Understanding the Clinical Significance of MUC5AC in Biliary Tract Cancers

Simple Summary Biliary tract cancers (BTC) are tumors arising from cells lining ducts in the liver that facilitate the transport of bile into the gastrointestinal tract. They are often divided into two broad groups, cholangiocarcinoma and gallbladder cancers. They are aggressive tumors with limited treatment options that are often ineffective and have bad outcomes. In current clinical practice, there are no good tests to identify these cancers in early stages and predict their aggressiveness or response to the available treatments. We shed light on the role of a glycoprotein, MUC5AC, in BTC including its potential impact on biliary cancer development. We discuss the clinical evidence surrounding the use of MUC5AC when detected in BTC patients’ tumor tissue and blood and its potential use in managing these cancers. Abstract Biliary tract cancers (BTC) arise from biliary epithelium and include cholangiocarcinomas or CCA (including intrahepatic (ICC) and extrahepatic (ECC)) and gallbladder cancers (GBC). They often have poor outcomes owing to limited treatment options, advanced presentations, frequent recurrence, and poor response to available systemic therapy. Mucin 5AC (MUC5AC) is rarely expressed in normal biliary epithelium, but can be upregulated in tissues of benign biliary disease, premalignant conditions (e.g., biliary intraepithelial neoplasia), and BTCs. This mucin’s numerous glycoforms can be divided into less-glycosylated immature and heavily-glycosylated mature forms. Reported MUC5AC tissue expression in BTC varies widely, with some associations based on cancer location (e.g., perihilar vs. peripheral ICC). Study methods were variable regarding cancer subtypes, expression positivity thresholds, and MUC5AC glycoforms. MUC5AC can be detected in serum of BTC patients at high concentrations. The hesitancy in developing MUC5AC into a clinically useful biomarker in BTC management is due to variable evidence on the diagnostic and prognostic value. Concrete conclusions on tissue MUC5AC are difficult, but serum detection might be relevant for diagnosis and is associated with poor prognosis. Future studies are needed to further the understanding of the potential clinical value of MUC5AC in BTC, especially regarding predictive and therapeutic value.


Introduction
Biliary tract cancers (BTC) are tumors arising from the biliary epithelium [1]. They are often subclassified into gallbladder cancer (GBC) and cholangiocarcinoma (CCA), the latter of which is subdivided into intrahepatic (ICC) and extrahepatic cholangiocarcinoma (ECC); ECC can be further subdivided into perihilar and distal [1,2]. These cancers are relatively uncommon with around 200,000 cases reported worldwide in 2017 [3][4][5]. However, there Mucins have been extensively studied in the gastrointestinal tract, where each organ relies on different mucins for transport, secretion, and protection against bacteria [47]. Human mucins can be divided into two categories: gel-forming mucins which are secreted to form a mucus layer (including MUC2, MUC5AC, MUC5B, and MUC6) and transmembrane mucins which are expressed on the apical surface of epithelial cells (including MUC1, MUC3, MUC4, MUC13, and MUC17) [48]. MUC4 is a ligand for erythroblastic oncogene B2 (ErbB2) receptor tyrosine, and MUC1 affects the immune response to tumor cells and aids in metastasis [37,[49][50][51]. Expression of both mucins is often associated with poor outcomes. MUC5B expression was studied in many GB pathologies (stones, infection, and inflammation), but its clinical significance in GBC or BTC has not been determined yet [52][53][54]. MUC2 has an anti-inflammatory effect, and its expression does not seem to affect the outcomes in BTCs [50,55]. MUC6 is a "protective mucin", a good prognostic marker, and is often detected in well-differentiated tumors [50,56].
Secreted mucins in humans can undergo O-glycosylation. During this modification, various monosaccharides are attached to immature mucins intracellularly via enzymes through the Golgi and endoplasmic reticulum. Mucins subsequently reach the apical surface and are secreted in mature heavily-glycosylated form [74]. Research in human colorectal adenocarcinoma cell lines has shown that MUC5AC molecules similarly undergo an assembly process in cells to proceed from non-glycosylated immature monomers to glycosylated dimers to fully glycosylated mature oligomers which are secreted [75].
