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Biology of Disease

The natural course of CG is characterized by a high tendency for invasion, spread, and metastasis (Fahim et al. 1962). Most of the patients with CG present with advanced disease at the time point of diagnosis, precluding curative resection resulting in dismal prognosis (Dutta 2012). In some series, almost half of patients showed metastatic disease at the time point of diagnosis (Hamrick et al. 1982). The overall 5-year survival for patients with CG having undergone R0 curative resection was reported to range from 21 % to 69 % in diverse studies (review: Lai and Lau 2008).

Local Invasion

Perineural and intraneural invasions are important modes of local spread in CG. The incidence ranged from 22 % to more than 50 % (Vadheim et al. 1944; Kuwayti et al. 1957; Fahim et al. 1962; Ouchi et al. 1987; Nagakawa et al. 1993). Perineural cancer invasion has to be distinguished from perineural invasion observed in benign conditions, such as adenomyomatous hyperplasia (Albores-Saavedra et al. 2007) and pyloric gland metaplasia (Albores-Saavedra and Henson 1999). There is a correlation between the prevalence of perineural invasion and higher histologic grade (Fahim et al. 1962). CG often extends beyond the organ’s boundaries and invades the gallbladder bed, the liver, soft tissues surrounding the bile ducts, and the hepatoduodenal ligament. In bile ducts located to the hepatoduodenal ligament, perineural invasion in the setting of bile duct infiltration is a sign of aggressive tumor disease (Kaneoka et al. 2003). Pathogenetically, expression of the neural cell adhesion molecule/NCAM by carcinoma cells seems to play an important role as a neurotrophic mechanism (Seki et al. 1995). The direct extension of CG tumor cells from the primary tumor to adjacent organs and structures is only partially elucidated. One mechanism involves mucosal and submucosal lymphatic vessel networks that connect neighboring organs. In patients with CG, cancer cells spread through lymphatic vessels in the submucosal layer of the common bile duct (Chikamoto et al. 2009). CG frequently invades blood vessels, in particular veins. The anatomy of the venous drainage of the gallbladder has been studied in detail (Habighorst et al. 1965; Halvorsen and Myking, 1971 9). The first authoritative investigation based on injection methods dates from 1932 (Karlmark 1932). This author demonstrated that the venules and small veins of the gallbladder form a continuous plexus localized to the adventitia of the organ. This plexus is, via venous bridges, connected with a similar plexus of the extrahepatic bile ducts. The plexus system is in turn drained by a variable number of cholecystic veins showing diverse types of arrangement. On the hepatic side of the gallbladder, the number of cholecystic veins ranges from 2 to 20, and these veins terminate in the quadrate lobe of the liver adjacent to the gallbladder bed. In contrast, the peritoneal side of the gallbladder shows usually only one cholecystic vein (rarely two). This vein drains either into the liver or into the periductal venous plexus, which in turn is connected to the veins of the quadrate lobe. In part of cases, this vein drains into the portal vein (review: Fahim et al. 1962). The significance of portal vein drainage is underlined by the observation of dilated gallbladder veins or gallbladder varices in the setting of portal hypertension (Marchal et al. 1985; Ralls et al. 1988; Kainberger et al. 1990). Veins that enter the quadrate lobe form a plexus of small vessel that finally drain into the hepatic vein of this lobe (Petrén 1932). Small veins of the cystic duct join at the neck of the gallbladder to form either single or double cystic veins which accompany the cystic duct to connect to the periductal veins (review: Fine 1997). This anatomical substrate suggests an important pathway for CG spread both into the region of the extrahepatic bile ducts and the hepatoduodenal ligament and the right-sided liver. In a study of 155 cases of CG, involvement of gallbladder vessels was identified in 13.9 %, invasion involving venules having a diameter of 500–1,200 μm to larger veins with a thick muscular wall. In large veins, traffic of cancer cells through the wall seems to take place via transmural invasion and/or extension along vasa vasorum (Fahim et al. 1962). CG cancer cells can spread through lymphatic vessels in the submucosa of the common bile duct (Chikamoto et al. 2009).

