Diagnosis of cholangiocarcinoma in primary sclerosing cholangitis

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1 Expert Review of Gastroenterology & Hepatology ISSN: (Print) (Online) Journal homepage: Diagnosis of cholangiocarcinoma in primary sclerosing cholangitis Seyed Alireza Taghavi, Ahad Eshraghian, Ramin Niknam, Gholam Reza Sivandzadeh & Kamran Bagheri Lankarani To cite this article: Seyed Alireza Taghavi, Ahad Eshraghian, Ramin Niknam, Gholam Reza Sivandzadeh & Kamran Bagheri Lankarani (2018): Diagnosis of cholangiocarcinoma in primary sclerosing cholangitis, Expert Review of Gastroenterology & Hepatology, DOI: / To link to this article: Published online: 21 May Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at

2 EXPERT REVIEW OF GASTROENTEROLOGY & HEPATOLOGY REVIEW Diagnosis of cholangiocarcinoma in primary sclerosing cholangitis Seyed Alireza Taghavi, Ahad Eshraghian, Ramin Niknam, Gholam Reza Sivandzadeh and Kamran Bagheri Lankarani Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran ABSTRACT Introduction: Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the hepatobiliary system characterized by chronic inflammation, progressive fibrosis, stricture formation and destruction of extrahepatic and intrahepatic bile ducts. Areas covered: The increased incidence of cholangiocarcinoma (CCA) in PSC has been well documented and can be explained by the continuous inflammation in the biliary tree leading to an enhanced dysplasia carcinoma sequence. Although PSC patients may progress to liver cirrhosis; CCA most commonly occurs between the ages of 30 and 45 years when cirrhosis has not yet developed. Therefore, CCA in patients with PSC occurs earlier than in patients without PSC. Expert commentary: Despite improvement in diagnostic methods and devices, the dilemma of diagnosing CCA in patients with PSC has not been solved yet and needs further investigation. ARTICLE HISTORY Received 5 January 2018 Accepted 2 May 2018 KEYWORDS Primary sclerosing cholangitis; cholangiocarcinoma 1. Introduction Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of hepatobiliary system, eventually leading to cholestasis, liver cirrhosis, and death at a median of years from the time of diagnosis [1]. It is characterized by chronic inflammation, progressive fibrosis, stricture formation, and destruction of extrahepatic and intrahepatic bile ducts [1]. The increased incidence of cholangiocarcinoma (CCA) in PSC has been well documented and can be explained by the continuous inflammation in the biliary tree leading to an enhanced dysplasia carcinoma sequence. In Europe, PSC is the most common risk factor for CCA [2]. The annual incidence of CCA in patients withpscis1.5%withanestimatedlifelongriskabout15 20% [3]. Although PSC patients may progress to liver cirrhosis, CCA most commonly occurs between the ages of 30 and 45 years when cirrhosis has not yet developed. Therefore, CCA in patients with PSC occurs up to 20 years earlier than those without PSC [4]. In most cases, diagnosis of CCA is made in less than years after the diagnosis of PSC [5]. Although colorectal cancer, mostly in association with inflammatory bowel disease (IBD), is more common in PSC patients, CCA is a greater threat to long-term survival due to its poor prognosis [6]. Depending on the location of the malignant transformation, a 5-year survival of less than 5% and a median survival of months have been reported in patients [7]. Surgical resection is the only curative treatment; unfortunately, most of the patients are not eligible for surgery at the time of diagnosis due to early disease progression [8]. Five-year survival after liver transplantation for PSC is around 85% when CCA is absent, but when CCA is present, the tumor will recur in almost all cases [9]. The concept of dysplasia carcinoma sequence in CCA by analogy to the same process in colorectal cancer indicates the possibility of early diagnosis of malignancy or premalignant lesions if the suitable tests were available. The distinction between malignant and inflammatory strictures is challenging in PSC because the inflammation associated with PSC complicates cytological assessment, and access to the bile duct is limited for cytological and tissue acquisition [10]. The so-called dominant stricture, defined as a stricture with a diameter of 1.5 mm in the common bile duct or 1 mm in the hepatic ducts, is often regarded as a possible indicator of the developmentofmalignancyinpsc. However, dominant strictures are rather common and are more often benign rather than malignant [11]. Demonstration of a mass lesion with typical imaging features is the only observation that has a high sensitivity and specificity for the diagnosis of CCA [12]. However, mass lesions are unusual in