The various forms of MUC5AC (heavily glycosylated vs. less glycosylated) can be detected with different antibodies. CLH2 antibodies against the core tandem repeat region of MUC5AC have been shown to bind weakly to heavily glycosylated mature MUC5AC (and strongly to immature or less glycosylated MUC5AC) in epithelial cytoplasm and are considered antibodies against "immature" MUC5AC [76][77][78][79]. Another antibody, M5P-b1, is also against the core tandem repeat region and stains epithelial cytoplasm similar to CLH2. M5P-b1 will be considered as binding immature MUC5AC in this review [80]. On the other hand, antibodies which bind strongly to both heavily-glycosylated and less-glycosylated MUC5AC are considered antibodies against "mature" MUC5AC. These include MAN-5ACI, Lum5-1 EU-batch, 21M1, and 45M1 [76,81,82]. For some antibodies (such as MSVA-109, polyclonal antibody (Dako, Germany), and manufactured ELISA kits), it is unknown whether they bind immature or mature MUC5AC (see Table S1 in Supplementary Materials for a list of relevant MUC5AC antibodies for this review) [38,83,84].
In the literature, aberrant glycan variants of MUC5AC are associated with adenocarcinomas from various tissues (stomach, ampulla of Vater, colon, lung, breast, and ovary) [66]. In another study, mucinous ovarian cancers are associated with blood-group-ABH glycan variants of MUC5AC compared to control ovaries or serous ovarian cancers (using PLA verified with mass spectrometry) [85]. Aberrant glycosylation of MUC5AC has also been studied in pancreatic cancer (with various techniques including immunohistochemistry, mass spectrometry, and serum ELISA) where certain variants of MUC5AC are associated with malignant transformation and promotion of carcinogenesis [60][61][62][63][64][65].
The remainder of this review will focus on the potential role of MUC5AC in biliary tissues and BTC.
The literature suggests that MUC5AC is rarely expressed in the normal epithelium of biliary ducts or peribiliary mucous glands but is frequently expressed in normal gallbladder surface epithelium. Importantly, this distinction is independent of whether immature or mature MUC5AC antibodies are used [37,40,80,83,90,[93][94][95][96].

Benign Biliary Disease
MUC5AC is expressed or upregulated in many benign biliary disease tissues, as detected using immature CLH2 or M5P-b1 antibodies, mature 45M1 antibodies, or polyclonal Dako antibodies, including primary sclerosing cholangitis (PSC), hepatolithiasis, recurrent pyogenic cholangitis, bile duct adenomas, gallbladder adenomas, and chronic cholecystitis [80,84,90,94,[96][97][98][99][100]. There might be a relatively higher expression detected in biliary epithelial cells (bile duct or gallbladder) than peribiliary mucus glands or goblet cells (as seen in cholecystitis with 45M1 antibodies or hepatolithiasis/biliary obstruction tissues with M5P-b1 antibodies) [80,97]. At first glance, MUC5AC upregulation seems to be more consistent in studies on chronic cholecystitis (93-94.3% positivity in three studies using various antibodies) than studies on hepatolithiasis (17%, 40%, and 89% in three studies, respectively, using immature antibodies) [80,84,[97][98][99][100]. However, no studies directly compared MUC5AC expression levels between benign obstructive diseases of the bile ducts versus gallbladder to see if there are any statistically significant differences between these two classes. Lastly, the research on serum or bile MUC5AC levels in benign biliary disease (not shown in Table 1) is somewhat limited. One study using mature Lum5-1 EU-batch antibodies via Western Blot found that MUC5AC was present in the bile of patients with PSC and other benign biliary diseases, but much lower in the serum (although statistical comparisons were not drawn) [37]. Another study using USCN Life Science ELISA kit on serum found that serum MUC5AC levels were higher in patients with benign biliary disease than in healthy controls but did not perform statistical comparisons to see if the difference was significant [38].