Lymph Node Metastasis

Lymph node metastasis is a characteristic feature of CG that represents a significant prognostic factor. The incidence of lymph node metastasis in CG varies from 35 % to 75 % in diverse studies (Cooper 1937; Vadheim et al. 1944; Jones 1950; Willis 1953). Lymphatic spread may show a distinct distribution pattern, with the pericholedochal nodes being the most commonly affected, followed by the cystic node. Lymph node metastasis by CG cab develops in the absence of involvement of the liver (Cappell and Tudhope 1934). This distinct pattern results from the specific anatomy of the lymphatic drainage of the gallbladder. By means of Prussian blue injection into human fetuses 8–9 months old, Clermont (1909) had studied the lymphatic vessels of the gallbladder and gallbladder-associated lymph nodes in detail. The gallbladder wall harbors lymphatic plexus that traverses the entire wall and enters lymphatic collecting trunks that are mainly located on the inferior surface of the gallbladder. They are arranged like the letter “N,” as they are present on both the left and the right borders and diagonally toward the left side of the gallbladder neck (Fahim et al. 1962). The left-sided collecting trunks empty into the cystic node which is situated in the acute angle formed between the cystic duct and the common hepatic duct, while the right-sided collecting trunks pass along the right border of the gallbladder neck and extend uninterrupted to empty into the pericholedochal nodes (reviewed by Fahim et al. 1962). Also the lymph from the cystic node ends in the pericholedochal nodes. The latter are usually two lymph nodes, termed the node of the hiatus and the superior pancreaticoduodenal node. The node of the hiatus was a constant finding in Clermont’s investigation. It is situated to the right of the common duct, its lower pole being at the level of the attachment of the lesser omentum behind the superior portion of the duodenum. The node of the hiatus also receives lymph from the extrahepatic bile ducts and from the right liver lobe. Three to four efferent lymphatic vessels of the node of the hiatus continue to reach the superior pancreaticoduodenal node, which is located along the superior surface of the pancreas to the right of the common duct (summarized in Fahim et al. 1962). Efferent lymphatics of this node continue either to the preaortic peri-celiac nodes or to the three to four posterior pancreaticoduodenal nodes which are located around the vessel arcade in the posterior pancreaticoduodenal groove. From here, the lymph flows to the nodes around the superior mesenteric artery. Clermont (1909) found lymph nodes along the hepatic pedicle in relation to the hepatic artery, draining the left liver lobe only, but he did not find any Quénu-type hepatic hilar lymph nodes (proposed to exist by Edouard André Victor Alfred Quénu, 1852–1944, French surgeon active in anatomy and surgical pathology). Clermont’s studies referred to human fetuses and infants and the situation in adults may differ from findings obtained in the prenatal and pediatric age group. The lymphatic system of the gallbladder was subject to more recent investigations on adult individuals. Ito and coworkers (1991) divided gallbladder lymphatic drainage into three pathways, i.e., the cholecysto-retropancreatic pathway (the main pathway), having two routes, namely, one running spirally and posteriorly from the anterior surface of the common bile duct to the right and the other one running almost straight down from the posterior surface of the common bile duct. These routes end at a retroportal lymph node, termed by the authors the principal retroportal node. A second route connected the gallbladder with the celiac nodes, and a third ran in front of the portal vein to the superior mesenteric nodes.

There is a relationship between the T stage and the incidence of locoregional lymph node metastases. In one study, none of 15 patients with stage pT1 tumors had lymph node metastasis, while 60 of 96 patients with pT2-4 tumor disease had lymph node metastases. pT3-4 tumors displayed more lymph node involvement and significantly higher N2:N1 ratios (2.5) than pT2 tumors. Patients with N2 disease had a worse prognosis than those with N1 (Tsukada et al. 1997). Overexpression of microRNA-155, associated with inflammation-induced carcinogenesis, in CG is closely correlated with the emergence of lymphatic metastasis and poor prognosis. In vitro assays revealed that aberrant expression of miRNA-155 enhanced CG cell proliferation and invasion (Kono et al. 2013).

Invasion of the Liver and Intrahepatic Metastasis

Among early sites of metastasis, the liver is often involved. In fact, hepatic metastasis is the most frequent mode of recurrence of advanced CG after radical resection, and microscopic liver metastases in CG are an independent prognostic factor (Endo et al. 2004). Infiltration and/or metastatic involvement of the liver was detected in up to 89 % of cases (Lam 1940; Vadheim et al. 1944; Glenn and Hays 1954; Burdette 1957; Sons et al. 1985). It may, however, sometimes be difficult to distinguish hepatic metastases in segments close to the gallbladder from direct invasion of the liver substance. The pathways leading to true hepatic metastases have been discussed, emphasis being placed on the anatomic relationships between gallbladder veins and the anterior portal vein (Shirai et al. 1995; Yoshimitsu et al. 1997, 2001; Ohtsuka et al. 1998; Lin et al. 2005; Table 1). The extent of microscopic angiolymphatic portal tract invasion correlated with the gross depth of direct invasion of the liver (Shirai et al. 1995).