early-stage CCA and can still be absent even in advanced stages of the disease [13]. Incidental CCA is discovered in 9 21% of liver explants in patients undergoing livertransplantationforpsc[14]. In fact, more hepatobiliary tumors are detected in the liver explants from patients with PSC than those were detected during pretransplant evaluation [14]. This further demonstrates the shortfalls of the current methods in identification of CCA, especially in the early stages. As the success rates of surgical resection and liver transplantation for locally advanced CCA improve, the need to diagnose early CCA becomes imperative [15]. An effective surveillance regimentodetectccaatatreatablestageinpatientswithpsc remains highly sought since no clinically proven modality for early accurate diagnosis has been identified. Herein, we review the current diagnostic methods for detection of CCA in patients with PSC. CONTACT Ramin Niknam niknamramin@yahoo.com Gastroenterohepatology Research Center, Namazi Hospital, PO Box: , Shiraz, Iran 2018 Informa UK Limited, trading as Taylor & Francis Group

3 2 S. A. TAGHAVI ET AL. 2. Current diagnostic methods for the diagnosis of CCA in PSC 2.1. Endoscopic retrograde cholangiopancreatography (ERCP)-based modalities Diagnostic ERCP ERCP is considered the gold standard method for delineation of the bile ducts [16]. Definitive cholangiographic findings suggestive of malignancy such as polypoid lesions, new onset or progressive stricture formation, and marked bile duct dilatation are relatively rare, especially in early stages of the disease [17]. ERCP is also limited by a 3 8% incidence of complications and an inability to visualize the ducts proximal to high-grade stenoses [18,19]. Furthermore, it is well established that ERCP alone cannot reliably differentiate between different etiologies of biliary stricture including CCA, IgG4- associated cholangiopathy, or PSC [20]. Bile duct brush cytology, fluorescence in situ hybridization (FISH), probe-based confocal laser endomicroscopy, and cholangiospcoy with random biopsies are ERCP-based methods applied for diagnosis of CCA in PSC patients Brush cytology of bile ducts The most common tissue sampling method during ERCP in patients with PSC is brushing of the bile ducts. Biliary brushing is a safe and inexpensive method for detection of malignancy during ERCP; however, it has its own limitations. Accuracy of bile duct brushing and cytologic examination has been evaluated in previous studies with a reported sensitivity ranging from 8 80% and a specificity of % [21 25]. These discordant findings have several explanations. Severity of accompanying inflammation, location and size of tumor, previous episodes of cholangitis, and stenting are all interfere with cytological evaluation [26,27]. A meta-analysis of observational studies has reported a sensitivity of 43% and specificity of 97% for brush cytology as a diagnostic method for early detection of CCA in patients with PSC [28]. The lower sensitivity indicates that addition of other diagnostic modalities to brushing may improve accuracy of this test. Repeated biliary brushing [29], submission of the entire brush on the histologic slide [30], correlation with serum biomarkers like carcinoembryonic antigen (CEA) and CA 19-9 [31,32], DNA analysis [33], and FISH [34] have been reported to improve sensitivity of brushing in different studies FISH To improve diagnostic accuracy of brush cytology in the diagnosis of CCA, FISH assay has been applied on biliary brushing samples to detect abnormal loss or gains in chromosomes 3, 7, 17 and the p16 gene in chromosome 9 (9p21) by using four centromeric probes [35]. The results of FISH are interpreted as normal, trisomy, tetrasomy, or polysomy. While trisomy and tetrasomy are not independent predictors of CCA, polysomy is a strong predictor of CCA, especially in patients with a dominant stricture or patients with elevated serum CA 19-9 levels [36]. In a meta-analysis, the sensitivity and specificity of polysomy in FISH were reported to be 51% and 93%, respectively [37]. It is estimated that FISH analysis permits the detection of an additional 14% cases of CCA in patients with PSC who had normal cytology [37]. Serial reports of polysomy in PSC patients without positive cytology or a mass lesion on noninvasive imaging pose a diagnostic challenge. In a prospective study, the cumulative incidence of CCA in patients with PSC and serial polysomy was 75% compared to 18% in those with only one report of polysomy [38]. This highlights the necessity of repeated ERCP and biliary brushing for cytology and FISH to increase sensitivity of detection of CCA in patients with PSC. The same study also found multifocal polysomy to be a stronger predictor of CCA when