Prerpints.org logo
Preprint
Review

Recent Advancement in Diagnosis of Biliary Tract Cancer through Pathological and Molecular Classifications

Altmetrics

Downloads

159

Views

52

Comments

0

A peer-reviewed article of this preprint also exists.

This version is not peer-reviewed

Submitted:

23 February 2024

Posted:

23 February 2024

You are already at the latest version

Alerts
Abstract
Biliary tract cancers, including intrahepatic, perihilar, and distal cholangiocarcinomas, as well as gallbladder cancer, are a heterogeneous group of cancers with distinct molecular characteristics in each anatomic and pathological subtype. The pathological classification of BTC includes grossly classifiable growth patterns, such as mass-forming, periductal infiltrating, and intraductal growing. The small duct and large duct types of intrahepatic cholangiocarcinoma have been recently introduced in the WHO classification. Typical clinical presentations and various radiological, endoscopic, and molecular modalities for diagnosis are described in detail. To overcome the limitations of traditional tissue acquisition, new diagnostic modalities are being investigated. In fact, the treatment landscape is evolving rapidly owing to the emergence of distinct subgroups harboring unique molecular alterations with corresponding targeted therapies. In addition, we highlight the key points in the diagnostic process in real-world clinical practice for biliary tract cancer.
Keywords: 
Subject: Medicine and Pharmacology  -   Gastroenterology and Hepatology

1. Introduction

Biliary tract cancer (BTC) encompasses a range of invasive adenocarcinomas, including cholangiocarcinomas (arising in the intrahepatic, perihilar, or distal biliary tree), and gallbladder cancers. Cholangiocarcinomas arising from the bile ducts proximal to the second-order ducts are classified as intrahepatic cholangiocarcinoma(iCCA), those originating between the second-order ducts and the insertion of the cystic duct are termed perihilar cholangiocarcinoma (pCCA; previously referred to as Klatskin tumors), and those arising from the bile ducts distal to the insertion of the cystic duct are termed distal cholangiocarcinoma (dCCA). Extrahepatic cholangiocarcinoma refers to pCCA and dCCA collectively [1]. Gallbladder cancer (GBC) originates either from the gallbladder itself or from the cystic duct.
BTCs exhibit heterogeneous clinical manifestations, molecular characteristics, and biological behaviors, depending on their anatomical, pathological, and molecular classifications. In recent years, increasing genomic research has begun to uncover the molecular underpinnings of BTC and offer many potential treatments, ushering in a new era in precision medicine. However, in addition to understanding the clinicopathologic development of each BTC subtype, there must be an individualized assessment of each subtype and an effort to overcome clinical diagnostic hurdles. Here, we review the current imaging and histologic diagnosis of BTC, along with future perspectives of molecular diagnosis to approach precision medicine in BTC.

2. Pathologic Classification

2.1. Pathoologic Classification and Precancerous Leions of Cholangiocarcinoma

iCCA is grossly classifiable in three growth patterns (Table 1): mass-forming (78% of cases) presents as a mass lesion within the hepatic parenchyma; periductal infiltrating (16% of cases) characterized by infiltration along the bile ducts and portal tracts, causing strictures and thickening of the affected bile ducts and dilatation of the peripheral bile ducts; intraductal growing (6% of cases) consists of a polypoid or papillary tumor within the dilated bile ducts [2]. pCCA and dCCA have similar growth patterns: flat or nodular sclerosing type (73% of cases, corresponding to features of periductal infiltrating) and intraductal papillary type (27% of cases) [3].
Conventional iCCA can be further categorized into two main histological types based on the level or size of the affected duct. Small duct type and large duct type iCCA have been recently introduced in the WHO classification [4]. Small duct-type iCCA (36-84% of cases) is composed of small-sized tubular growth of cuboidal or low-columnar tumor epithelial cells with little or no mucin production. Large duct-type iCCA (8-60% of cases) is characterized by mucin-producing columnar cells forming irregularly shaped and sized tubules or gland-like structures, which are usually accompanied by a highly invasive growth pattern and a desmoplastic reaction [4,5,6].
Different carcinogenesis and progression pathways may give rise to different morphological subtypes of cholangiocarcinoma. For example, canals of Hering and interlobular bile ducts can represent the cell-of-origin for small duct-type iCCA, which develops into a mass-forming pattern in the background of chronic hepatitis and cirrhosis [7], whereas the peribiliary glands are likely the origin of large duct-type iCCA, pCCA, and dCCA, which lead to periductal-infiltrating lesions related to biliary inflammation, such as hepatolithiasis, parasite infection in bile ducts, or primary sclerosing cholangitis (PSC) [8]. Notably, the intraductal growing pattern represents a distinct pathway from large bile ducts and is often associated with a more favorable prognosis.
The 2010 WHO classification proposed three types of precancerous lesions of the biliary tract: flat type (biliary intraepithelial neoplasia, BilIN), papillary type (intraductal papillary neoplasm of the bile duct, IPNB), and cystic type (mucinous cystic neoplasm, MCN). Recently, intraductal tubular neoplasm of the bile duct (ITNB) was proposed as another candidate for preneoplastic lesions; however, its advanced form remains unclear [9]. IPNB may be associated with intraductal growing type of iCCA and intraductal papillary type of pCCA and dCCA, whereas BilIN may be followed by a periductal infiltrating (iCCA) and a flat or nodular sclerosing (pCCA and dCCA) pattern [10]. MCN can result in MCN associated with an invasive carcinoma that may progress to a cystic lesion with a grossly surrounding nodular lesion. No precursor lesions have yet been defined for mass-forming iCCA [3].

2.2. Pathologic Classification and Precancerous Leions of Gallbladder Cancer

Adenocarcinoma is the main histological classification of GBC (approximately 90% of cases) [11]. Grossly, GBC can exhibit infiltrative, nodular, papillary, or a combination of these morphologies. In addition, there are three premalignant lesions of gallbladder adenocarcinoma: adenoma, BilIN, and intracystic papillary neoplasm (ICPN). BilIN is invisible on gross inspection, but can be microscopically identified around invasive tumors or chronic cholecystitis. ICPN is grossly identified as an exophytic polypoid mass or diffuse friable thickening of the mucosa and is composed of mucinous epithelial cells with papillary and tubular arrangements [12]. Dysplasia of the BilIN and ICPN is generally categorized using a three-tiered system, and high-grade dysplasia is placed in the same group as carcinoma in situ. The current definitions of Adenoma and ICPN are unclear and require revised diagnostic criteria to ensure consistency and accuracy of diagnoses.

3. Clinical Presentation

The presence and features of clinical symptoms depend on the anatomical location of the primary tumor and associated metastasis. Patients with extrahepatic cholangiocarcinoma typically become symptomatic when biliary obstruction caused by the disease results in jaundice. Patients with iCCA are less likely to experience jaundice and instead exhibit non-specific symptoms, such as dull right upper quadrant pain or unexplained weight loss. Approximately 20-25% of patients are asymptomatic, with the lesions detected incidentally [13]. Patients with early GBC are also usually asymptomatic and are often diagnosed incidentally through preoperative imaging studies or intra- or post-operative examinations.
Laboratory studies are generally non-diagnostic; elevated alkaline phosphatase or serum bilirubin levels may be related to biliary obstruction. Serum tumor markers such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) are frequently elevated, but do not provide diagnostically useful results due to their lack of specificity and sensitivity [14,15,16]. However, in the presence of an established diagnosis, these tumor markers can provide information on the response to treatment in addition to prognostic information.
Many patients initially undergo transabdominal ultrasonography to assess the biliary tree, and the results may aid in identifying the location of the lesion: an abrupt change in extrahepatic duct diameter with intrahepatic and extrahepatic biliary dilatation (dCCA case), intrahepatic ductal dilatation with normal-caliber extrahepatic ducts (pCCA case); mass lesions, occasionally in a non-cirrhotic liver, without radiographic characteristics of hepatocellular carcinoma (HCC) (iCCA case); or a protruding mass in the gallbladder, which sometimes extends directly into the liver bed (GBC case). Although BTC is frequently suspected based on ultrasound findings, it is crucial to conduct additional imaging studies, such as computed tomography (CT), magnetic resonance imaging (MRI)/ magnetic resonance cholangiopancreatography (MRCP), endoscopic retrograde cholangiopancreatography (ERCP), endoscopic ultrasound (EUS), and positron emission tomography (PET)-CT to verify the diagnosis and treatment plan. Imaging studies aim to rule out benign tumors or gallstones as potential causes, establish the extent and location of the primary tumor, and detect whether metastases have developed.

4. Diagnostic Tool

4.1. CT and MRI

Computed tomography (CT) is the standard modality for the diagnosis and staging of BTC. It provides a comprehensive evaluation of the primary tumor, its relationship with adjacent structures (specifically portal vein and hepatic artery involvement, determining resectability), and potential thoracic and abdominal spread [17]. MRI has a similar accuracy to CT for diagnosis and staging, but it offers specific sequences such as diffusion-weighted imaging (DWI) and the potential for performing MRCP, which is essential for pCCA staging [18].

4.1.1. Radiologic Findings of Mass-Forming Cholangiocarcinoma

The most common imaging pattern of mass-forming iCCA on both CT and MRI is arterial peripheral rim enhancement with centripetal progressive homogeneous contrast agent uptake until the delayed phase or stable uptake during the late dynamic phases [18]. In hepatobiliary phase of gadoxetic acid-enhanced MRI, most mass-forming iCCA exhibits ‘EOB-cloud’ described as cloud-like mild central hyperintensity and peripheral hypointensity in the tumor [19]. However, as no specific radiological pattern exists, histopathological or cytological results are mandatory to confirm the diagnosis.
The main differential diagnoses for mass-forming iCCA include HCC, metastatic adenocarcinoma, inflammatory pseudotumors, and angiosarcoma. HCC, the most common primary hepatic malignancy, should be differentiated due on different prognoses and treatments. Early arterial enhancement and washout of contrast are the main patterns in favor of HCC, and capsular retraction and peripheral bile duct dilatation are suggestive of iCCA. The target sign on DWI, defined as central hypointensity and a peripheral hyperintense rim, helps in the recognition of iCCA from HCC [20]. However, scirrhous HCC may closely resemble mass-forming iCCA on imaging, making it difficult to differentiate them and necessary for tissue diagnosis. Metastatic adenocarcinoma can show many typical findings of iCCA, including central hypointensity or intrahepatic bile duct dilatation. It can also be difficult to differentiate based on histopathology and requires special immunohistochemical studies [21]. Therefore, when approaching a suspected mass-forming iCCA, it is essential to exclude extrahepatic primary malignancies, especially colorectal cancer.