MUC5AC Expression in BTC Tissue
Studies analyzing MUC5AC expression in BTC tissues via IHC are summarized in Table 2. Among studies which analyzed CCA tissues using immature CLH2 antibodies, the percentage of MUC5AC-positive tumors ranged from 8% to 100% [44,50,83,90,93,99,100,[102][103][104][105]. For studies using antibodies against mature MUC5AC (MAN-5ACI, 45M1, or novel S121), the percentage of MUC5AC-positive CCA tumors ranged from 26.5% to 93% [40,44,95,106]. Only one study used both immature (CLH2) and mature (45M1) antibodies on the same tissue (ICC in this case) [44]. The results showed that 34 tumors were stained with both antibodies, 6 were stained with only CLH2, and 6 were stained with only 45M1, revealing no difference in staining frequency between the two antibodies. However, a difference in the staining location was noted (cytoplasm for CLH2 and cytoplasm/biliary lumen/extracellular stroma for 45M1 antibodies). Another study using MSVA-109 antibodies (unknown maturity) found 21% of CCA to be MUC5AC-positive [83]. Interestingly, MUC5AC tissue expression in CCA may vary depending on site. Three studies found that MUC5AC was more frequently expressed in perihilar or hilar (adjacent to the hilum) ICC than peripheral (non-hilar) ICC [44,104,105]. Another study found that ECCs expressed more MUC5AC than ICCs (70.6% vs. 47.1%) [50]. These results suggest that perihilar ICC and ECC may be more likely than peripheral ICC to express or upregulate MUC5AC, perhaps due to an unknown difference in location-specific carcinogenesis mechanisms.  Tumors with 2+ intensity in >70% or 3+ intensity in >30% of cells considered strongly positive; B = The number of MUC5ACpositive lesions did not differ significantly between BilIN and CCA (p = 0.38 between low-grade and high-grade BilIN, p = 0.40 between high-grade BilIN and CCA, and p = 0.62 between low-grade BilIN and CCA); C = A higher degree of MUC5AC expression was observed in ECCs than ICCs (p = 0.026); D = MUC5AC was strongly expressed in all IPNLs and was expressed in 75% of well-differentiated and 33% of moderately-differentiated non-IG-ICCs. No poorly differentiated non-IG-ICCs exhibited MUC5AC expression; E = MUC5AC expression was significantly higher in CCA than normal bile ducts (p < 0.05); F = MUC5AC expression is more frequently observed in ICC with BilIN (83%) and ICC with IPNB than non-neoplastic epithelium (p < 0.001); G = Hilar ICC more often expressed MUC5AC than peripheral ICC (p < 0.0001); H = Hilar CCA defined as predominantly involving the right and left hepatic ducts and junction, whereas more proximal tumors were considered ICC. Perihilar ICC is defined as a ductal morphology and minor tubular components (if present) only at the tumor-liver interface. ICC beyond these criteria is called peripheral ICC; K = MUC5AC expressed more frequently in hilar CCA and perihilar ICC than peripheral ICC (p < 0.05); L = Hilar ICC classified as tumors as involving the second branch of the bile duct localized in the hilar portion of the liver. Peripheral ICC classified as tumors in the hepatic periphery; M = MUC5AC expression more frequent in hilar than peripheral ICC (p < 0.0001) when visualized with 45M1 or CLH2 antibodies; N = MUC5AC expression significantly lower in GBC than normal gallbladder mucosa or gallbladder adenomas (p < 0.01); P = MUC5AC expression significantly less frequent in GBC (18 GBC + 3 BilIN) (28.57%) than chronic cholecystitis (87.19%) (p < 0.001); Q = MUC5AC expression rates significantly lower in GBC than in chronic cholecystitis (p < 0.01), peritumoral tissues (p < 0.01), or adenomatous polyp (p < 0.05).