compared to the unifocal polysomy. This signifies the importance of brushing of multiple sites of the biliary tree regardless of the presence of dominant strictures [39]. In summary, FISH polysomy increases the sensitivity of cytology and plays an important role in the further evaluation of patients but, at the same time, decreases the specificity. It is, therefore, more suitable as a confirmatory test in those patients with a high suspicion of CCA rather than as a screening test in patients with a low pretest probability of the disease [40]. However, results of some studies favor using serial polysomy for screening of CCA in patients with PSC. Quinn et al. showed that PSC patients with serial polysomy were more likely to diagnose with CCA than PSC patients with isolated polysomy [41]. Based on these results, PSC patients with serial polysomy may benefit from early evaluation for liver transplantation Trans-papillary endobiliary forceps biopsy The sensitivity of this technique for detection of biliary malignancy may exceed 80%. Navaneethan et al. reported a sensitivity of 30% and specificity of 100% for forceps biopsy to detect CCA [42]. The sensitivity of brush cytology and forceps biopsy in combination exceeds the sensitivity of either technique alone. In one cohort with 27 confirmed cases of CCA, cytology and biopsy samples alone each identified 44% of cases, whereas an accurate diagnosis was made in 86% of cases for which both cytology and biopsy samples were available. If both brush cytology and forceps biopsy were negative, but there is still suspicion for malignancy, repeat ERCP with combined brushing and biopsy should be considered Cholangioscopy Cholangioscopy is a method that provides direct visualization of biliary tree and has been used for diagnosis of malignant strictures [43]. The sensitivity and specificity of cholangioscopic-guided biopsies for detection of malignant strictures in patients without PSC is 92% and 93%, respectively [44]. The utility of this method in patients with PSC is challenging. In patients with PSC, there is fear of development of cholangitis after insertion of cholangioscope and the overall sensitivity/specificity of the method has been reported to be lower compared to those without PSC [45] Intraductal confocal endomicroscopy Probe-based confocal laser endomicroscopy (PCLE) is an in vivo examination of bile duct epithelium using microscopic images produced by a confocal miniprobe [46]. This method is based on the different distribution of fluorescein contrast in normal and malignant tissues after intravenous injection of

4 EXPERT REVIEW OF GASTROENTEROLOGY & HEPATOLOGY 3 the contrast media [47]. Criteria for normal bile ducts, inflammatory changes, and malignant transformations have been described (Paris criteria) [48]. The sensitivity and specificity of this method for characterization of intermediate biliary stricture have been reported to be 89% and 71%, respectively, compared to the index pathology [49]. PCLE has been used in PSC patients with dominant biliary stricture to look for neoplasia. Sensitivity, specificity positive predictive value (PPV), and negative predictive value (NPV) of this method for detection of neoplastic process were 100%, 61.1%, 22.2%, and 100%, respectively [50]. More studies are needed to further investigate the role of this method for diagnosis of CCA in patients with PSC Imaging techniques Cross-sectional liver imaging Ultrasound (US), computed tomography (CT) scan, and magnetic resonance imaging (MRI) all suffer some downfall in detection of CCA in patients with PSC; the prevalence of definitive signs of malignancy which can be detected by these methods is small which leads to low sensitivities, especially in those with early malignancies. The sensitivity of US, CT, and MRI, when only definitive signs of malignancy are present, considered are 10%, 25%, and 32%, respectively, which increase to 57%, 75%, and 63%, respectively, if any possible sign of malignancy is considered resulting in an overall PPV for US, CT, and MRI of 48%, 38%, and 40%, respectively, indicating a large number of false-positive results [51]. The low performance of cross-sectional imaging studies in CCA may be related to the varying growth patterns in CCA. CCA often grows longitudinally along the bile duct in an intraductal or periductal infiltrating manner, rather than radially away from the bile duct producing a visible mass. Presence of the perihilar lymphadenopathy has also been investigated as a mean of detection of CCA via cross-sectional studies in patients with PSC. Although multiple perihilar lymph nodes (LNs) are found more frequently in patients with CCA (58% versus 37%), neither the size nor the number of perihilar LNs allowed a definitive