Table 1 Patterns of gallbladder carcinoma spread to the liver
Full size table

Kondo and coworkers (2002) identified six types of CG spread. In the hepatic bed type, a large mass in the fundus and body of the gallbladder invaded the liver substance through the gallbladder bed. In the hepatic hilum type, CG of the gallbladder neck infiltrates the hepatic hilum causing obstructive jaundice. In the bed and hilum type, huge masses occupying the entire gallbladder involve both the gallbladder bed and the hepatic hilum. In the lymph node type, enlarged metastatic lymph nodes are the most prominent feature. In the cystic duct type, a small mass arising from the cystic duct involves the common bile duct. In the localized type, tumor spread is localized to the gallbladder and presents at the earliest stage of any type (Kondo et al. 2002). In another study, the mode of hepatic spread was classified into three patterns, i.e., direct invasion through the gallbladder bed, portal tract invasion, and true hepatic metastatic nodules. A significant proportion of cases with portal tract invasion revealed invasion of hepatic lymphatic vessels as shown by D2-40/podoplanin immunohistochemistry (Wakai et al. 2010).

Intraductal Spread of Gallbladder Carcinoma

CG can spread through intraluminal invasion of the extrahepatic biliary tract. It was found in up to 4 % of cases (Fahim et al. 1962). Rarely, CG showed intraluminal implantation into bile ducts (so-called implantation metastasis or formation of intraluminal tumor “thrombi” or “emboli”; Hollings 1963; Al-Qudah 1994; Evans 1994; Midorikawa et al. 2000). In case intraluminal CG metastasis involves the confluence or the common bile duct, obstructive jaundice ensues (Hollings 1963; Midorikawa et al. 2000).

Distant Metastasis

CG produces a distinct pattern of distant metastases, although extraabdominal metastases are overall uncommon in CG. The most common metastatic site is the lung (Tokunaga et al. 2005; Jeyaraj et al. 2013), followed by the central nervous system (Takano et al. 1991; Tayo et al. 2005), ovaries (Singh et al. 2010), soft tissues, and breast (Khangembam et al. 2013). In rare instances, pulmonary metastasis of CG presents with a lepidic growth patterns, i.e., cancer cells lining alveolar spaces (Tokunaga et al. 2005). Similar to carcinomas of the extrahepatic bile ducts, CG can produce ovarian metastases that sometimes present as Krukenberg tumors (Andrieux et al. 1972; Chicos et al. 2007). Solitary metastases to the skeleton occur, but are rather uncommon (Rominger et al. 1961; Prakash et al. 2010; Chaudhari et al. 2014; Puranik et al. 2014). Multiple osseous metastases are even less common (Commandre et al. 1965; Misra et al. 1997). Subcutaneous metastasis is also rare in CG, but may present as a rapidly progressive cancer disease (Heavey et al. 2014). Other rare sites comprise the orbita (Puglisi et al. 2005), heart (Gunjiganvi et al. 2013), adrenal gland (Sahoo et al. 2014), and uterus (Martinez-Roman et al. 2005; Kefeli et al. 2009).

The mechanisms leading to early and widespread metastasis in CG are only partially known. CG shows an upregulation of the prometastatic microRNA-20, closely associated with local cancer invasion and distant metastasis. A markedly increased level of TGF-beta1 is responsible for the elevation of microRNA-20a, which in turn promotes epithelial-mesenchymal transition thought to promote metastasis (Chang et al. 2013).