4.1.2. Radiologic Findings of Periductal-Infiltrating Cholangiocarcinoma

This is the most common growth pattern in pCCA and dCCA, which presents as a narrowed biliary duct with irregular circumferential wall thickening (usually ≥ 5 mm) and upstream biliary tree dilatation. These tumors slowly enhance to a peak in the delayed phase; however, they are rarely hypervascular and enhance in the arterial phase [22]. When infiltration is nodular, the bile ducts appear protuberant, whereas they appear narrowed and stretched when infiltrated diffusely [23].
PSC, Mirizzi syndrome, portal biliopathy, IgG4-related sclerosing cholangitis, benign idiopathic stricture, and hepatobiliary sarcoidosis or lymphoma can mimic periductal infiltrating cholangiocarcinoma. PSC typically presents as multiple intra- and extrahepatic biliary strictures with a beaded appearance on MRCP. These strictures are often short, with only mild dilatation of the intervening segments observed [24]. On MRCP of Mirizzi syndrome, an abrupt stricture of the common hepatic duct, along with a normal common bile duct, is evident, with an impacted gallstone located in the neck of the distended gallbladder [25]. Portal biliopathy refers to narrowing of the extrahepatic biliary tract secondary to extrahepatic portal venous obstruction, which results from the compressive effect of portal vein collaterals or ischemic damage. Imaging studies revealed a circumferential, long, and smooth stricture of the common bile duct accompanied by the presence of collaterals and choledochal varices. [26]. IgG4-related sclerosing cholangitis is a persistent inflammatory disorder of the biliary system that is most commonly associated with other manifestations of IgG4-related disease. IgG4-related sclerosing cholangitis shows circumferential symmetric wall thickening of the bile ducts, frequently involving the extrahepatic segments, with smooth outer and inner margins, visible lumen in the thickened segments, and delayed homogenous contrast enhancement [27]. Nine out of ten cases exhibit pancreatic involvement, which typically presents with diffuse or focal pancreatic enlargement, a peripheral capsule-like rim, and a pancreatic duct stricture [28]. The diagnosis of IgG4-related sclerosing cholangitis is established by a combination of characteristic imaging features, serum IgG4 antibody levels, histologic findings, and response to steroid therapy [29].

4.1.3. Radiologic Findings of Intraductal-Growing Cholangiocarcinoma

The intraductal type of cholangiocarcinoma has been reported in 8-18% of all types of cholangiocarcinoma [18]. These tumors present as polypoid or sessile masses confined within the bile duct with proximal ductal dilatation due to occlusion or excessive mucin production. These lesions exhibit imaging characteristics similar to those of mass-forming types, displaying a heterogeneous enhancement that begins early and reaches its peak in the delayed phase. A markedly dilated intrahepatic bile duct segment may mimic a cystic mass such as cystadenoma, cystadenocarcinoma, or liver abscess [30].

4.1.4. Radiologic Findings of Gallbladder Cancer

GBC can appear as a polypoid mass protruding into the lumen or completely filling it, focal or diffuse wall thickening, or as a large mass in the gallbladder fossa with an unidentifiable gallbladder [31]. The presence of features such as an increased frequency of lymph node enlargement, more extensive wall thickness, focal irregularity in wall thickness, and less gallbladder distention are indicative of GBC complicated by cholecystitis rather than simple cholecystitis [32].

4.2. PET-CT

PET-CT can be used to complement CT and MRI to provide additional information about lymph node involvement, the presence of distant metastasis, and postoperative recurrence. In fact, preoperative PET scanning leads to a change in surgical management in approximately one-fourth of cases, mainly by detecting occult distant metastases. However, because of its low specificity, it is not sufficient for the diagnosis of primary lesions, and cytological or histological confirmation is still necessary [33].

4.2. EUS

EUS can visualize the local extent of the primary tumor and the status of the regional lymph nodes, particularly in cases where dCCA lesions are suspected. EUS-guided fine needle aspiration (FNA) of tumors and enlarged lymph nodes can also be performed. EUS-FNA has a higher sensitivity for detecting malignancies in distal tumors than ERCP with brushings [34]. EUS is less effective in imaging and staging proximal bile duct lesions than distal lesions, and clinical experience with this technique is limited [35].
EUS is also considered a useful modality both in the detection and differential diagnosis of gallbladder polyps and in staging early GBC. In particular, EUS is helpful in assessing the depth of tumor invasion in the gallbladder wall [36,37] and in defining lymph node involvement in the portal hepatis or peripancreatic regions). Although some authors reported accurate and safe results of EUS-FNA for GB wall lesions [38], this procedure poses a potential risk of bile leakage after gallbladder biopsy.

4.3. ERCP or Percutaneous Tranhepatic Cholangiography (PTC)

Preoperative cholangiography performed using ERCP or PTC may be indicated either diagnostically or therapeutically for patients with biliary obstruction. Recently, MRCP or CT scanning, which is non-invasive and highly accurate, has largely replaced invasive cholangiography for diagnostic purposes.

4.3.1. Intraductal Ultrasound (IDUS)

IDUS uses a small wire-guided ultrasound catheter that provides high-resolution images, enabling precise evaluation of the biliary tract during ERCP. IDUS is useful for characterizing malignant biliary strictures and for the local staging of cholangiocarcinoma. It can detect early lesions in the biliary tree, determine the longitudinal tumor extent, and identify tumor extension into adjacent organs (e.g., pancreas) and major vessels (e.g., portal vein and hepatic artery) [39,40,41]. In contrast to EUS, IDUS is often better able to evaluate the proximal biliary system and surrounding structures such as the right hepatic artery, portal vein, and hepatoduodenal ligament. However, IDUS limits the evaluation of more distant tissues or lymph nodes and cannot be used to perform FNA.

4.3.2. Peroral Cholangioscopy (POC)

POC (direct visualization of the bile ducts using a very thin cholangioscope during ERCP) can be used to evaluate indeterminate biliary strictures (e.g., strictures that cannot be diagnosed as benign or malignant using sampling techniques such as brush cytology or biopsy) by targeted biopsies of bile duct lesions [42,43,44,45]. It can also be used to investigate equivocal fluoroscopy findings during ERCP, assess the extent of cholangiocarcinoma prior to surgery, and identify invisible stones by conventional cholangiography. "Tumor vessels" may be observable during POC in patients with cholangiocarcinoma, which are characterized by irregularly dilated and tortuous blood vessels. Other characteristic findings suggesting malignancy include nodules or masses, infiltrative or ulcerative strictures, and papillary or villous mucosal projections [46]. A recent study reported 100% sensitivity and 89.5% specificity for visual impression during POC [47].

4.3.3. Tissue Acquisition

ERCP or PTC-guided biopsies or brush cytology are the traditional standard methods for the tissue diagnosis of periductal infiltrating or intraductal growing cholangiocarcinoma. Although brush cytology has high specificity, its low sensitivity is a major limitation (e.g., 97% specificity and 43% sensitivity for detecting cholangiocarcinoma in patients with PSC) [48]. The addition of endoscopic biopsy of malignant strictures increases the diagnostic accuracy to only 43–88 percent [49,50,51]. These tests may be useful in diagnostic evaluation if they are positive, but cannot rule out malignancy if they are negative.
Fluorescence in situ hybridization (FISH) is a cytological test using labeled deoxyribonucleic acid (DNA) probes to detect abnormal loss or gain of chromosomes or chromosomal loci on cells routinely collected by the brush technique, which may also improve the sensitivity of brush cytology [52]. A meta-analysis of FISH has demonstrated that this is highly specific (pooled 70%), but with limited sensitivity (68%) for the diagnosis of cholangiocarcinoma in patients with PSC [53].
Another auxiliary technique for improving the diagnostic ability of bile cytology is the implementation of a new scoring system for evaluating cytologic results. Hayakawa et al. proposed a scoring system based on four cytological findings, including abnormal chromatin, irregularly arranged nuclei, irregularly overlapped nuclei, and irregular cluster margins. The scoring system yielded an area under the receiver operating characteristic (ROC) curve (AUC) of 0.981 with a sensitivity of 87% and specificity of 98% [54]. Another study reported that the diagnostic sensitivity of bile cytology increased from 31.6% to 80.3% after combined p53 immunostaining [55].
Recently, various emerging analytical methods for extracellular vesicles (EVs), nucleic acids, proteins, and metabolites in bile have been developed as potential biomarkers for BTC diagnosis [56]. For example, circular RNA (Circ-CCAC1) in serum-derived or bile-derived EVs has a diagnostic role, with an AUC of 0.857 [57]. In a prospective study of bile samples, K-ras mutations detected in bile cell-free DNA indicated the possibility of cholangiocarcinoma in high-risk lesions such as PSC [58]. A study screened four DNA methylation biomarkers (COD1, CNRIP1, SEPT9, and VIM) based on DNA methylation analysis of ERCP brush samples and achieved 85% sensitivity and 98% specificity with an AUC of 0.944 [59]. Based on this study, the role of a four-gene methylation panel in bile was investigated to predict early diagnosis of BTC in patients with PSC. The results showed that the AUC for predicting the diagnosis of cholangiocarcinoma in PSC patients within one year was between 0.84 and 0.98, with a sensitivity of 67%–96% and a specificity of 93%–98% [60]. sB7-H3, a cancer-related immune protein, is upregulated in the bile of patients with malignant biliary obstruction including BTC and pancreatic cancer. The ROC-AUC for the diagnosis of malignant biliary obstruction was 0.878, and the sensitivity and specificity were 81.2% and 81.6%, respectively [61]. Another study revealed the potential of bile multi-omics analysis, including metabolomics, for molecular diagnosis of GBC, which combined lipidomics and metagenomics in bile to characterize microbial and lipid alterations associated with the development of GBC. Based on the random forest classifier model, this study established a diagnostic model containing eight lipid substances that can effectively be used to diagnose GBC from gallstones or healthy groups with an AUC of 1 [62]. Table 2 summarizes previous studies on bile EVs, nucleic acids, and protein detection for the diagnosis of BTC (Table 2).