differentiation to be made between the presence and absence of CCA [52,53] FDG-PET F-18 flurodeoxyglucose (FDG) positron emission tomography (F-18 FDG-PET) has an established role in the diagnosis, staging, and follow-up of patients with a number of solid tumors [54]. F-18 FDG-PET has been used for detection of metastases from CCA; however, the accuracy of this method for the diagnosis of CCA in patients with PSC has not been established yet [55]. The sensitivity of this method for detection of malignant strictures in PSC patients has been reported to be as high as 90%. The specificity is lower and reported as 65% to 85% based on different standardized uptake values [55]. In a prospective study of patients with PSC and dominant strictures, Sangfelt et al. reported a sensitivity, specificity, PPV, and NPV of 89%, 92%, 62%, and 98% of F-18 FDG- PET for detection of malignant strictures [56]. In summary, the role of FDG- PET in diagnosis of CCA in patients with PSC (especially the infiltrative type in PSC) needs more clarification. In addition, false-negative (because of hypocellularity) and false-positive (cholangitis, stent) results can also occur. It does however, appear to have a role in the detection of occult metastasis from CCA if CT has been nondiagnostic [57] Endoscopic ultrasound (EUS) Another endoscopic method which can be used for diagnosis of CCA is EUS with fine needle aspiration (FNA). It should also be noted that EUS-FNA is generally used in patients with a suspicious lesion found with another modality of imaging and its ability in detection of early CCA is therefore uncertain. In a meta-analysis, the sensitivity of EUS-FNA for diagnosis of CCA was reported to be 66% with a pooled likelihood ratio of 0.34 [58]. The sensitivity, PPV, and NPV of EUS-guided biopsy of those patients with suspected hilar CCA were 79%, 100%, and 42%, respectively [59]. Since the peritoneal cavity is traversed in this method, the possibility of tumor seeding is raised [60]. Although it is suggested that FNA may not affect the diseasefree survival in CCA patients in this particular series, the median disease-free survival had been 17.8 months [61], and it is therefore unlikely that any effect of tumor seeding on survival will be shown. Other articles have suggested that trans-peritoneal biopsy of the CCA may lead to disease dissemination [62]. It should be noted that in this article, biopsies obtained by either EUS (transluminal) and trans-peritoneal biopsies were included. In conclusion, although EUS-FNA of the primary lesion has good sensitivity and can add to the diagnostic ability of the ERCP, seeding is a real concern and with improving long-term posttransplant survivals reported in PSC patients with early CCA, the risk of dissemination cannot be overlooked. On the other hand, EUS-FNA can be of great help in PSC patients with hilar lymphadenopathy. Up to 70% of the PSC patients may have hilar lymphadenopathy [63] and although most of these LNs are benign in nature, they can also represent metastatic disease originating in the bile duct [63,64]. Since the presence of metastatic malignancy in the hilar LNs essentially precludes the possibility of curative treatment, EUS- FNA can elucidate the nature of lymphadenopathy with no additional harms [65]. It should be noted that except for case reports and occasional case series, the diagnostic yield of EUS- FNA in this setting has not adequately studied yet [58] Magnetic resonance cholangiopancreatography (MRCP) MRCP is a good noninvasive imaging modality for the evaluation of biliary and pancreatic ducts. MRCP has been used for the detection of hilar CCA and extrahepatic CCA [66]. Hilar CCA usually appears as a hilar mass on MRCP and extrahepatic CCA usually appears as a diffuse stricturing of the bile ducts with or without mass formation [67]. While MRCP is useful for the diagnosis of PSC, it seems to be an inaccurate method to differentiate malignant from benign strictures in these patients [68] Magnetic resonance spectroscopy (MRS) MRS is a technique with the potential to detect small and early biochemical changes associated with diseases [69]. A single study compared patients with PSC alone, PSC with CCA, benign biliary lesions without PSC and CCA without PSC, using MRS. Bile of CCA patients differed from the benign