In studies that analyzed GBC using immature CLH2 antibodies, the percentage of MUC5AC-positive tumors ranged from 16.7% to 81.8% [50,94,98,107]. One study that analyzed GBC with mature 45M1 antibodies found 80% of GBC overall to be MUC5ACpositive, and another using polyclonal Dako antibodies (unknown whether immature or mature) observed this in 51.9% of GBC [84,97]. Finally, one study which used mature 21M1 antibodies on general BTC (CCA and GBC tissues) found only 10% of samples to be MUC5AC-positive [37].
Evidently, there is a wide variability in the literature regarding reported tissueexpression levels of MUC5AC in BTC. Although there are ample differences in study methods such as the cancer subtype analyzed, MUC5AC glycoform targeted, or positivity threshold selected, there is no clear association between these factors and the variability of the evidence.

MUC5AC in Biliary Pathogenesis and Carcinogenesis
The role of MUC5AC in biliary tumorigenesis and metastasis is unclear but we obtained an insight into it through the work of Silsirivanit et al. [42]. They developed a novel monoclonal antibody CA-S27 from pooled CCA tissues that bound to a Lewis-a (Le(a))associated glycan conjugated to mature MUC5AC [42]. High levels of CA-S27-MUC5AC (i.e., mature MUC5AC) were detected in CCA patients, and had reliable diagnostic and prognostic value (see Table 3 and Table 5). The same experiment showed that suppression of CA-S27-MUC5AC expression in CCA cell lines significantly reduced proliferation, adhesion, migration, and invasion. This study's results possibly suggest that mature MUC5AC may be involved in pathways in CCA to promote carcinogenesis and metastasis and is similar to its established effect in other tumors [58,108]. The interactions of MUC5AC with other molecules/mutations in biliary pathologies and cancers are summarized in Figure 1 and discussed below. Aquaporin-1 (AQP-1) has been proposed as a regulator of MUC5AC expression in ICC [109]. AQP-1 is an aquaporin channel that serves as a water transporter for bile in normal cholangiocytes [110]. They are involved in bile secretion and microbial infections. AQP-1 is upregulated in biliary dysplasia but downregulated in invasive ICC. Decreased AQP-1 expression in ICC was associated with increased MUC5AC expression (detected with CLH2 immature antibodies). Additionally, decreased AQP-1 was associated with lymph node metastasis and increased MUC5AC was associated with decreased survival. The authors hypothesized that the downregulation of AQP-1 induces MUC5AC expression in invasive ICC and suggested that AQP-1 may serve a direct role in ICC carcinogenesis. Notably, AQP-1 has been potentially linked with carcinogenesis in other cancers, including lung and colon [111,112].
Research has also connected ecotropic virus integration site 1 protein homolog (EVI-1, a transcription factor) with MUC5AC expression in ICC [113]. EVI-1 upregulation was seen in half of ICC tumors and all IPNBs. EVI-1-positive ICC is a more aggressive disease with advanced stage at diagnosis and decreased survival. EVI-1-positive ICC was more likely to express MUC5AC. An anti-EVI-1 molecule called pyrrole-imidazole polyamide PIP1 was recently designed which inhibits EVI-1 in vitro, and the study authors suggested PIP1 should be investigated as a possible targeted treatment for EVI-1-positive ICC. This could point to a potential role for the measurement of EVI-1 and subsequent EVI-1-targeted treatment in MUC5AC-positive ICC patients.
Trefoil Factor 1 ((TFF1); an estrogen-responsive signaling protein) and MUC5AC were upregulated and correlated in hepatolithiasis, biliary dysplasia, and CCA [114,115]. When TTF1 was applied to CCA cell lines, invasion was stimulated. Additionally, when cell lines were grown in media lacking estrogen agonists, TFF1 expression decreased. The results suggest that TFF1/MUC5AC interactions may be important in the pathogenesis of CCA and suggest a potential role for treatment of TFF1-positive CCA with estrogen antagonists. Further research could investigate a possible utility for measuring TFF1 expression in MUC5AC-positive CCA.