5. Pathologic and Molecular Diagnosis

5.1. Pathologic Diagnosis

Pathologic diagnosis can be assessed using a variety of methods in patients suspected to have BTC (ERCP or PTC-guided biopsy or brush cytology, EUS-FNA, ultrasonography/CT/MRI-guided biopsy), but obtaining tissue may be difficult, especially in patients with perihilar lesions. In cases of potentially resectable tumors with typical findings of malignant biliary obstruction, solitary intrahepatic mass, or early GBC confined to the gallbladder, surgery can be performed without preoperative pathologic diagnosis. In patients with biliary obstruction due to pCCA and dCCA without extraductal metastasis, ERCP or PTC-guided biopsies, or brush cytology are preferred and should be conducted to ensure adequate tissue for pathological diagnosis and molecular profiling. EUS-FNA may be an alternative method to obtain biopsies of regional lymph nodes (if enlarged) or distally located tumors and may be considered if ERCP or PTC-guided biopsies are negative or inconclusive. In addition, EUS-FNA or ultrasonography/CT/MRI-guided biopsy via the transperitoneal approach rarely results in seeding of the biopsy tract with tumor cells [106]. Therefore, it is necessary to establish tissue diagnosis prior to surgery in a multidisciplinary setting.
Pathological diagnosis is important in the following situations: clinically indeterminate strictures, patients requiring diagnostic documentation before nonsurgical treatment, or situations where a physician or patient is reluctant to undergo surgery without a tissue diagnosis [107]. Conversely, tissue diagnosis is not mandatory for unresectable patients who are scheduled to receive only palliative management, such as biliary drainage.

5.2. Molecular Diagnosis

Molecular profiling is recommended for advanced diseases and is considered suitable for systemic treatments [108]. Small duct-type iCCA are enriched for actionable targets, such as IDH 1/2 mutations (15%–20%) and FGFR-2 fusions (10%-20%). Large duct-type iCCA tumorigenesis frequently involves KRAS (15%–30%) and TP53 mutations (10%–40%). GBC, pCCA, and dCCA are characterized by a high frequency of KRAS mutations (30–45%), ERBB2 amplification (15–20%), and low frequency of IDH 1/2 or FGFR2 fusions. Although rare, gene rearrangements such as NTRK, ROS1, or ALK fusions have been identified in BTC. All subtypes of BTC harbor similar rates of BRAF alteration (3–5%), homologous recombination deficiency (5–15%), and microsatellite instability-high (MSI-H)/mismatch repair (MMR)-deficient (dMMR; 2–5%) [109,110,111]. Although distinct genetic and epigenetic profiles have been identified for each BTC subtype, subgroups with driver mutations amenable to targeted therapy are generally mutually exclusive from one another.
Parallel tests of several genes using focused next-generation sequencing (NGS) are preferred over single-gene sequencing. NGS can be performed on formalin-fixed and paraffin-embedded tumor tissues and is well suited for tissue biopsies. Alternatively, liquid biopsies using cell-free circulating DNA may be considered if insufficient tumor tissue is available for NGS [108]. MSI status can be evaluated by immunohistochemical (IHC) staining for MMR proteins, including MLH1, MSH2, MSH6, and PMS2. Instead, DNA-based assays can be used to analyze the composition and length of microsatellites. The preferred methods for NGS, IHC staining, or RNA sequencing depend on the target and availability of materials (tissue or circulating tumor DNA).

6. Approach to the Patient

6.1. Suspected iCCA

When an intrahepatic lesion is suspected, cross-sectional imaging (multiphasic contrast-enhanced CT or MRI) is performed to differentiate between HCC and mass-forming iCCA. However, the classical radiologic features of iCCA were present in only 70% of cases [19], and some small mass-forming iCCA may mimic HCC, which hyperehances during the arterial phase and demonstrates washout during the delayed phase. If the initial imaging test is non-diagnostic, other imaging modalities (CT or MRI) can be conducted. Biopsy or surgery of the lesion is performed if the diagnosis remains uncertain.
The issue is further complicated by the fact that some intrahepatic tumors can contain elements of both cholangiocarcinoma and HCC in the same nodule, termed mixed hepatocellular-cholangiocellular carcinomas [7]. Studies have suggested that these tumors have a distinct appearance in cross-sectional imaging studies. A strongly enhancing rim and irregular shape on gadoxetic acid-enhanced MRI are indicative of mixed hepatocellular-cholangiocellular carcinoma, while a lobulated shape, weak rim, and target appearance suggest a mass-forming iCCA [112]. The target appearance can also help differentiate mixed hepatocellular-cholangiocellular carcinomas from atypical hypovascular HCC [113]. Additionally, the presence of liver capsule retraction and biliary dilatation in the vicinity of the intrahepatic lesion can raise suspicion for a diagnosis of iCCA; however, biopsy may be needed to confirm the diagnosis. These mixed tumors were staged as iCCA and not as HCC.
IHC staining of tissue biopsies is required to differentiate iCCA from metastatic lesions and mixed hepatocellular-cholangiocellular carcinoma. Tumors negative for TTF-1 (lung), CDX2 (colon), and DPC4 (pancreas) and positive for AE1/AE3, CK7, and CK20 (biliary epithelium) were suggestive of iCCA [114].

6.2. Suspeected pCCA

Careful evaluation with cross-sectional imaging studies (enhanced MRI with MRCP was preferred over CT) and EUS helps delineate the tumor location, size, morphology, hepatic artery or portal vein involvement, volume of potential liver remnant, lymph node involvement, and presence of distant metastasis. If imaging studies and/or tissue samples are highly suggestive of pCCA, the tumor staging proceeds directly. In cases where the diagnosis is uncertain, we typically proceed with an ERCP procedure that includes brush cytology (with or without IDUS). Where available, POC can be performed to evaluate the bile ducts. Alternatively, MRI- or CT-guided biopsy can be performed if a mass lesion is observed on imaging, although there is a small risk of needle tract seeding. If the diagnosis remains doubtful, surgery may be required to confirm it.

Patients with PSC

PSC is one of the most common risk factors for BTC, and the lifetime incidence of cholangiocarcinoma in PSC has been reported to be 5%–10% [115,116]. Cholangiocarcinoma in PSC usually infiltrates and appears as the formation of progressive strictures in perihilar areas [116,117]. In such cases, patients may have a dominant benign biliary stricture that is difficult to differentiate from cholangiocarcinoma. Mass lesions are seldom identified on imaging, and patients often do not develop significant intrahepatic biliary ductal dilation. However, detection of a new parenchymal lesion adjacent to the bile ducts, appearance of a new bile duct dilatation in a short interval, and disproportionate regional/segmental bile duct dilatation are among the warning signs of developing cholangiocarcinoma in these patients.
In patients with PSC with suspected cholangiocarcinoma, CA 19-9 levels above 129 U/mL are 79% sensitive and 98% specific for confirming the diagnosis [118]. However, the positive predictive value (the likelihood that a patient with PSC and a CA 19-9 level ≥ 129 unit/mL has cholangiocarcinoma) was only 57 percent.
The initial step of work-up is MRCP to evaluate the segmental extent of ductal involvement, search for intrahepatic metastases, and identify aberrant ductal anatomy. If the MRCP is nondiagnostic or if a dominant stricture is identified, we will obtain an ERCP or PTC with brush cytology, which is almost 100% specific; however, even with FISH, only 40%–70% is sensitive for the detection of cholangiocarcinoma in patients with PSC [119,120]. In cases of negative cytologic results, MRI/MRCP and/or ERCP plus CA 19-9 should be repeated within 3-6 months.

6.3. Suspected dCCA

On cross-sectional imaging, dCCA may be seen as an abrupt stenosis with proximal biliary dilatation, where a nodular mass or concentric or asymmetric thickening of the bile duct with enhancement typically coexist. In uncommon cases of thickening or stricture of the distal bile ducts without a mass, it is difficult to differentiate them from benign strictures. While ERCP has traditionally dominated the initial workup of dCCA owing to tissue sampling for diagnosis and biliary decompression, EUS has recently become the preferred method for direct visualization and sampling of the distal bile duct. ERCP carries a risk of ascending cholangitis by injecting contrast, whereas EUS-FNA poses a risk of seeding the biopsy tract. If radiographic findings are sufficiently diagnostic for dCCA, such that a negative biopsy would be characterized as a potential false-negative and the tumor appears resectable, biopsy is not indicated.

7. Conclusions

BTC is a heterogeneous disease of cancer that arises from the biliary tree. Although they have historically been classified as a single disease, extensive molecular characterization has recently led to more informative anatomical, pathological, and molecular classification of BTC. The development of radiologic and endoscopic tools for accurate diagnosis strengthens our understanding of BTC carcinogenesis. Precision medicine for BTC patients is facilitated by pathological and molecular profiling. We anticipate that advancements in diagnostic and personalized strategies for BTC management will lead to improved patient outcomes in the near future.

Author Contributions

Conceptualization, S.H.L. and S.Y.S.; methodology, S.H.L.; investigation, S.H.L.; writing—original draft preparation, S.H.L.; writing—review and editing, S.Y.S.; and supervision, S.Y.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding

Institutional Review Board Statement

Not applicable

Informed Consent Statement

Not applicable.