Implant Metastasis

Implant metastasis of CG can develop in cholecystectomy scars (Merz et al. 1993). In some patients, metastases of CG have been found at the port site (Karayannakis and Knight 1997; Reber et al. 1998; Ohmura et al. 1999; Winston et al. 1999; Nakagawa et al. 2000), trocar site (Copher et al. 1995), or umbilicus (Clair et al. 1993; Kessler and Mihaljevic 1994; Jacobi et al. 1995; Jeon et al. 1999) following laparoscopic cholecystectomy. The incidence of recurrence of incidental CG at the port site following laparoscopic cholecystectomy was 14 % in one study and was similar whether the primary tumor was confined to the gallbladder (T1/T2) or locally advanced (T3/T4; Z’graggen et al. 1998). In an international survey, it was found port site recurrence was identified in 17.1 % of 409 patients with a median of 180 days following laparoscopic cholecystectomy for nonapparent CG (Paolucci et al. 1999). Laparoscopic cholecystectomy with the finding of unsuspected CG may be followed by cutaneous seeding (Kim and Roy 1994), and laparoscopic removal of CG may cause peritoneal cancer dissemination (Pezet et al. 1992; Sailer et al. 1995; Marmorale et al. 1998; Ohtani et al. 1998; Shirai et al. 1998). Cancer spread to the peritoneum in the setting of laparoscopic cholecystectomy was mainly found in the presence of T3 tumors (Wysocki and Krzywon 2000). Rarely, CG gave rise to umbilical metastasis, alias Sister Mary Joseph’s nodule, in the absence of laparoscopic surgery (Rousselot et al. 1964; Bork et al. 2002; Renner and Sticherling 2007). Sister Mary Joseph’s nodule refers to a palpable tumor nodule bulging into the umbilicus due to a metastasis from an abdominal or pelvic malignancy. The eponym is related to Sister Mary Joseph Dempsey (1856–1939), surgical assistant of Dr. William J. Mayo at St. Mary’s Hospital in Rochester, who drew Dr. Mayo’s attention to the phenomenon of umbilical metastasis, the metastatic pathway probably involving lymphatics along the obliterated umbilical vein. Laparoscopic cholecystectomy can also be complicated by parietal seeding of carcinoma (Barsoum and Windsor 1992) or by seeding into the gallbladder fossa (Evrard et al. 1996). Seeding of CG along the intervention tract was observed following percutaneous transhepatic choledochoscopy (Yamakawa et al. 1983).

Prognosticators

A growing list of factors affecting prognosis of CG have been identified, relating to growth factors and their receptors, factors regulating proliferation and apoptosis, adhesion molecules, factors regulating cell motility and locomotion, enzymes involved in tissue invasion, and angiogenic factors (review: Gomez-Roel et al. 2007).

Stage

Tumor stage (T stage) and lymph node metastases (N stage) are important prognosticators in CG. The T category is a decisive factor for predicting outcome (Kayahara and Nagakawa 2007; Cho et al. 2012). Early CG, limited in its extension to the intramucosal compartment (pT1a), is usually an incidental finding, but this stage is associated with good prognosis (Albores-Saavedra et al. 2011). The presence of lymph node metastases is an important predictor of outcome, whereby the number in involved lymph nodes independently determines prognosis after tumor resection (Sakata et al. 2010; Shirai et al. 2012). Distant positive lymph nodes have a particularly adverse effect on outcome (Meng et al. 2011). There is increasing evidence that the lymph node ratio (LNR) is an important prognostic factor for CG patients. LNR was a powerful predictor of disease-free survival in curatively resected patients, specifically in stage IIIB disease (Negi et al. 2011; Choi et al. 2013). In another investigation, however, the number of positive lymph nodes better predicted patient outcome after resection than LNR or location of positive nodes (Shirai et al. 2012). The presence of lymph node metastases in CG is associated with expression of the high molecular weight glycoprotein, DF3 (MUC 1). Lymph node metastasis was frequently found in the cytoplasmic DF3- and stromal DF3-positive CG, suggesting that DF3 expression plays important roles in cancer cell growth and metastasis of CG (Kashiwagi et al. 2000). The prognostic significance of nodal micrometastases in CG is not yet known in detail, but was shown to have an impact on survival (Sasaki et al. 2006) or even be an independent prognosticator for poor outcome (Nagakura et al. 2001). In one investigation, nodal micrometastases were found in 2.3 % of lymph nodes that were macrometastasis negative at conventional examination. Micrometastases correlated with non-micro-lymph node metastases on conventional tissue sections and were a significant prognostic factor, although they may reflect cancer cell spread to the whole body rather than an initial event of cancer spread (Tanabe et al. 2012). Distant metastases are a decisive factor for prognosis in patients with CG. Hepatic metastasis is the most common mode of recurrence of advanced CG after radical resection. Both macroscopic and microscopic liver metastases are involved in progressive disease and dismal prognosis.

Radicality of Resection

The presence or absence of carcinoma in the resection margin significantly affects outcome (Chakravarty et al. 2009). Carcinoma spread to the cystic duct is a prognosticator which indicates poor prognosis in CG patients, probably also linked to a high proportion of associated perineural invasion and lymph node metastasis (Nakata et al. 2007). It should be mentioned that high-grade dysplasia may rarely occur in the cystic duct margin in the absence of invasive CG (Bickenbach et al. 2011). Intraoperative frozen section is a common method to evaluate resection margins in the setting of cholecystectomies performed for CG, but has some interpretational drawbacks due to the presence of gallbladder structures that may be confounded with cancer, including glands and (atypical) Rokitansky-Aschoff sinuses. It has been demonstrated that frozen section and permanent diagnoses of the bile duct margin in CG may be inconsistent in up to 25 % of cases due to overdiagnosis in frozen sections and the “appearance” of carcinoma in permanent histology slides (Yamaguchi et al. 2005).