MUC5AC expression in BTC precursor neoplasms was linked with mutations in KRAS (a signaling protein in the RAS/MAPK pathway) and the upregulation of the Wnt/β-catenin pathway [87,116]. Research has also linked the Wnt pathway to upregulation of MUC5AC in cultured biliary cells [117]. Treatment of cells with micro-RNA miR93 to repress the Wnt pathway led to decreased MUC5AC. The RAS/MAPK and Wnt/β-catenin pathways are involved in cell proliferation and homeostasis, and have also been linked to carcinogenesis in many gastrointestinal cancers. Therefore, a potential association with MUC5AC expression in BTC precursor neoplasms and biliary cell lines could point to interactions of MUC5AC with the cellular machinery involved in biliary carcinogenesis [118,119]. Research has also linked mutations in chromatin modifiers (ARID1A, BAP1, and KMT2C) with MUC5AC-positive IPNBs which are similar to mutations seen in CCA, which points to the possible aberrant epigenetic regulation of MUC5AC in BTC [116].
Cholelithiasis is a known risk factor for BTC [120]. A study showed that cholesterol crystals are associated with the upregulation of inflammasomes and increased expression of MUC5AC in gallbladder tissue from cholelithiasis patients [121]. The results indicated that MUC5AC secretion could be decreased in cultured cholesterol-exposed biliary cells through three ways: by inhibiting inflammasomes with siRNAs, inhibiting IL-1 receptor, or inhibiting caspase-1 with Ac-YVAD. These results point to links between cholestasis and MUC5AC expression that could potentially be relevant for using MUC5AC to track inflammation in patients with cholestasis and stratifying their risk for BTC.
Chronic infections and hepatolithiasis are known risk factors for BTC, and these conditions can be associated with chronic exposure of the biliary tract to bacterial lipopolysaccharide (LPS) [122][123][124]. Research on biliary cells has shown that LPS exposure is associated with the upregulation of MUC5AC, possibly through the upregulation of p38-mitogenactivated protein kinase (p38 MAPK) and the downregulation of micro-RNA miR-130b (which disinhibits transcription factor Sp1 which binds the MUC5AC promoter) [125][126][127]. Treatment of LPS-exposed cultured biliary cells with small interference RNA (siRNA) to silence p38 MAPK led to the downregulation of MUC5AC and inflammatory cytokines. Inhibition of cyclo-oxygenase 2 (COX-2) or antagonism of prostaglandin E2 (PGE2) in LPS-treated cultured biliary cells were associated with reduced MUC5AC expression [128]. PGE2 caused upregulation of p38 MAPK, and treatment with a p38 MAPK inhibitor correlated to reduced MUC5AC. MUC5AC and PGE2 were elevated in the bile of hepatolithiasis patients. The literature illuminates the important roles of LPS and p38 MAPK in pathways of biliary inflammation, which correlates with MUC5AC expression.
EGFR (epithelial growth factor receptor) is an important player in normal cell proliferation, but mutations or upregulation in EGFR are associated with many cancers [129]. LPS upregulates EGFR activation in human-cultured biliary epithelial cells [130]. When EGFR activation was inhibited, MUC5AC was downregulated. Treatment of rats affected by chronic proliferative cholangitis ((CPC); a hyperproliferative disorder connected with carcinogenesis) with an anti-EGFR monoclonal antibody (panitumumab) was associated with decreased EGFR expression, lower MUC5AC expression, and decreased hyperproliferation [131]. These results suggest a possible connection between EGFR and MUC5AC expression in biliary cells, and point to a possible role for EGFR-inhibitors in MUC5ACpositive biliary proliferative disorders, although more human research is needed.
Overall, the exact mechanisms of MUC5AC contributing to BTC carcinogenesis are currently unknown. More research is needed to understand it better.