Data Availability Statement

Data sharing is not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Razumilava, N.; Gores, G.J. Cholangiocarcinoma. Lancet 2014, 383, 2168–2179. [Google Scholar] [CrossRef]
  2. Nakanuma, Y.; Sato, Y.; Harada, K.; Sasaki, M.; Xu, J.; Ikeda, H. Pathological classification of intrahepatic cholangiocarcinoma based on a new concept. World J Hepatol 2010, 2, 419–427. [Google Scholar] [CrossRef]
  3. Nakanuma, Y.; Kakuda, Y. Pathologic classification of cholangiocarcinoma: New concepts. Best Pract Res Clin Gastroenterol 2015, 29, 277–293. [Google Scholar] [CrossRef]
  4. Sigel, C.S.; Drill, E.; Zhou, Y.; Basturk, O.; Askan, G.; Pak, L.M.; Vakiani, E.; Wang, T.; Boerner, T.; Do, R.K.G.; et al. Intrahepatic cholangiocarcinomas have histologically and immunophenotypically distinct small and large duct patterns. American Journal of Surgical Pathology 2018, 42, 1334–1345. [Google Scholar] [CrossRef]
  5. Hayashi, A.; Misumi, K.; Shibahara, J.; Arita, J.; Sakamoto, Y.; Hasegawa, K.; Kokudo, N.; Fukayama, M. Distinct clinicopathologic and genetic features of 2 histologic subtypes of intrahepatic cholangiocarcinoma. Am J Surg Pathol 2016, 40, 1021–1030. [Google Scholar] [CrossRef]
  6. Chung, T.; Rhee, H.; Nahm, J.H.; Jeon, Y.; Yoo, J.E.; Kim, Y.J.; Han, D.H.; Park, Y.N. Clinicopathological characteristics of intrahepatic cholangiocarcinoma according to gross morphologic type: Cholangiolocellular differentiation traits and inflammation- and proliferation-phenotypes. HPB (Oxford) 2020, 22, 864–873. [Google Scholar] [CrossRef]
  7. Komuta, M.; Govaere, O.; Vandecaveye, V.; Akiba, J.; Van Steenbergen, W.; Verslype, C.; Laleman, W.; Pirenne, J.; Aerts, R.; Yano, H.; et al. Histological diversity in cholangiocellular carcinoma reflects the different cholangiocyte phenotypes. Hepatology 2012, 55, 1876–1888. [Google Scholar] [CrossRef] [PubMed]
  8. Cardinale, V.; Wang, Y.; Carpino, G.; Reid, L.M.; Gaudio, E.; Alvaro, D. Mucin-producing cholangiocarcinoma might derive from biliary tree stem/progenitor cells located in peribiliary glands. Hepatology 2012, 55, 2041–2042. [Google Scholar] [CrossRef] [PubMed]
  9. Katabi, N.; Torres, J.; Klimstra, D.S. Intraductal tubular neoplasms of the bile ducts. American Journal of Surgical Pathology 2012, 36, 1647–1655. [Google Scholar] [CrossRef] [PubMed]
  10. Nakanuma, Y.; Jang, K.T.; Fukushima, N.; Furukawa, T.; Hong, S.M.; Kim, H.; Lee, K.B.; Zen, Y.; Jang, J.Y.; Kubota, K. A statement by the japan-korea expert pathologists for future clinicopathological and molecular analyses toward consensus building of intraductal papillary neoplasm of the bile duct through several opinions at the present stage. J Hepatobiliary Pancreat Sci 2018, 25, 181–187. [Google Scholar] [CrossRef] [PubMed]
  11. Henson, D.E.; Albores-Saavedra, J.; Corle, D. Carcinoma of the gallbladder. Histologic types, stage of disease, grade, and survival rates. Cancer 1992, 70, 1493–1497. [Google Scholar] [CrossRef]
  12. Adsay, V.; Jang, K.T.; Roa, J.C.; Dursun, N.; Ohike, N.; Bagci, P.; Basturk, O.; Bandyopadhyay, S.; Cheng, J.D.; Sarmiento, J.M.; et al. Intracholecystic papillary-tubular neoplasms (icpn) of the gallbladder (neoplastic polyps, adenomas, and papillary neoplasms that are >/=1.0 cm): Clinicopathologic and immunohistochemical analysis of 123 cases. Am J Surg Pathol 2012, 36, 1279–1301. [Google Scholar] [CrossRef]
  13. Alvaro, D.; Bragazzi, M.C.; Benedetti, A.; Fabris, L.; Fava, G.; Invernizzi, P.; Marzioni, M.; Nuzzo, G.; Strazzabosco, M.; Stroffolini, T.; et al. Cholangiocarcinoma in italy: A national survey on clinical characteristics, diagnostic modalities and treatment. Results from the "cholangiocarcinoma" committee of the italian association for the study of liver disease. Dig Liver Dis 2011, 43, 60–65. [Google Scholar] [CrossRef] [PubMed]
  14. Strom, B.L.; Maislin, G.; West, S.L.; Atkinson, B.; Herlyn, M.; Saul, S.; Rodriguez-Martinez, H.A.; Rios-Dalenz, J.; Iliopoulos, D.; Soloway, R.D. Serum cea and ca 19-9: Potential future diagnostic or screening tests for gallbladder cancer? Int J Cancer 1990, 45, 821–824. [Google Scholar] [CrossRef]
  15. Ritts, R.E., Jr.; Nagorney, D.M.; Jacobsen, D.J.; Talbot, R.W.; Zurawski, V.R., Jr. Comparison of preoperative serum ca19-9 levels with results of diagnostic imaging modalities in patients undergoing laparotomy for suspected pancreatic or gallbladder disease. Pancreas 1994, 9, 707–716. [Google Scholar] [CrossRef]
  16. Kim, H.J.; Kim, M.H.; Myung, S.J.; Lim, B.C.; Park, E.T.; Yoo, K.S.; Seo, D.W.; Lee, S.K.; Min, Y.I. A new strategy for the application of ca19-9 in the differentiation of pancreaticobiliary cancer: Analysis using a receiver operating characteristic curve. Am J Gastroenterol 1999, 94, 1941–1946. [Google Scholar] [CrossRef]
  17. Joo, I.; Lee, J.M.; Yoon, J.H. Imaging diagnosis of intrahepatic and perihilar cholangiocarcinoma: Recent advances and challenges. Radiology 2018, 288, 7–13. [Google Scholar] [CrossRef]
  18. Jhaveri, K.S.; Hosseini-Nik, H. Mri of cholangiocarcinoma. J Magn Reson Imaging 2015, 42, 1165–1179. [Google Scholar] [CrossRef]
  19. Kim, S.H.; Lee, C.H.; Kim, B.H.; Kim, W.B.; Yeom, S.K.; Kim, K.A.; Park, C.M. Typical and atypical imaging findings of intrahepatic cholangiocarcinoma using gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid-enhanced magnetic resonance imaging. J Comput Assist Tomogr 2012, 36, 704–709. [Google Scholar] [CrossRef] [PubMed]
  20. Park, H.J.; Kim, Y.K.; Park, M.J.; Lee, W.J. Small intrahepatic mass-forming cholangiocarcinoma: Target sign on diffusion-weighted imaging for differentiation from hepatocellular carcinoma. Abdom Imaging 2013, 38, 793–801. [Google Scholar] [CrossRef]
  21. Somoracz, A.; Tatrai, P.; Horvath, G.; Kiss, A.; Kupcsulik, P.; Kovalszky, I.; Schaff, Z. Agrin immunohistochemistry facilitates the determination of primary versus metastatic origin of liver carcinomas. Hum Pathol 2010, 41, 1310–1319. [Google Scholar] [CrossRef]
  22. Manfredi, R.; Masselli, G.; Maresca, G.; Brizi, M.G.; Vecchioli, A.; Marano, P. Mr imaging and mrcp of hilar cholangiocarcinoma. Abdom Imaging 2003, 28, 319–325. [Google Scholar] [CrossRef]
  23. Vanderveen, K.A.; Hussain, H.K. Magnetic resonance imaging of cholangiocarcinoma. Cancer Imaging 2004, 4, 104–115. [Google Scholar] [CrossRef] [PubMed]
  24. Kim, J.H.; Byun, J.H.; Kim, S.Y.; Lee, S.S.; Kim, H.J.; Kim, M.H.; Lee, M.G. Sclerosing cholangitis with autoimmune pancreatitis versus primary sclerosing cholangitis: Comparison on endoscopic retrograde cholangiography, mr cholangiography, ct, and mri. Acta Radiol 2013, 54, 601–607. [Google Scholar] [CrossRef] [PubMed]
  25. Choi, B.W.; Kim, M.J.; Chung, J.J.; Chung, J.B.; Yoo, H.S.; Lee, J.T. Radiologic findings of mirizzi syndrome with emphasis on mri. Yonsei Med J 2000, 41, 144–146. [Google Scholar] [CrossRef]
  26. Chattopadhyay, S.; Nundy, S. Portal biliopathy. World J Gastroenterol 2012, 18, 6177–6182. [Google Scholar] [CrossRef] [PubMed]
  27. Madhusudhan, K.S.; Das, P.; Gunjan, D.; Srivastava, D.N.; Garg, P.K. Igg4-related sclerosing cholangitis: A clinical and imaging review. AJR Am J Roentgenol 2019, 213, 1221–1231. [Google Scholar] [CrossRef]
  28. Krasinskas, A.M.; Raina, A.; Khalid, A.; Tublin, M.; Yadav, D. Autoimmune pancreatitis. Gastroenterol Clin North Am 2007, 36, 239–257, vii. [Google Scholar] [CrossRef]
  29. Finkelberg, D.L.; Sahani, D.; Deshpande, V.; Brugge, W.R. Autoimmune pancreatitis. N Engl J Med 2006, 355, 2670–2676. [Google Scholar] [CrossRef]
  30. Lim, J.H.; Yi, C.A.; Lim, H.K.; Lee, W.J.; Lee, S.J.; Kim, S.H. Radiological spectrum of intraductal papillary tumors of the bile ducts. Korean J Radiol 2002, 3, 57–63. [Google Scholar] [CrossRef]
  31. Kumar, A.; Aggarwal, S. Carcinoma of the gallbladder: Ct findings in 50 cases. Abdom Imaging 1994, 19, 304–308. [Google Scholar] [CrossRef]
  32. Liang, J.L.; Chen, M.C.; Huang, H.Y.; Ng, S.H.; Sheen-Chen, S.M.; Liu, P.P.; Kung, C.T.; Ko, S.F. Gallbladder carcinoma manifesting as acute cholecystitis: Clinical and computed tomographic features. Surgery 2009, 146, 861–868. [Google Scholar] [CrossRef] [PubMed]
  33. Lamarca, A.; Barriuso, J.; Chander, A.; McNamara, M.G.; Hubner, R.A.; D, O.R.; Manoharan, P.; Valle, J.W. (18)f-fluorodeoxyglucose positron emission tomography ((18)fdg-pet) for patients with biliary tract cancer: Systematic review and meta-analysis. J Hepatol 2019, 71, 115–129. [Google Scholar] [CrossRef]