5 4 S. A. TAGHAVI ET AL. group by lower peaks corresponding to phosphatidylcholine and taurine, and it was possible to distinguish CCA from benign conditions. Using the four spectral regions and boots trap-derived coefficients, two spectra from benign non-psc patients were misclassified as malignant [70]. There was a good degree of similarity between the benign reference and the PSC spectra, whereas the CCA spectrum was different from the two benign conditions [70]. This resulted in an overall sensitivity of 88.9%, specificity of 87.1%, and an accuracy of 87.8% in distinguishing between CCA and non-cca cases [70] Conventional serum tumor markers Carbohydrate antigen (CA) 19-9 is one of the most commonly used serum markers for the diagnosis of CCA. This is a sialylated Lewis blood group antigen discovered in 1979 primarily as an antigen in a colorectal cancer cell line [71]. CA 19-9 has been used for the detection of CCA in patients with PSC. In one study, the optimal cutoff value for serum CA 19-9 was 20 U/mL, which yielded a sensitivity of 78% and specificity of 67%, PPV of 23%, and NPV of 96% [31]. Increasing the cutoff value to 125, while increasing the PPV to 100%, decreases the sensitivity to 13% and at the same time prevents the detection of most early (and therefore treatable) CCA. The area under curve of 0.79% in the ROC curve for this marker shows a modest power for this marker as a screening test [72]. But even with cutoff values as high as 129 U/mL, 37% of those with positive values do not have CCA [72] and values of up to 10,000 U/mL have been reported in PSC patients with benign strictures with a dramatic drop to normal after endoscopic treatment [73]. The level of CA 19-9 seems to correlate with the stage of the disease [74], which raises questions about its suitability as a screening tool for early CCA. When used as a predictive tool in 75 patients with PSC under surveillance, the serum CA 19-9 level only identified, only one in four patients who developed [75]. The performance of CA 19-9 in PSC patients awaiting liver transplantation is less favorable; using a CA 19-9 threshold of two times the upper normal limit provides an overall sensitivity of 50%, specificity of 54.5%, and PPV of 16.6% [76]. It should be noted that levels of CA 19-9 can rise during episodes of intervening acute cholangitis [77]. It is also noteworthy to know that patients with negative Lewis antigen, representing 7% of the general population, are negative for CA 19-9 irrespective of disease status. CEA is a glycoprotein tumor marker which is used as a tumor marker in gastric, colon, and pancreatic cancers [78]. CEA has a lower diagnostic yield than CA 19-9 in CCA. Serum CEA levels greater than 5 ng/ml have a sensitivity of 33 68% and specificity of 82 89%. Combined or serial testing of CA 19-9 and CEA might increase sensitivity, but with a resultant increase in falsepositive results. The usefulness of CEA for prediction of prognosis of patients with CCA is also controversial. Interleukin-6 (IL-6) is a human bile duct epithelium growth factor. A cutoff level of 25.8 pg/ml resulted in sensitivity of 73%, specificity of 92%, PPV of 87%, and NPV of 87% in CCA patients compared with HCC and healthy controls [79]. Serum levels correlated with tumor thickness (as assessed by intraductal ultrasonography) and decreased significantly following photodynamic therapy. At present, there is no evidence showing that measurement of tumor markers is useful for monitoring development of CCA in PSC. 3. Translational/future diagnostic methods 3.1. Proteomics Background The proteome was originally used to describe the protein complement expressed by a tissue or cell type, but it can also be applied to biological fluids and subcellular compartments. The proteome is a complex mixture of proteins from products of different genes, as well as genetic and epigenetic phenomena which may result in the production of different proteins from the same gene [80]. In a proteomics-based approach for biomarker discovery, samples are selected to address the clinical question, proteins are separated by use of specific complementary techniques, data are analyzed to identify potential differences in expression of proteins or biomarkers, suitable assays are developed, results are validated, and ultimately large-scale clinical trials are done [80]. Bile is a complex biologic fluid which contains up to 2552 proteins [81]. Bile is produced in the liver and stored in the gallbladder. Pathological changes in the biliary system can therefore be reflected in the bile composition. It can therefore serve as a source of biomarkers for CCA diagnosis. The sources of proteins in bile include plasma, hepatocytes, and cholangiocytes lining the biliary tree. Proteins with diverse functions have been reported in bile including multiple proteins with possible links to malignant conditions [76]. Recent improvements in proteomic technologies have made the analysis of complex biological matrices possible. Bile, however, is one of the less studied body fluids mainly because of the high concentrations of interfering substances hampering a comprehensive analysis [82]. Myristoylated alanine-rich C kinase substrate (MARCKS), EPCAM (a trans-membrane glycoprotein), and carcinoembryonic antigen-related cellular adhesion molecule 6 (CEACAM6) (all associated with CCA) were identified in an analysis of the bile proteome [81] Proteomic studies on CCA Tissue studies and cell lines. Investigative proteomic studies identified actinin-1, actinin-4, protein DJ-1, and cathepsin B [83], as well as Golgi membrane protein 1 or GP73 (GOLM1) [84] as potential biomarkers in CCA tissues. A proteomic study on secretory product of CCA and hepatocellular carcinoma cell lines identified lipocalin 2 as a potential biomarker for CCA [85] Biological fluids. Although bile as a proximal fluid should be a potential source of markers for CCA, only a few proteomic studies have been done on human bile [86,87]. A small study on four samples from patients with CCA was able to identify 378 proteins and 741 products of 813 unique genes [87]. Another study on patients with biliary strictures of different etiologies identified overexpression of CEACAM6 and membrane-bound mucin-1 (MUC1) (CA 19-9) in bile samples of patients with pancreatic cancer and CCA [88]. Neutrophils gelatinase-associated lipocalin was identified in another

6 EXPERT REVIEW OF GASTROENTEROLOGY & HEPATOLOGY 5 study as a potential marker for malignant pancreatobiliary diseases [89]. CEACAM1, CA125, and MUC2 are cell-derived proteins that have also been found in bile of patients with CCA. Specificity and sensitivity of 78% and 84%, respectively, in detection of CCA are reported in one of the studies. It is of note that 8 out of 10 samples of patients with CCA complicating PSC were identified [86]. In a very recent proteomic study, serum extracellular vesicles obtained from cholangiocytes of patients with CCA had higher levels of oncogenic proteins than normal cholangiocytes [90]. Recently, there are increasing interests in using micrornas for diagnosis of different diseases including autoimmune diseases and cancer. It has been suggested that PSC patients with CCA have different micrornas in serum and bile compared to PSC patients without CCA [91]. Another recent study showed that expression of microrna- 222 and micrornas-483-5p in serum are different among patients with PSC and patients with CCA [92]. Further refinement and expansion of such approaches should make discovery of novel biomarkers for CCA a real possibility. Lastly, it is the evolving role of liver transplantation in CCA patients with excellent outcomes [93]. The role of liver transplantation is highlighted especially among CCA in the setting of PSC. It has been suggested that more than 60% of patients with PSC have foci of bile duct dysplasia [94]. Wu et al. reported good disease-free survival after liver transplantation for patients with PSC and CCA [95]. Liver transplantation with chemoradiotherapy has been reported for treatment of hilar CCA [9]. Therefore, liver transplantation can be considered for PSC patients with CCA. These studies swing between high specificity low detection rates and high sensitivity high false-positive rates states depending on the cutoff, which appears to be inherent to the nature of the tumor and abilities of the imaging modalities. 4. Diagnostic approach to PSC patient with suspected CCA The summary of modalities of diagnosis is outlined in Tables 1 and 2. A reasonable algorithm for a diagnostic approach to PSC patients with suspected CCA is depicted in Figure 1. The diagnostic approach is different based on the site of the suspected lesion (intrahepatic versus perihilar and distal extrahepatic). The initial clinical presentation and radiographic findings are clues to the location of the lesion. Although the primary imaging modality is usually trans-abdominal US, additional imaging studies are usually necessary for definite diagnosis and guiding future management. 5. Expert commentary CCA is the most dreadful malignant complication of PSC because of its poor prognosis. CCA in patients with PSC is occurred up to 20 years earlier than those without PSC and in most cases diagnosis of CCA is made in less than years after diagnosis of PSC. CCA will have significant negative impact on survival of PSC patients depending on the location of the malignant transformation. Surgical resection is disappointing since most of patients are not eligible for surgery at the time of diagnosis. The concept of existence of a dysplasia carcinoma sequence in CCA by analogy to the same process in colorectal cancer can indicate the possibility of early diagnosis of malignancy or premalignant lesions if the suitable tests will be available. The distinction between malignant and inflammatory strictures is confounded in PSC because the inflammation associated with PSC complicates cytological assessment, and