Tissue MUC5AC Testing
The ambiguous sensitivity of MUC5AC for BTC in tissue samples can be inferred from the numerous BTC tissue studies mentioned earlier, where the percentage of MUC5ACpositive tumors for CCA and GBC was highly variable, ranging from 8% to 100% (CCA) and 16.67% to 90% (GBC), depending on tumor subtype and MUC5AC antibodies (see Table 1). Many studies did not compare the frequency of MUC5AC expression between BTC, other diseases, and/or healthy subjects. However, differences in MUC5AC expression between BTC and other conditions have occasionally been studied in recent years.
Tissue MUC5AC testing, specifically immature MUC5AC (CLH2-reactive), can distinguish malignant tissues (CCA) from healthy controls but not from benign or premalignant designs such as PSC, BilIN, and IPNB [90,93,99]. As mentioned earlier, several studies on gallbladder tissues (with immature CLH2 or unknown-maturity polyclonal Dako antibodies) found significantly lower MUC5AC expression in invasive GBC than gallbladder polyps, adenomas, or chronic cholecystitis [84,94,98]. In one study, mature MUC5AC (21M1-reactive) was detected in just 10% of BTC tissues but not in any healthy biliary tissues [37]. Another study using CLH2 antibodies found that pancreatic ductal carcinoma tissues expressed MUC5AC significantly more frequently than ICC [75]. In addition, a 2004 study using CLH2 antibodies on various carcinoma tissues found that MUC5AC was variably expressed in different gastrointestinal cancers (26% of colorectal, 67% of esophageal, 45% of CCA, 73% of pancreatic ductal, and 55% of stomach carcinomas) but did not draw statistical comparisons between these cancers [76].
Overall, there is obvious variability in detected MUC5AC expression in BTC tissues and unclear usefulness in differentiating BTC from other conditions in tissue samples. However, research is limited with variable study methods, and therefore more studies are needed to elucidate the potential diagnostic relevance of tissue MUC5AC expression for BTC.

Serum MUC5AC Testing
Detection of MUC5AC in serum or bile has been widely studied in recent years for diagnostic purposes in BTC (Table 3). A 2016 systematic review and meta-analysis looked at serum MUC5AC across six studies [36][37][38]40,95,132,133]. This meta-analysis found the pooled area under the curve (AUC) for diagnostic performance of serum MUC5AC for distinguishing BTC from a variety of conditions with statistical significance (including normal subjects, benign biliary diseases, and non-biliary GI cancers) was 0.9138 indicating excellent performance (the range of reported AUC values among the studies was from 0.814 to 0.97). Authors suggested serum MUC5AC could be used to confirm BTC with excellent performance when an indeterminate biliary lesion is found on imaging. However, their data also suggested that serum MUC5AC could miss early CCA. Similar findings were reported in another meta-analysis from 2019 (which included one extra study) [41,134].
Notably, one study also examined bile MUC5AC and noted serum MUC5AC was higher in CCA than benign biliary disease and bile MUC5AC was higher in benign biliary disease [132]. The authors found that a serum/bile MUC5AC ratio demonstrated greater diagnostic performance (AUC 0.97) for CCA than serum MUC5AC alone (AUC 0.82). Finally, three other studies with varying results are described in Table 3 [42,43,135]. Of note, two analyzed serum biomarker panels which showed potential promise for distinguishing CCA from PSC or healthy patients [43,135]. Meanwhile, whilst serum MUC5AC may potentially have relevance as a future diagnostic tool for BTC (either alone or in a panel), there is a variability in performance across the literature, which needs to be examined.   