  34. Abu-Hamda, E.M.; Baron, T.H. Endoscopic management of cholangiocarcinoma. Semin Liver Dis 2004, 24, 165–175. [Google Scholar] [CrossRef] [PubMed]
  35. Mohamadnejad, M.; DeWitt, J.M.; Sherman, S.; LeBlanc, J.K.; Pitt, H.A.; House, M.G.; Jones, K.J.; Fogel, E.L.; McHenry, L.; Watkins, J.L.; et al. Role of eus for preoperative evaluation of cholangiocarcinoma: A large single-center experience. Gastrointest Endosc 2011, 73, 71–78. [Google Scholar] [CrossRef]
  36. Fujita, N.; Noda, Y.; Kobayashi, G.; Kimura, K.; Yago, A. Diagnosis of the depth of invasion of gallbladder carcinoma by eus. Gastrointest Endosc 1999, 50, 659–663. [Google Scholar] [CrossRef]
  37. Sadamoto, Y.; Kubo, H.; Harada, N.; Tanaka, M.; Eguchi, T.; Nawata, H. Preoperative diagnosis and staging of gallbladder carcinoma by eus. Gastrointest Endosc 2003, 58, 536–541. [Google Scholar] [CrossRef] [PubMed]
  38. Wu, L.M.; Jiang, X.X.; Gu, H.Y.; Xu, X.; Zhang, W.; Lin, L.H.; Deng, X.; Yin, Y.; Xu, J.R. Endoscopic ultrasound-guided fine-needle aspiration biopsy in the evaluation of bile duct strictures and gallbladder masses: A systematic review and meta-analysis. Eur J Gastroenterol Hepatol 2011, 23, 113–120. [Google Scholar] [CrossRef] [PubMed]
  39. Kuroiwa, M.; Tsukamoto, Y.; Naitoh, Y.; Hirooka, Y.; Furukawa, T.; Katou, T. New technique using intraductal ultrasonography for the diagnosis of bile duct cancer. J Ultrasound Med 1994, 13, 189–195. [Google Scholar] [CrossRef]
  40. Tamada, K.; Ueno, N.; Ichiyama, M.; Tomiyama, T.; Nishizono, T.; Wada, S.; Oohashi, A.; Tano, S.; Aizawa, T.; Ido, K.; et al. Assessment of pancreatic parenchymal invasion by bile duct cancer using intraductal ultrasonography. Endoscopy 1996, 28, 492–496. [Google Scholar] [CrossRef]
  41. Choi, E.R.; Chung, Y.H.; Lee, J.K.; Lee, K.T.; Lee, K.H.; Choi, D.W.; Choi, S.H.; Heo, J.S.; Jang, K.T.; Park, S.M.; et al. Preoperative evaluation of the longitudinal extent of borderline resectable hilar cholangiocarcinoma by intraductal ultrasonography. J Gastroenterol Hepatol 2011, 26, 1804–1810. [Google Scholar] [CrossRef]
  42. Shah, R.J.; Langer, D.A.; Antillon, M.R.; Chen, Y.K. Cholangioscopy and cholangioscopic forceps biopsy in patients with indeterminate pancreaticobiliary pathology. Clin Gastroenterol Hepatol 2006, 4, 219–225. [Google Scholar] [CrossRef]
  43. Fukuda, Y.; Tsuyuguchi, T.; Sakai, Y.; Tsuchiya, S.; Saisyo, H. Diagnostic utility of peroral cholangioscopy for various bile-duct lesions. Gastrointestinal Endoscopy 2005, 62, 374–382. [Google Scholar] [CrossRef]
  44. Iqbal, S.; Stevens, P.D. Cholangiopancreatoscopy for targeted biopsies of the bile and pancreatic ducts. Gastrointest Endosc Clin N Am 2009, 19, 567–577. [Google Scholar] [CrossRef] [PubMed]
  45. Wen, L.J.; Chen, J.H.; Xu, H.J.; Yu, Q.; Liu, K. Efficacy and safety of digital single-operator cholangioscopy in the diagnosis of indeterminate biliary strictures by targeted biopsies: A systematic review and meta-analysis. Diagnostics (Basel) 2020, 10. [Google Scholar] [CrossRef] [PubMed]
  46. Seo, D.W.; Lee, S.K.; Yoo, K.S.; Kang, G.H.; Kim, M.H.; Suh, D.J.; Min, Y.I. Cholangioscopic findings in bile duct tumors. Gastrointest Endosc 2000, 52, 630–634. [Google Scholar] [CrossRef] [PubMed]
  47. Pereira, P.; Santos, S.; Morais, R.; Gaspar, R.; Rodrigues-Pinto, E.; Vilas-Boas, F.; Macedo, G. Role of peroral cholangioscopy for diagnosis and staging of biliary tumors. Dig Dis 2020, 38, 431–440. [Google Scholar] [CrossRef] [PubMed]
  48. Trikudanathan, G.; Navaneethan, U.; Njei, B.; Vargo, J.J.; Parsi, M.A. Diagnostic yield of bile duct brushings for cholangiocarcinoma in primary sclerosing cholangitis: A systematic review and meta-analysis. Gastrointest Endosc 2014, 79, 783–789. [Google Scholar] [CrossRef] [PubMed]
  49. Sugiyama, M.; Atomi, Y.; Wada, N.; Kuroda, A.; Muto, T. Endoscopic transpapillary bile duct biopsy without sphincterotomy for diagnosing biliary strictures: A prospective comparative study with bile and brush cytology. American Journal of Gastroenterology 1996, 91, 465–467. [Google Scholar]
  50. Rabinovitz, M.; Zajko, A.B.; Hassanein, T.; Shetty, B.; Bron, K.M.; Schade, R.R.; Gavaler, J.S.; Block, G.; Van Thiel, D.H.; Dekker, A. Diagnostic value of brush cytology in the diagnosis of bile duct carcinoma: A study in 65 patients with bile duct strictures. Hepatology 1990, 12, 747–752. [Google Scholar] [CrossRef]
  51. Ponchon, T.; Gagnon, P.; Berger, F.; Labadie, M.; Liaras, A.; Chavaillon, A.; Bory, R. Value of endobiliary brush cytology and biopsies for the diagnosis of malignant bile duct stenosis: Results of a prospective study. Gastrointest Endosc 1995, 42, 565–572. [Google Scholar] [CrossRef] [PubMed]
  52. Gonda, T.A.; Viterbo, D.; Gausman, V.; Kipp, C.; Sethi, A.; Poneros, J.M.; Gress, F.; Park, T.; Khan, A.; Jackson, S.A.; et al. Mutation profile and fluorescence in situ hybridization analyses increase detection of malignancies in biliary strictures. Clin Gastroenterol Hepatol 2017, 15, 913–919 e911. [Google Scholar] [CrossRef] [PubMed]
  53. Navaneethan, U.; Njei, B.; Venkatesh, P.G.; Vargo, J.J.; Parsi, M.A. Fluorescence in situ hybridization for diagnosis of cholangiocarcinoma in primary sclerosing cholangitis: A systematic review and meta-analysis. Gastrointest Endosc 2014, 79, 943–950 e943. [Google Scholar] [CrossRef] [PubMed]
  54. Hayakawa, C.; Hoshikawa, M.; Imura, J.; Ueno, T.; Koike, J. Bile cytology: A new scoring system for improving diagnostic accuracy. Diagn Cytopathol 2019, 47, 641–647. [Google Scholar] [CrossRef] [PubMed]
  55. Yeo, M.K.; Kim, K.H.; Lee, Y.M.; Lee, B.S.; Choi, S.Y. The usefulness of adding p53 immunocytochemistry to bile drainage cytology for the diagnosis of malignant biliary strictures. Diagn Cytopathol 2017, 45, 592–597. [Google Scholar] [CrossRef] [PubMed]
  56. Liu, F.; Hao, X.; Liu, B.; Liu, S.; Yuan, Y. Bile liquid biopsy in biliary tract cancer. Clin Chim Acta 2023, 551, 117593. [Google Scholar] [CrossRef] [PubMed]
  57. Xu, Y.; Leng, K.; Yao, Y.; Kang, P.; Liao, G.; Han, Y.; Shi, G.; Ji, D.; Huang, P.; Zheng, W.; et al. A circular rna, cholangiocarcinoma-associated circular rna 1, contributes to cholangiocarcinoma progression, induces angiogenesis, and disrupts vascular endothelial barriers. Hepatology 2021, 73, 1419–1435. [Google Scholar] [CrossRef]
  58. Kubicka, S.; Kuhnel, F.; Flemming, P.; Hain, B.; Kezmic, N.; Rudolph, K.L.; Manns, M.; Meier, P.N. K-ras mutations in the bile of patients with primary sclerosing cholangitis. Gut 2001, 48, 403–408. [Google Scholar] [CrossRef]
  59. Andresen, K.; Boberg, K.M.; Vedeld, H.M.; Honne, H.; Jebsen, P.; Hektoen, M.; Wadsworth, C.A.; Clausen, O.P.; Lundin, K.E.; Paulsen, V.; et al. Four DNA methylation biomarkers in biliary brush samples accurately identify the presence of cholangiocarcinoma. Hepatology 2015, 61, 1651–1659. [Google Scholar] [CrossRef]
  60. Vedeld, H.M.; Grimsrud, M.M.; Andresen, K.; Pharo, H.D.; von Seth, E.; Karlsen, T.H.; Honne, H.; Paulsen, V.; Farkkila, M.A.; Bergquist, A.; et al. Early and accurate detection of cholangiocarcinoma in patients with primary sclerosing cholangitis by methylation markers in bile. Hepatology 2022, 75, 59–73. [Google Scholar] [CrossRef]
  61. Liu, Y.; Cheng, C.; Bai, L.; Yao, F.; Shi, S.; Zhang, Y. Value of bile soluble b7h3 for the diagnosis of malignant biliary strictures: Results of a retrospective study. Surg Oncol 2019, 28, 195–200. [Google Scholar] [CrossRef]
  62. Sharma, N.; Yadav, M.; Tripathi, G.; Mathew, B.; Bindal, V.; Falari, S.; Pamecha, V.; Maras, J.S. Bile multi-omics analysis classifies lipid species and microbial peptides predictive of carcinoma of gallbladder. Hepatology 2022, 76, 920–935. [Google Scholar] [CrossRef]
  63. Li, L.; Masica, D.; Ishida, M.; Tomuleasa, C.; Umegaki, S.; Kalloo, A.N.; Georgiades, C.; Singh, V.K.; Khashab, M.; Amateau, S.; et al. Human bile contains microrna-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis (vol 60, pg 896, 2014). Hepatology 2014, 60, 2135–2135. [Google Scholar] [CrossRef] [PubMed]
  64. Ge, X.; Tang, L.; Wang, Y.; Wang, N.; Zhou, J.; Deng, X.; Zhong, Y.; Li, Q.; Wang, F.; Jiang, G.; et al. The diagnostic value of exosomal mirnas in human bile of malignant biliary obstructions. Dig Liver Dis 2021, 53, 760–765. [Google Scholar] [CrossRef] [PubMed]
  65. Pan, Y.; Shao, S.J.; Sun, H.; Zhu, H.F.; Fang, H.X. Bile-derived exosome noncoding rnas as potential diagnostic and prognostic biomarkers for cholangiocarcinoma. Front Oncol 2022, 12. [Google Scholar] [CrossRef] [PubMed]