access to the bile duct is limited for cytological and tissue acquisition. Dominant strictures, which may be a harbinger of malignancy, are rather common and are more often benign than malignant. Demonstration of a mass lesion with typical imaging features is the only observation that has a high sensitivity and specificity for the diagnosis of CCA. However, mass lesions are unusual in early-stage CCA and are not even that frequent in advanced stages. Incidental CCAs are discovered in 9 21% of liver explants in patients undergoing liver transplantation because of the PSC. In fact, more hepatobiliary tumors are detected in the liver explants from patients with PSC than those were detected during pretransplant evaluation. These issues altogether demonstrate the shortfalls of the current methods in identification of this malignancy especially in early stages. As the success rates of surgical resection and liver transplantation for locally advanced CCA improve, the need to diagnose early CCA becomes more imperative. An effective surveillance regimen to detect CCA at a treatable stage in patients with PSC remains highly sought since no clinically proven modality has been identified. Recent improvements in proteomic technologies made the analysis of complex biological matrices possible. Therefore, an interesting area of research is to analyze serum and bile proteomes in patients with PSC and CCA. 6. Five-year viewpoint Progress in diagnosis of CCA developing in the setting of PSC will most likely be made in three frontiers: 1. novel Serum, urine, and to a lesser degree, bile biomarkers: these potential markers are the easiest to obtain and therefore have huge potential implications for use as screening tools in this patient population. A few of these are already under investigation (circulating angiopoeitin-2, cytokeratin-19 fragments, and antiglycoprotein 2 IgA autoantibodies, microrna) and others most likely will be added through conventional marker discovery methods and newer methods as proteomicsbased research. 2. New approaches to enhance the accuracy of brush cytology tissue sampling such as optimization of fluorescence in situ hybridization(fish) probes and the assessment of genetic aberrations in the obtained samples will also be of great value, particularly for confirmation of diagnosis in those with a positive result on a screening test. 3. Advanced endoscopy-based techniques such as spyglass cholangioscopy and confocal laser endomicroscopy have shown promising results with respect to CCA detection. These techniques, however, will most likely be best suited for those with unclear diagnosis after the initial imaging and tests considering the difficulty and inherent risks of biliary endoscopy in patients with PSC.

7 6 S. A. TAGHAVI ET AL. Table 1. Cholangiographic, tissue sampling, imaging methods, and serum markers in diagnosis of CCA. Endoscopic methods Methods Advantages Disadvantages ERCP 17 The accepted gold standard for delineation of the bile ducts - Definitive cholangiographic findings of CCA are relatively rare - Limited by complications - Inability to visualize the ducts proximal to high-grade stenosis Brush cytology Very high specificity (97 100%) 13,18,21 - Sensitivity is less favorable (50 75%) 13,18,22 - Addition of DNA analysis and serum markers (CA 19-9, CEA) is of promise but needs further studies 23,24 FISH Detects an additional 14% of CCA in patients with PSC and normal - FISH polysomy increases the sensitivity of cytology but may decrease the specificity cytology 19,35,36 - Cannot be recommended as a screening test but rather a confirmatory test Endobiliary forceps biopsy Sensitivity may exceed 80% Combination of brush cytology and forceps biopsy is more sensitive Endoscopic ultrasound, cholangioscopy, and intraductal confocal microscopy (IDCM) 44,46,59,60 than either alone 42 Favorable sensitivity and specificity rates more than routine cholangiography and brushing were reported in limited studies available It is not possible to obtain an adequate tissue sample in a significant number of patients - Technically challenging - Available in limited centers - Generally used for suspicious lesions found in another modality rather than screening/early diagnosis purposes Noninvasive imaging techniques MRCP Noninvasive - Performance is rather dismal - Sensitivities of 11 14% if only the definitive signs of malignancy considered positive - Low PPVs 67 of 21 23% if all findings (possible or definitive) considered positive Cross-sectional liver Imaging (US, CT, and Noninvasive and easily available The prevalence of definitive signs of malignancy is small which leads to low sensitivities, especially in MRI) 51,52 FDG-PET scan 13,54,55 May be useful in diagnosis of malignancy in non-psc