and bile MUC4 analyses increased sensitivity for detecting BTC to 58% but decreased specificity to 87%; E = Serum MUC5AC levels significantly higher in BTC than BBD (p < 0.01); F = The median serum S121 value was elevated significantly in CCA patients compared to comparison groups (p < 0.001); G = Serum MUC5AC levels significantly higher in patients with CCA than those with BBD (cholangitis or cholelithiasis) (p = 0.0002); H = Bile MUC5AC levels were significantly higher in patients with BBD than CCA (p < 0.0001); K = Using serum/bile MUC5AC ratio, the AUC for differentiating CCA from cholangitis was 0.94 (95% CI 0.86-1.00; p < 0.0001), between CCA and cholelithiasis was 0.99 (95% CI, 0.98-1.00; p < 0.0001), and between cholangitis and cholelithiasis was 0.93 (95% CI, 0.82-1.00; p = 0.001); L = Serum MUC5AC levels were greater in patients with BTC compared with BBD (p < 0.01) and healthy patients (p < 0.01); M = Serum CA-S27 levels of CCA patients were significantly higher than controls (p < 0.001); N = Serum MUC5AC levels significantly higher in CCA than PSC (p < 0.001); P = A panel combining biomarkers PKM2, CYFRA21.1, MUC5AC, and GGT at 90% specificity gave 81.8% sensitivity and AUC 0.903 for differentiating CCA and PSC; Q = No statistically significant difference in serum MUC5AC between non-biliary GI cancer vs. normal (AUC 0.545 with cut-off of 128 ng/mL; p = 0.492) or CCA vs. non-biliary GI cancer (AUC 0.581 with cut-off of 90.51 ng/mL; p = 0.213); R = A combined panel with biomarkers S100A9, MUC5AC, angiopoietin-2, and CA19-9 to distinguish CCA vs. healthy gave a sensitivity of 90%, specificity of 95%, and AUC 0.975 (p < 0.0001); S = Serum MUC5AC levels were significantly higher in CCA patients than in normal healthy patients (p = 0.032).

Tissue MUC5AC Testing
Four tissue studies associated MUC5AC-positive tumors with decreased overall survival (three using immature CLH2 antibodies and one using both immature CLH2 and mature 45M1) [39,44,109,138]. Four studies found no association between MUC5AC and survival; two used immature CLH2 and two used mature antibodies (MAN-5ACI or 21M1) [37,50,106,107]. In contrast, three associated MUC5AC positivity with improved overall and/or disease-free survival, including one which only showed this association for perihilar ECC and no association for distal ECC [84,136,137]. Two of these analyses used immature CLH2 antibodies and one used polyclonal Dako (unknown MUC5AC maturity). Finally, two studies did not discuss survival outcomes relative to MUC5AC [40,94]. These studies are summarized above in Table 4.
One study using mature MAN-5ACI antibodies associated MUC5AC-positive tumors with more advanced TNM tumor staging and increased frequency of perineural invasion [106]. Two analyses (one with CLH2 and one with CLH2/45M1) found an association with increased frequency of lymph node metastasis [39,44]. One study (using CLH2) found association with higher T category (T2 or greater versus T1) [50]. On the other hand, two studies (one using immature CLH2 and one using unknown maturity polyclonal Dako antibodies) associated MUC5AC positivity with decreased tumor size, including one which only showed this association for perihilar ECC [84,136]. The study using polyclonal Dako also found associations with well-differentiated tumors and lower T category (T1 vs. T4). Six other studies that evaluated prognostic factors found no significant associations with MUC5AC positivity, and finally two studies did not discuss prognostic factors.
In the case reports with tissue MUC5AC analysis (not shown in Table 4), MUC5ACpositive IPNB precursor neoplasms have been associated with DIC/thrombosis as well as local recurrence of tumor after surgery and progression to delayed distant metastasis [139,140]. Another case report of a MUC5AC-negative ICC with local spread to the colon and distant brain metastases, which were surgically removed, noted no recurrence 7 years after the surgeries [141].
Overall, the literature is quite mixed on the prognostic relevance of tissue MUC5AC expression in BTC. There are a wide range of study methods (e.g., positivity thresholds), cancer subtypes, and MUC5AC glycoforms analyzed; however, it is unclear whether these differences could explain any of the variation in reported prognostic relevance. The next subsection will discuss prognostic impact of serum MUC5AC.

