European Crohn's and Colitis Organization (ECCO) guidelines and Montreal classification. Any patient with established PSC should undergo a full ileocolonoscopy with histology before concurrent IBD may be excluded.5.2.When assessing the existence of backwash ileitis, it is recommended that the ECCO definition be followed. This defines backwash ileitis as continuous extension of macroscopic or histologic inflammation from the cecum into the most distal ileum.6.Staging6.1.Disease staging of PSC is defined as the histologic progression from initial disease onset to cirrhosis.6.2.A clinically significant change in disease stage is defined by a 1-point change in the Ludwig or Nakanuma system or a 2-point change in the Ishak system.6.3.Histologic cirrhosis is a clinically significant endpoint.6.4.Cirrhosis (stage 5–6 by Ishak) in PSC can be defined by an LS of >14.4 kPa measured by VCTE.6.5.Cirrhosis (stage 5–6 or greater by Ishak) in PSC can be defined by an ELF score of >9.8 or FibroTest of >7.3.7.Clinical endpoints7.1.Liver transplant:Liver transplant is an appropriate endpoint in PSC clinical trials. However, divergent indications for transplant listing (end-stage disease, dysplasia, CCA, symptoms, intractable cholangitis) and method of transplantation (deceased vs living donation) should not be grouped into a single endpoint because of the wide heterogeneity of clinical practice.7.2.Liver-related death:Liver-related death in the setting of PSC is defined as death from clinically significant portal hypertension, synthetic liver dysfunction, or specific complications from biliary disease such as hepatobiliary infections.8.Cancer development8.1.Cholangiocarcinoma:8.1.1.CCA in PSC requires pathologic confirmation and is classified according to CCA in general.8.1.2.Dysplastic changes considered precursors to CCA in PSC are defined by standard criteria.8.2.Colorectal carcinoma:8.2.1.CRC in the setting of PSC is defined according to standard criteria.9.Pediatric PSC9.1.Pediatric PSC is not a specific disease phenotype. PSC in children should be described in terms of small vs large duct involvement, whether features of overlap with AIH are present or absent, and whether IBD is present or absent, as in adult patients.9.2.Terms such as juvenile sclerosing cholangitis and autoimmune sclerosing cholangitis should be avoided.9.3.When gGT is substituted for ALP, the consensus definitions in this document are applicable to children.10.Recurrent PSC10.1.The diagnosis of rPSC is based on the presence of compatible radiologic changes (using high-quality MR) with/or without histologic findings, along with the exclusion of other causes of similar findings. Open table in a new tab A particular challenge in assessing clinical symptoms in patients with PSC is the frequent concurrence of inflammatory bowel disease (IBD), which itself can influence the clinical course of PSC and may also account for a significant proportion of symptoms, especially during periods of IBD exacerbations.15Cheung A.C. Patel H. Meza-Cardona J. et al.Factors that influence health-related quality of life in patients with primary sclerosing cholangitis.Dig Dis Sci. 2016; 61: 1692-1699Crossref PubMed Scopus (1) Google Scholar Existing tools, including the PSC PRO, do not specifically account for symptoms attributable to IBD. Although the Simple Cholestatic Complaints Score did not show a difference in PSC patient symptoms according to the presence or absence of IBD, a systematic evaluation of IBD symptoms in patients with PSC has not been performed, which can result in confounding findings. Therefore, assessment of PSC symptoms should be performed alongside specifically designed tools to detect symptoms from IBD in those with PSC-IBD or other relevant coexistent conditions (Box 1, CS 1.2) This is critically important for clinical trials that aim to use PRO measures as endpoints in PSC. Cholestatic serum liver tests are a characteristic, but nonspecific, feature of biliary disease irrespective of the nature of biliary involvement. Alkaline phosphatase (ALP) as the hallmark of cholestasis is elevated in the vast majority of patients with PSC.16Poupon R. Liver alkaline phosphatase: a missing link between choleresis and biliary inflammation.Hepatology. 2015; 61: 2080-2090Crossref PubMed Scopus (75) Google Scholar Previously, it was thought that to make a diagnosis of PSC, elevation of ALP, or gamma-glutamyl transpeptidase (gGT) in children (see the "Pediatric Primary Sclerosing Cholangitis" section) was a prerequisite. Recent studies have shown that elevated cholestatic serum parameters need not always be present. For more background, see the Supplementary Material. Aminotransferases are often moderately elevated, whereas at diagnosis, serum bilirubin and albumin levels are usually normal.3Lindor K.D. Kowdley K.V. Harrison M.E. ACG clinical guideline: primary sclerosing cholangitis.Am J Gastroenterol. 