  66. Ge, X.; Wang, Y.; Nie, J.; Li, Q.; Tang, L.; Deng, X.; Wang, F.; Xu, B.; Wu, X.; Zhang, X.; et al. The diagnostic/prognostic potential and molecular functions of long non-coding rnas in the exosomes derived from the bile of human cholangiocarcinoma. Oncotarget 2017, 8, 69995–70005. [Google Scholar] [CrossRef]
  67. Ikeda, C.; Haga, H.; Makino, N.; Inuzuka, T.; Kurimoto, A.; Ueda, T.; Matsuda, A.; Kakizaki, Y.; Ishizawa, T.; Kobayashi, T.; et al. Utility of claudin-3 in extracellular vesicles from human bile as biomarkers of cholangiocarcinoma. Sci Rep-Uk 2021, 11. [Google Scholar] [CrossRef] [PubMed]
  68. Lee, J.G.; Leung, J.W.; Cotton, P.B.; Layfield, L.J.; Mannon, P.J. Diagnostic utility of k-ras mutational analysis on bile obtained by endoscopic retrograde cholangiopancreatography. Gastrointestinal Endoscopy 1995, 42, 317–320. [Google Scholar] [CrossRef] [PubMed]
  69. Saurin, J.C.; Joly-Pharaboz, M.O.; Pernas, P.; Henry, L.; Ponchon, T.; Madjar, J.J. Detection of ki-ras gene point mutations in bile specimens for the differential diagnosis of malignant and benign biliary strictures. Gut 2000, 47, 357–361. [Google Scholar] [CrossRef]
  70. Han, J.Y.; Ahn, K.S.; Kim, T.S.; Kim, Y.H.; Cho, K.B.; Shin, D.W.; Baek, W.K.; Suh, S.I.; Jang, B.C.; Kang, K.J. Liquid biopsy from bile-circulating tumor DNA in patients with biliary tract cancer. Cancers (Basel) 2021, 13. [Google Scholar] [CrossRef]
  71. Itoi, T.; Takei, K.; Shinohara, Y.; Takeda, K.; Nakamura, K.; Horibe, T.; Sanada, A.; Ohno, H.; Matsubayashi, H.; Saito, T.; et al. K-ras codon 12 and p53 mutations in biopsy specimens and bile from biliary tract cancers. Pathol Int 1999, 49, 30–37. [Google Scholar] [CrossRef] [PubMed]
  72. Müller, P.; Ostwald, C.; Püschel, K.; Brinkmann, B.; Plath, F.; Kröger, J.; Barten, M.; Nizze, H.; Schareck, W.D.; Hauenstein, K.; et al. Low frequency of p53 and ras mutations in bile of patients with hepato-biliary disease:: A prospective study in more than 100 patients. Eur J Clin Invest 2001, 31, 240–247. [Google Scholar] [CrossRef] [PubMed]
  73. Wang, Y.; Yamaguchi, Y.; Watanabe, H.; Ohtsubo, K.; Wakabayashi, T.; Sawabu, N. Usefulness of p53 gene mutations in the supernatant of bile for diagnosis of biliary tract carcinoma: Comparison with k- ras mutation. J Gastroenterol 2002, 37, 831–839. [Google Scholar] [CrossRef] [PubMed]
  74. Kinugasa, H.; Nouso, K.; Ako, S.; Dohi, C.; Matsushita, H.; Matsumoto, K.; Kato, H.; Okada, H. Liquid biopsy of bile for the molecular diagnosis of gallbladder cancer. Cancer Biol Ther 2018, 19, 934–938. [Google Scholar] [CrossRef] [PubMed]
  75. Klump, B.; Hsieh, C.J.; Dette, S.; Holzmann, K.; Kiesslich, R.; Jung, M.; Sinn, U. Promoter methylation of ink4a/arf as detected in bile-significance for the differential diagnosis in biliary disease (vol 9, pg 1773, 2003). Clin Cancer Res 2003, 9, 2877–2877. [Google Scholar]
  76. Zhang, Y.; Yang, B.; Du, Z.; Gao, Y.T.; Wang, Y.J.; Jing, X.; Bai, T. Identification and validation of specific methylation profile in bile for differential diagnosis of malignant biliary stricture. Clin Biochem 2010, 43, 1340–1344. [Google Scholar] [CrossRef] [PubMed]
  77. Shin, S.H.; Lee, K.; Kim, B.H.; Cho, N.Y.; Jang, J.Y.; Kim, Y.T.; Kim, D.; Jang, J.J.; Kang, G.H. Bile-based detection of extrahepatic cholangiocarcinoma with quantitative DNA methylation markers and its high sensitivity. J Mol Diagn 2012, 14, 256–263. [Google Scholar] [CrossRef]
  78. He, S.; Zeng, F.; Yin, H.; Wang, P.; Bai, Y.; Song, Q.; Chu, J.; Huang, Z.; Liu, Y.; Liu, H.; et al. Molecular diagnosis of pancreatobiliary tract cancer by detecting mutations and methylation changes in bile samples. EClinicalMedicine 2023, 55, 101736. [Google Scholar] [CrossRef]
  79. Shen, N.J.; Zhang, D.D.; Yin, L.; Qiu, Y.H.; Liu, J.; Yu, W.L.; Fu, X.H.; Zhu, B.; Xu, X.Y.; Duan, A.Q.; et al. Bile cell-free DNA as a novel and powerful liquid biopsy for detecting somatic variants in biliary tract cancer. Oncol Rep 2019, 42, 549–560. [Google Scholar] [CrossRef]
  80. Gou, Q.; Zhang, C.Z.; Sun, Z.H.; Wu, L.G.; Chen, Y.; Mo, Z.Q.; Mai, Q.C.; He, J.; Zhou, Z.X.; Shi, F.; et al. Cell-free DNA from bile outperformed plasma as a potential alternative to tissue biopsy in biliary tract cancer. Esmo Open 2021, 6. [Google Scholar] [CrossRef]
  81. Uchida, N.; Tsutsui, K.; Ezaki, T.; Fukuma, H.; Kobara, H.; Kamata, H.; Aritomo, Y.; Masaki, T.; Watanabe, S.; Kobayashi, S.; et al. Combination of assay of human telomerase reverse transcriptase mrna and cytology using bile obtained by endoscopic transpapillary catheterization into the gallbladder for diagnosis of gallbladder carcinoma. American Journal of Gastroenterology 2003, 98, 2415–2419. [Google Scholar] [CrossRef]
  82. Shigehara, K.; Yokomuro, S.; Ishibashi, O.; Mizuguchi, Y.; Arima, Y.; Kawahigashi, Y.; Kanda, T.; Akagi, I.; Tajiri, T.; Yoshida, H.; et al. Real-time pcr-based analysis of the human bile micrornaome identifies as a potential diagnostic biomarker for biliary tract cancer. Plos One 2011, 6. [Google Scholar] [CrossRef] [PubMed]
  83. Baraniskin, A.; Nopel-Dunnebacke, S.; Schumacher, B.; Gerges, C.; Bracht, T.; Sitek, B.; Meyer, H.E.; Gerken, G.; Dechene, A.; Schlaak, J.F.; et al. Analysis of u2 small nuclear rna fragments in the bile differentiates cholangiocarcinoma from primary sclerosing cholangitis and other benign biliary disorders. Dig Dis Sci 2014, 59, 1436–1441. [Google Scholar] [CrossRef]
  84. Voigtlander, T.; Gupta, S.K.; Thum, S.; Fendrich, J.; Manns, M.P.; Lankisch, T.O.; Thum, T. Micrornas in serum and bile of patients with primary sclerosing cholangitis and/or cholangiocarcinoma. Plos One 2015, 10, e0139305. [Google Scholar] [CrossRef] [PubMed]
  85. Han, H.S.; Kim, M.J.; Han, J.H.; Yun, J.; Kim, H.K.; Yang, Y.; Kim, K.B.; Park, S.M. Bile-derived circulating extracellular mir-30d-5p and mir-92a-3p as potential biomarkers for cholangiocarcinoma. Hepatob Pancreat Dis 2020, 19, 41–50. [Google Scholar] [CrossRef] [PubMed]
  86. Rose, J.B.; Correa-Gallego, C.; Li, Y.; Nelson, J.; Alseidi, A.; Helton, W.S.; Allen, P.J.; D’Angelica, M.I.; DeMatteo, R.P.; Fong, Y.M.; et al. The role of biliary carcinoembryonic antigen-related cellular adhesion molecule 6 (ceacam6) as a biomarker in cholangiocarcinoma. Plos One 2016, 11. [Google Scholar] [CrossRef] [PubMed]
  87. Farina, A.; Dumonceau, J.M.; Antinori, P.; Annessi-Ramseyer, I.; Frossard, J.L.; Hochstrasser, D.F.; Delhaye, M.; Lescuyer, P. Bile carcinoembryonic cell adhesion molecule 6 (ceam6) as a biomarker of malignant biliary stenoses. Bba-Proteins Proteom 2014, 1844, 1018–1025. [Google Scholar] [CrossRef]
  88. Liu, Y.; Sun, J.; Zhang, Q.; Jin, B.; Zhu, M.; Zhang, Z. Identification of bile survivin and carbohydrate antigen 199 in distinguishing cholangiocarcinoma from benign obstructive jaundice. Biomark Med 2017, 11, 11–18. [Google Scholar] [CrossRef]
  89. Matsuda, A.; Kuno, A.; Kawamoto, T.; Matsuzaki, H.; Irimura, T.; Ikehara, Y.; Zen, Y.; Nakanuma, Y.; Yamamoto, M.; Ohkohchi, N.; et al. Agglutinin-positive mucin 1 is a sensitive biliary marker for human cholangiocarcinoma. Hepatology 2010, 52, 174–182. [Google Scholar] [CrossRef]
  90. Matull, W.R.; Andreola, F.; Loh, A.; Adiguzel, Z.; Deheragoda, M.; Qureshi, U.; Batra, S.K.; Swallow, D.M.; Pereira, S.P. Muc4 and muc5ac are highly specific tumour-associated mucins in biliary tract cancer. Br J Cancer 2008, 98, 1675–1681. [Google Scholar] [CrossRef]
  91. Danese, E.; Ruzzenente, O.; Ruzzenente, A.; Iacono, C.; Bertuzzo, F.; Gelati, M.; Conci, S.; Bendinelli, S.; Bonizzato, G.; Guglielmi, A.; et al. Assessment of bile and serum mucin5ac in cholangiocarcinoma: Diagnostic performance and biologic significance. Surgery 2014, 156, 1218–1224. [Google Scholar] [CrossRef]
  92. Koopmann, J.; Thuluvath, P.J.; Zahurak, M.L.; Kristiansen, T.Z.; Pandey, A.; Schulick, R.; Argani, P.; Hidalgo, M.; Iacobelli, S.; Goggins, M.; et al. Mac-2-binding protein is a diagnostic marker for biliary tract carcinoma. Cancer 2004, 101, 1609–1615. [Google Scholar] [CrossRef] [PubMed]
  93. Navaneethan, U.; Gutierrez, N.G.; Jegadeesan, R.; Venkatesh, P.G.; Poptic, E.; Liu, X.; Sanaka, M.R.; Jang, S.; Vargo, J.J.; Parsi, M.A. Vascular endothelial growth factor levels in bile distinguishes pancreatic cancer from other etiologies of biliary stricture: A pilot study. Dig Dis Sci 2013, 58, 2986–2992. [Google Scholar] [CrossRef] [PubMed]