Serum MUC5AC Testing
As seen in Table 5, six serum analyses (five using mature MUC5AC antibodies and one using a manufactured ELISA kit) associated increased or positive MUC5AC with decreased survival in BTC [37,38,[40][41][42]133]. One (using CSB-E10109h Cusabio of unknown MUC5AC maturity) found no significant association with survival [135]. The latter noted that a combined panel of four biomarkers including MUC5AC was associated with decreased survival. Among six analyses using mature MUC5AC antibodies, associations were also found between increased MUC5AC and more advanced TNM staging (three studies), larger tumor size (one study), and increased frequency of lymph node metastasis (one study) [38,41,133]. The relative consistency of associations across the literature between increased serum MUC5AC, decreased survival, and poor prognostic factors could potentially indicate a prognostic role for serum MUC5AC in BTC. However, data are limited, and future study is necessary to clarify its reliability and usefulness.

Discussion, Future Directions
MUC5AC might be a potentially useful biomarker for the diagnosis and prognostic outlook of BTC, although more research is needed. While it is rarely expressed in normal biliary epithelial cells, it is expressed or upregulated in benign biliary disease, premalignant precursors, and BTC. MUC5AC is upregulated in the serum of BTC patients, and many studies suggest that serum MUC5AC might be used to differentiate BTC from a variety of other conditions with excellent diagnostic performance (although the data on diagnostic performance of tissue MUC5AC are variable and relatively lacking). Data also suggest that while tissue MUC5AC currently has unclear associations with prognosis, serum MUC5AC levels may be associated with worse prognostic factors and decreased survival in BTC. MUC5AC may also be involved in biliary carcinogenesis (either indirectly or directly) via various biochemical interactions, especially in its mature glycosylated forms.
Given the dire need for effective systemic treatments against BTC, prospective biomarkers such as MUC5AC also need to be evaluated in relation to impact on treatment selection and response. Within the literature there is a lack of studies analyzing the effect of MUC5ACpositivity on BTC treatment; consequently, MUC5AC has no demonstrated predictive value in BTC treatment. However, some research is available that outlines future directions. One study on tumor-associated antigen (TAA) epitopes for stimulating cytotoxic T lymphocyte (CTL) responses in CCA patients showed there were 18 TAA-derived epitopes that were associated with CTL response, including a MUC5AC epitope [142]. Survival was significantly increased in CCA patients who had at least two TAA-specific CTL responses compared to one or no responses. The authors used a set of criteria to determine which epitopes would be potential candidates for epitope therapy to stimulate CTL anti-cancer responses in CCA, and they found that MUC5AC was included on this candidate list. TAA-derived epitopes have been studied in several other types of cancer as potential vaccine therapies to stimulate anti-tumor immune response [1,[143][144][145][146]. The results of the CCA study point to a need for further studies on the possible utility of MUC5AC epitope vaccine treatment against BTC. Another question that remains to be answered is the possible role of anti-MUC5AC antibody treatments. Although this has not yet been studied as a treatment for BTC, one study (discussed earlier) provides potential clues for a starting point. The serum levels of MUC5AC detected with novel antibody CA-S27 (against mature MUC5AC) were linked with decreased survival in BTC patients, and CA-S27-MUC5AC is possibly directly involved in BTC carcinogenesis [42]. Given that the in vitro inhibition of CA-S27-MUC5AC with neutralizing antibodies in CCA cell lines led to decreased invasion and growth, this points to a need for further research on the effects of anti-MUC5AC antibodies on CCA cell lines and perhaps eventually for BTC patients.

Conclusions
In summary, although the literature identifies potentially promising associations for serum MUC5AC in the diagnosis and prognosis of BTC, MUC5AC cannot yet be used in clinical practice as a diagnostic or prognostic marker based on the variability and limitations of the available evidence. The variations in the MUC5AC glycoforms used, study populations (e.g., BTC vs. ICC-only vs. ECC-only vs. CCA vs. GBC) analyzed, other study methods utilized, and outcomes reported among the studies makes it difficult to draw concrete conclusions. Future studies must address these issues, and work on understanding the potential predictive and therapeutic value of MUC5AC in BTC.