2015; 110: 646-659Crossref PubMed Scopus (358) Google Scholar Aminotransferase activity of more than 5 times the upper limit of normal (ULN) and a serum IgG of >2 × ULN should raise the suspicion of coexisting autoimmune hepatitis (AIH) and should trigger consideration of liver biopsy to identify histologic features that would support a diagnosis of AIH or PSC/AIH overlap, also called variant syndrome (Box 1, CS 2.1). This has management consequences, because patients with PSC/AIH commonly respond favorably to immunosuppressive medication such as corticoids and thiopurines, although not as well as patients with isolated AIH.17Boberg K.M. Chapman R.W. Hirschfield G.M. et al.Overlap syndromes: the International Autoimmune Hepatitis Group (IAIHG) position statement on a controversial issue.J Hepatology. 2011; 54: 374-385Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar In contrast to primary biliary cholangitis (PBC) and AIH, there is no pattern of autoantibody reactivity in patients with PSC of relevance to diagnosis or treatment. Positive titers of autoantibodies are common in PSC, including antinuclear antibody (8%–77%), smooth muscle antibody (0%–83%), and atypical perinuclear antibody (26%–94%).18Prideaux L. De Cruz P. Ng S.C. et al.Serological antibodies in inflammatory bowel disease: a systematic review.Inflamm Bowel Dis. 2012; 18: 1340-1355Crossref PubMed Scopus (139) Google Scholar,19Moiseev S. Cohen Tervaert J.W. Arimura Y. et al.2020 International consensus on ANCA testing beyond systemic vasculitis.Autoimmun Rev. 2020; 19: 102618Crossref PubMed Scopus (18) Google Scholar However, these are also frequently present in AIH, and perinuclear antineutrophil cytoplasmic antibodies can be found in ulcerative colitis (UC) in 41%–73% and in Crohn's disease in 6%–38%.17Boberg K.M. Chapman R.W. Hirschfield G.M. et al.Overlap syndromes: the International Autoimmune Hepatitis Group (IAIHG) position statement on a controversial issue.J Hepatology. 2011; 54: 374-385Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 18Prideaux L. De Cruz P. Ng S.C. et al.Serological antibodies in inflammatory bowel disease: a systematic review.Inflamm Bowel Dis. 2012; 18: 1340-1355Crossref PubMed Scopus (139) Google Scholar, 19Moiseev S. Cohen Tervaert J.W. Arimura Y. et al.2020 International consensus on ANCA testing beyond systemic vasculitis.Autoimmun Rev. 2020; 19: 102618Crossref PubMed Scopus (18) Google Scholar Serum IgG4 levels can be elevated in patients with PSC, but this has no validated bearing on diagnosis or disease severity (Box 1, CS 2.2). For distinction from IgG4-sclerosing cholangitis, see the Supplementary Material. Biliary imaging is integral to the diagnosis and classification of PSC. MRCP has been established as the noninvasive imaging of choice.3Lindor K.D. Kowdley K.V. Harrison M.E. ACG clinical guideline: primary sclerosing cholangitis.Am J Gastroenterol. 2015; 110: 646-659Crossref PubMed Scopus (358) Google Scholar,4European Association for the Study of the LiverEASL clinical practice guidelines: management of cholestatic liver diseases.J Hepatol. 2009; 51: 237-267Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar Regarding the utility of magnetic resonance imaging (MRI) in PSC, the reader is referred to a recent publication from the IPSCSG Working Group on MRI in PSC.20Schramm C. Eaton J. Ringe K.I. et al.Recommendations on the use of magnetic resonance imaging in PSC-A position statement from the International PSC Study Group.Hepatology. 2017; 66: 1675-1688Crossref PubMed Scopus Google Scholar A of features are described in large duct PSC, including or within the and/or biliary of the intrahepatic ducts with of the biliary of the and changes not of PSC, and from causes of secondary sclerosing cholangitis in the absence of can be difficult. in the bile ducts on cholangiography are thought to be highly specific for PSC. in the common and common bile the are usually seen in disease and are reported in of cases in a large of C.Y. et al.Natural history of primary sclerosing cholangitis and prognostic of cholangiography in a 51: PubMed Scopus (0) Google Scholar small duct PSC cholangiography is normal is an in PSC. recent have reported on the history of small cohorts of small duct PSC patients to large duct PSC was described in and after a of years of in K. Eaton et resonance imaging features of primary sclerosing 2020; PubMed Scopus Google K.I. Bergquist A. H. et features and MRI progression of small duct primary sclerosing cholangitis J 2020; Full Text Full Text PDF PubMed Scopus (0) Google Scholar (Box 1, CS was no with MRI findings and progression to large duct PSC in either in the bile ducts are a common and clinically relevant of the chronic inflammatory process that in the biliary in PSC. a of large duct PSC patients with the incidence of the development of can be to to approximately in patients with large duct A. P. et al.Development of bile duct in patients with primary sclerosing cholangitis with outcome after endoscopic Hepatol. Full Text Full Text PDF PubMed Scopus Google Scholar extensive in the Supplementary the a of criteria that into the definition of a as in of is called a when it the criteria before MRCP or with of in or first-order intrahepatic not and worsening of cholestatic symptoms within 2 biliary or and/or of × ULN and recent for × P. et of in primary sclerosing cholangitis: outcome after Full Text Full Text PDF PubMed Scopus Google and/or within for × or of >2 × ULN no within is

Vasopressin regulates the growth of the biliary epithelium in polycystic liver disease