  94. Ayaru, L.; Stoeber, K.; Webster, G.J.; Hatfield, A.R.; Wollenschlaeger, A.; Okoturo, O.; Rashid, M.; Williams, G.; Pereira, S.P. Diagnosis of pancreaticobiliary malignancy by detection of minichromosome maintenance protein 5 in bile aspirates. Br J Cancer 2008, 98, 1548–1554. [Google Scholar] [CrossRef] [PubMed]
  95. Sato, Y.; Harada, K.; Sasaki, M.; Yasaka, T.; Nakanuma, Y. Heat shock proteins 27 and 70 are potential biliary markers for the detection of cholangiocarcinoma. Am J Pathol 2012, 180, 123–130. [Google Scholar] [CrossRef] [PubMed]
  96. Shen, J.; Wang, W.Z.; Wu, J.D.; Feng, B.; Chen, W.; Wang, M.; Tang, J.C.; Wang, F.Q.; Cheng, F.; Pu, L.Y.; et al. Comparative proteomic profiling of human bile reveals ssp411 as a novel biomarker of cholangiocarcinoma. Plos One 2012, 7. [Google Scholar] [CrossRef] [PubMed]
  97. Budzynska, A.; Nowakowska-Dulawa, E.; Marek, T.; Boldys, H.; Nowak, A.; Hartleb, M. Differentiation of pancreatobiliary cancer from benign biliary strictures using neutrophil gelatinase-associated lipocalin. J Physiol Pharmacol 2013, 64, 109–114. [Google Scholar] [PubMed]
  98. Zabron, A.A.; Horneffer-van der Sluis, V.M.; Wadsworth, C.A.; Laird, F.; Gierula, M.; Thillainayagam, A.V.; Vlavianos, P.; Westaby, D.; Taylor-Robinson, S.D.; Edwards, R.J.; et al. Elevated levels of neutrophil gelatinase-associated lipocalin in bile from patients with malignant pancreatobiliary disease. Am J Gastroenterol 2011, 106, 1711–1717. [Google Scholar] [CrossRef]
  99. Chiang, K.C.; Yeh, T.S.; Wu, R.C.; Pang, J.H.S.; Cheng, C.T.; Wang, S.Y.; Juang, H.H.; Yeh, C.N. Lipocalin 2 (lcn2) is a promising target for cholangiocarcinoma treatment and bile lcn2 level is a potential cholangiocarcinoma diagnostic marker. Sci Rep-Uk 2016, 6. [Google Scholar] [CrossRef]
  100. Sato, Y.; Harada, K.; Sasaki, M.; Nakanuma, Y. Clinicopathological significance of s100 protein expression in cholangiocarcinoma. J Gastroenterol Hepatol 2013, 28, 1422–1429. [Google Scholar] [CrossRef]
  101. Laohaviroj, M.; Potriquet, J.; Jia, X.; Suttiprapa, S.; Chamgramol, Y.; Pairojkul, C.; Sithithaworn, P.; Mulvenna, J.; Sripa, B. A comparative proteomic analysis of bile for biomarkers of cholangiocarcinoma. Tumour Biol 2017, 39, 1010428317705764. [Google Scholar] [CrossRef]
  102. Chen, C.Y.; Lin, X.Z.; Wu, H.C.; Shiesh, S.C. The value of biliary amylase and hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein i (hip/pap-i) in diagnosing biliary malignancies. Clin Biochem 2005, 38, 520–525. [Google Scholar] [CrossRef]
  103. Chen, C.Y.; Tsai, W.L.; Wu, H.C.; Syu, M.J.; Wu, C.C.; Shiesh, S.C. Diagnostic role of biliary pancreatic elastase for cholangiocarcinoma in patients with cholestasis. Clin Chim Acta 2008, 390, 82–89. [Google Scholar] [CrossRef] [PubMed]
  104. Dhar, D.K.; Damink, S.W.M.O.; Brindley, J.H.; Godfrey, A.; Chapman, M.H.; Sandanayake, N.S.; Andreola, F.; Mazurek, S.; Hasan, T.; Malago, M.; et al. Pyruvate kinase m2 is a novel diagnostic marker and predicts tumor progression in human biliary tract cancer. Cancer 2013, 119, 575–585. [Google Scholar] [CrossRef] [PubMed]
  105. Han, J.Y.; Ahn, K.S.; Baek, W.K.; Suh, S.I.; Kim, Y.H.; Kim, T.S.; Kang, K.J. Usefulness of bile as a biomarker via ferroptosis and cysteine prenylation in cholangiocarcinoma; role of diagnosis and differentiation from benign biliary disease. Surgical Oncology-Oxford 2020, 34, 174–181. [Google Scholar] [CrossRef] [PubMed]
  106. Razumilava, N.; Gleeson, F.C.; Gores, G.J. Awareness of tract seeding with endoscopic ultrasound tissue acquisition in perihilar cholangiocarcinoma. Am J Gastroenterol 2015, 110, 200. [Google Scholar] [CrossRef] [PubMed]
  107. Pelsang, R.E.; Johlin, F.C. A percutaneous biopsy technique for patients with suspected biliary or pancreatic cancer without a radiographic mass. Abdominal Imaging 1997, 22, 307–310. [Google Scholar] [CrossRef]
  108. Vogel, A.; Bridgewater, J.; Edeline, J.; Kelley, R.K.; Klumpen, H.J.; Malka, D.; Primrose, J.N.; Rimassa, L.; Stenzinger, A.; Valle, J.W.; et al. Biliary tract cancer: Esmo clinical practice guideline for diagnosis, treatment and follow-up. Ann Oncol 2023, 34, 127–140. [Google Scholar] [CrossRef]
  109. Borger, D.R.; Tanabe, K.K.; Fan, K.C.; Lopez, H.U.; Fantin, V.R.; Straley, K.S.; Schenkein, D.P.; Hezel, A.F.; Ancukiewicz, M.; Liebman, H.M.; et al. Frequent mutation of isocitrate dehydrogenase and in cholangiocarcinoma identified through broad-based tumor genotyping. Oncologist 2012, 17, 72–79. [Google Scholar] [CrossRef]
  110. Nakamura, H.; Arai, Y.; Totoki, Y.; Shirota, T.; Elzawahry, A.; Kato, M.; Hama, N.; Hosoda, F.; Urushidate, T.; Ohashi, S.; et al. Genomic spectra of biliary tract cancer. Nat Genet 2015, 47, 1003–1010. [Google Scholar] [CrossRef]
  111. Valle, J.W.; Kelley, R.K.; Nervi, B.; Oh, D.Y.; Zhu, A.X. Biliary tract cancer. Lancet 2021, 397, 428–444. [Google Scholar] [CrossRef]
  112. Hwang, J.; Kim, Y.K.; Park, M.J.; Lee, M.H.; Kim, S.H.; Lee, W.J.; Rhim, H.C. Differentiating combined hepatocellular and cholangiocarcinoma from mass-forming intrahepatic cholangiocarcinoma using gadoxetic acid-enhanced mri. J Magn Reson Imaging 2012, 36, 881–889. [Google Scholar] [CrossRef]
  113. Chong, Y.S.; Kim, Y.K.; Lee, M.W.; Kim, S.H.; Lee, W.J.; Rhim, H.C.; Lee, S.J. Differentiating mass-forming intrahepatic cholangiocarcinoma from atypical hepatocellular carcinoma using gadoxetic acid-enhanced mri. Clin Radiol 2012, 67, 766–773. [Google Scholar] [CrossRef] [PubMed]
  114. Weber, S.M.; Ribero, D.; O’Reilly, E.M.; Kokudo, N.; Miyazaki, M.; Pawlik, T.M. Intrahepatic cholangiocarcinoma: Expert consensus statement. Hpb 2015, 17, 669–680. [Google Scholar] [CrossRef] [PubMed]
  115. Claessen, M.M.; Vleggaar, F.P.; Tytgat, K.M.; Siersema, P.D.; van Buuren, H.R. High lifetime risk of cancer in primary sclerosing cholangitis. J Hepatol 2009, 50, 158–164. [Google Scholar] [CrossRef] [PubMed]
  116. Chapman, R.; Fevery, J.; Kalloo, A.; Nagorney, D.M.; Boberg, K.M.; Shneider, B.; Gores, G.J.; American Association for the Study of Liver, D. Diagnosis and management of primary sclerosing cholangitis. Hepatology 2010, 51, 660–678. [Google Scholar] [CrossRef] [PubMed]
  117. Tischendorf, J.J.; Hecker, H.; Kruger, M.; Manns, M.P.; Meier, P.N. Characterization, outcome, and prognosis in 273 patients with primary sclerosing cholangitis: A single center study. Am J Gastroenterol 2007, 102, 107–114. [Google Scholar] [CrossRef] [PubMed]
  118. Levy, C.; Lymp, J.; Angulo, P.; Gores, G.J.; Larusso, N.; Lindor, K.D. The value of serum ca 19-9 in predicting cholangiocarcinomas in patients with primary sclerosing cholangitis. Dig Dis Sci 2005, 50, 1734–1740. [Google Scholar] [CrossRef] [PubMed]
  119. Moreno Luna, L.E.; Kipp, B.; Halling, K.C.; Sebo, T.J.; Kremers, W.K.; Roberts, L.R.; Barr Fritcher, E.G.; Levy, M.J.; Gores, G.J. Advanced cytologic techniques for the detection of malignant pancreatobiliary strictures. Gastroenterology 2006, 131, 1064–1072. [Google Scholar] [CrossRef]
  120. Boberg, K.M.; Jebsen, P.; Clausen, O.P.; Foss, A.; Aabakken, L.; Schrumpf, E. Diagnostic benefit of biliary brush cytology in cholangiocarcinoma in primary sclerosing cholangitis. J Hepatol 2006, 45, 568–574. [Google Scholar] [CrossRef]
Table 1. Clinicopathological features of cholangiocarcinoma.
Table 1. Clinicopathological features of cholangiocarcinoma.
Title 1 Growth pattern Precancerous lesion Main etiology
iCCA – small duct type Mass-forming None Chronic hepatitis
Cirrhosis
iCCA – large duct type Periductal infiltrating BilIN Hepatolithiasis
Liver flukes
PSC
Intraductal growing IPNB, MCN, ITNB
pCCA - dCCA Flat or nodular sclerosing BilIN
Intraductal papillary IPNB, MCN, ITNB
iCCA, intrahepatic cholangiocarcinoma; BilIN, biliary intraepithelial neoplasia; IPNB, intraductal papillary neoplasm of the bile duct; MCN, mucinous cystic neoplasm; ITNB, intraductal tubular neoplasm of the bile duct; pCCA, perihilar cholangiocarcinoma; dCCA, distal cholangiocarcinoma; PSC, primary sclerosing cholangitis.
Table 2. Diagnostic studies of bile EVs, nucleic acids, and proteins.
Table 2. Diagnostic studies of bile EVs, nucleic acids, and proteins.
Biomarkers n ROC-AUC Sensitivity (%) Specificity (%) Reference
Exosomal cargoes
MicroRNA (miR-191, miR-486-3p, miR-1274b, miR-16 & miR-484)
MicroRNA (miR-483-5p, miR-126-3p)
MicroRNA (miR-141-3p, miR-200a-3p, miR-200c-3p, miR-200b-3p and ENST00000588480.1)
LncRNA (ENST00000588480.1 & ENST00000517758.1)
Circle-RNA (circ-CCAC1)
Protein (Claudin-3/CLDN3)
96
92
100