patients with biliary strictures, Serum markers CA Most investigated - Noninvasive - Easily available those with early malignancies The low performance of sectional imaging studies in CCA may be related to its longitudinal growth pattern along the bile duct rather than radially away from it, with less chance of producing a visible mass Low sensitivity (attributed to hypocellularity of the tumors) and low specificity (attributed to the inflammation) in PSC strictures - Levels can rise during episodes of acute cholangitis - Patients with negative Lewis antigen (7% of the general population) are also negative for CA Levels seem to correlate with the stage of the disease, questioning its use as a screening tool for early CCA - The raise in CA 19-9 can be a function of stricture itself rather than CCA; extremely high values are reported in PSC patients with benign strictures with dramatic drop to normal after endoscopic treatment - General performance of this test as a screening tool in differentiation of benign versus malignant strictures is modest (AOC for ROC curve = 0.79) - When used as a predictive tool, only 25% of CCA patients could be identified over 8 years - Poor performance in pretransplant PSC patients Carcinoembryonic antigen (CEA) 78 Noninvasive, easily available Sensitivity of 33 68% and specificity of 82 89% (cutoff > 5 ng/ml), lower than CA 19-9 for CCA Investigational markers 2,4,79 Promising early results in a limited number of subjects New markers, further investigation are needed before their use can be suggested IL-6

8 EXPERT REVIEW OF GASTROENTEROLOGY & HEPATOLOGY 7 Table 2. Combining diagnostic methods in diagnosis of CCA. Methods Characteristics Comments MRCP plus CA Sensitivity = 100, Specificity = 38 All associated with variable increase in sensitivity but decreased specificity and PPV CT scan plus CA Sensitivity = 100, Specificity = 38 Ultrasound plus CA Sensitivity = 91, Specificity = 62 CA 19-9 and CEA or serial testing for Might increase sensitivity tumor markers 31 Combination of US, CT, or ERCP 12 May miss CCA in 31% of the patients Figure 1. Algorithm for diagnostic approach to primary sclerosing cholangitis patient with suspected cholangiocarcinoma a)suspected cholangiocarcinma (CCA) is defined as presence of any of the following: rapid clinical deterioration with jaundice, weight loss, and abdominal discomfort. b)suggestive CCA: Imaging findings suggestive of extrahepatic CCA (intrahepatic versus perihilar and distal extrahepatic) including mass lesion, vascular encasement, thickening of the bile duct) and/or CA 19-9 > 129 u/ml. c)dominant stricture is a stenosis with a diameter of 1.5 mm in the common bile duct or 1 mm in the hepatic ducts. Strictures can occur at the biliary hilum or anywhere along the common hepatic or common bile ducts. d)ercp with brushings for cytology (including FISH studies, cholangiospcoy, IDUS where available). ERCP findings suggestive of CCA includs polypoid lesions, new onset or progressive stricture formation. e)pet scan may be performed if the clinical suspicion for a CCA is high. Although the role of PET in the diagnosis of CCA in PSC remains controversial. Key issues Although PSC patients may progress to liver cirrhosis; CCA most commonly occurs in ages between 30 and 45 years when cirrhosis has not yet developed. CCA in patients with PSC is occurred up to 20 years earlier than those without PSC. The so called dominant stricture, regarded as a possible indicator of development of malignancy in PSC is rather common and is more often benign rather malignant. An effective surveillance regimen to detect CCA at a treatable stage in patients with PSC remains highly sought since no clinically proven modality has been identified. Bile duct brush cytology, fluorescence in situ hybridization (FISH), probe based confocal laser endomicroscopy and cholangiospcoy with random biopsies are ERCP-based methods applied for diagnosis of CCA in PSC patients. Cholangioscpy is a method that provides direct visualization of biliary tree and has been used for diagnosis of malignant strictures with a rather good sensitivity and specificity for diagnosis of CCA in PSC patients. Progress in diagnosis of CCA in the setting of PSC will most likely be made with novel serum, urine and bile biomarkers, new approaches to enhance the accuracy of brush cytology tissue sampling such as optimization of fluorescence in situ hybridization (FISH) probes and the assessment of genetic aberrations in the obtained samples and advanced endoscopy-based techniques such as spyglass cholangioscopy and confocal laser endomicroscopy. Funding This article was not funded. Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the

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