91
0.81, 0.74
0.757~0.869

0.709		 0.67
0.811, 0.73
0.63~0.83

0.829		 0.69
0.811, 0.865
0.6~0.867

0.589		 [63]
[64]
[65]

[66]
316
20 		0.857
0.945
0.875
0.875		 [57]
[67]
DNA
K-ras mutation
K-ras mutation
KRAS
K-ras mutation & p53 mutation
K-ras mutation & p53 mutation
K-ras mutation & p53 mutation
TP53, ERBB2, and KRAS
KRAS, TP53, CDKN2A, SMAD4, BRAF
Promotor methylation INK4a/ARF
Promotor methylation of COD1, CNRIP1, SEPT9 & VIM
Methylation of DKK3, p16, SFRP2, DKK2, NPTX2 and ppENK
CCND2, CDH13, GRIN2B, RUNX3, and TWIST1
Gene mutations in KRAS, TP53, SMAD4, and CDNK2A; Methylation changes in SOX17, 3-OST-2, NXPH1, SEPT9 and TERT
150 tumor-related genes (Wildly target)
520 tumor-related genes (Wildly target)
RNA
Human telomerase reverse transcriptase mRNA
miR-9, miR-145, miR-944
RNU2-1f
miR-412, miR640, miR-1537 & miR-3189
miR-30d-5, miR-92a-3p
20
115
46
43
109
50
49
42
60
243
80
125
241

10
28

20
18
23
83
106 		0.667

0.738
0.742
0.564/ 0.508
0.783, 0.750
0.733
0.955
0.737/ 0.715
0.84~0.98
0.775







0.765~0.975
0.856
0.78~0.81
0.730, 0.652		 0.33
0.25
0.476
0.526
0.279/ 0.047
0.567, 0.5
0.467
0.909
0.536/ 0.462
0.67~0.96
0.773
0.71~0.83
0.92

0.947
0.955

0.833

0.67
0.5~0.67
0.811, 0.657		 1
0.96
1
0.958
0.848/ 0.970
1, 1
1
1
0.937/ 0.969
0.93~0.98
0.778
0.94
0.98

0.999


1

0.91
0.89~0.92
0.605, 0.667		 [68]
[69]
[70]
[71]
[72]
[73]
[74]
[70]
[75]
[60]
[76]
[77]
[78]

[79]
[80]

[81]
[82]
[83]
[84]
[85]
Protein
CEACAM6
CEACAM6
SVV, CA199
MUC1
MUC4
MUC5AC
Mac-2BP
VEGF
MCM2, MCM5
HSP27 & HSP70
SSP411
NGAL
NGAL
LCN2/NGAL
S100P
sB7-H3
α-1-antitrpysin
Amylase
PE-3B/amylase
M2-PK
GSH, hydrogen peroxide, GPx, Fe2+, FNTA
73
41
102
68
134
46
78
53
42
20
67
40
38
144
24
323
8
239
68
167
46 		0.74
0.92
0.78, 0.75
0.85

0.85
0.70
0.89
0.80
0.86, 0.81
0.913*
0.74
0.76
0.81
0.861
0.878
0.833
0.751
0.877

0.683~0.852		 87.5
83.3
67.3, 96.4
90.0
27
75
69
99.3

90, 80
90.0
77.3
94
87
92.9
81.2
80
66
81.8
90.3
67.9~100		 69.1
93.1
80.9, 46.7
76.3
93
76.9
67
88.9

90, 80
83.3
77.2
55
75
70
81.6
75
74
89.3
84.3
52.9~76.5		 [86]
[87]
[88]
[89]
[90]
[91]
[92]
[93]
[94]
[95]
[96]
[97]
[98]
[99]
[100]
[61]
[101]
[102]
[103]
[104]
[105]
* Serum samples for ROC analysis.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Copyright: This open access article is published under a Creative Commons CC BY 4.0 license, which permit the free download, distribution, and reuse, provided that the author and preprint are cited in any reuse.
Prerpints.org logo

Preprints.org is a free preprint server supported by MDPI in Basel, Switzerland.

facebook logotwitter logolinkedin logo
MDPI Initiatives
Important Links
Subscribe

Disclaimer

Terms of Use

Privacy Policy

© 2024 MDPI (Basel, Switzerland) unless otherwise stated