Incidental Versus Non-incidental CG

A significant fraction of CGs are incidental tumors detected in cholecystectomy specimens obtained via resection of suspected benign disease. Patients with incidental CG (IGC) had a longer survival rate compared to patients with non-incidental CG (NIGC). In one study, the majority of patients with potentially curable disease had IGC (D’Hondt et al. 2013).

Histologic Type

With few exceptions, the histologic type of CG did not affect biology of disease (Glenn and Hays 1954). In a comparative study, well-differentiated or moderately differentiated tubular adenocarcinomas were associated with a longer survival time than poorly differentiated carcinomas, the latter also having a higher incidence of hematogenous metastases (Egawa et al. 2004). Most CG shows the histology of adenocarcinoma, and the relatively few neoplasms with distinct morphologies different from ordinary adenocarcinoma mainly comprise papillary and colloid carcinomas. In comparison with all other histologic types, papillary CG had the most favorable prognosis, in some series found in patients with long survival (Fahim et al. 1963; Frank and Spjut 1967; Hart et al. 1972; Ouchi et al. 1986; Henson et al. 1992).

Histologic Grade

Dedifferentiation is often observed in CG and is associated with poor prognosis. In an ECOG study on 30 patients with advanced CG divided into either low-grade or high-grade lesions, the 13-week low-grade CG patient survival was significantly longer than the 7-week high-grade CG patient survival (Johnson et al. 1987). In part of CG, less differentiated tumor cells form isolated single cells or clusters of fewer than five cancer cells at the invasive front. Similar to the respective situation in colorectal carcinomas, this phenomenon is termed tumor budding, a lesion that reflects prognosis, particularly for T2 tumors (Kai et al. 2011).

Invasion Patterns

The presence of perineural invasion is a negative prognosticator in patients with CG (Nagakawa et al. 1993; Yamaguchi et al. 2002; Noshiro et al. 2003; Nakata et al. 2007). In one study of 68 patients with CG, perineural invasion was demonstrated in 71 % of patients. The incidence of invasion was correlated with invasion of extrahepatic bile ducts, which was the only independent factor associated with perineural involvement. The 5-year survival rate of patients with perineural invasion was 7 % in comparison with 71 % of patients without detectable perineural invasion (Yamaguchi et al. 2002). Bile duct involvement portends poor prognosis in resected CG (Chan et al. 2005; Eil et al. 2013). In early CG, intraepithelial extension into Rokitansky-Aschoff sinus is an adverse prognostic factor (Roa et al. 2013). As in other carcinomas, vascular invasion is a negative prognostic factor in CG. Expression of vascular endothelial growth factor-C/VEGF-C was expressed in 63 % of CG, and this expression was associated with lymphatic vessel invasion and lymph node metastasis, suggesting that VEGF-C is involved in tumor progression through lymphangiogenesis and facilitation of lymphatic vessel invasion (Nakashima et al. 2003). Invasion of the hepatic artery is an important prognostic factor in patients with CG, as infiltration of this artery confers high risk and a poorer prognosis (Kobayashi et al. 2012).

Oncogene and Tumor Suppressor Gene Expression and Factors Involved in Chromatin and DNA Metabolism

A significant proportion of CG expresses p53. Fifty-eight percent of these cancers were immunoreactive for p53 in one analysis, seen more often in moderately or poorly differentiated neoplasms (Washington and Gottfried 1996). No significant correlation was found between p53 overexpression and T stage, lymph node metastasis, prognosis, or recurrence in patients with CG, but p53 overexpression was correlated with aneuploidy (Ajiki et al. 1996). p53 nuclear overexpressions in CG were not related to cancer differentiation, depth of gallbladder wall invasion, or patient survival (Hidalgo Grau et al. 2004). Lack of correlation between p53 expression in CG and survival was found in another investigation (Washington and Gottfried 1996). On the other hand, other investigations reported a role of p53 gene mutation in the transition from premalignancy to malignancy (Ajiki et al. 1996; Moreno et al. 2005) and a correlation between p53 expression in CG and poor survival (Takagawa et al. 2005). An important regulator of chromatin, involved in silencing tumor suppressor genes, is the histone deacetylase (HDAC) system. The Myc oncogene pathway seems to be involved in carcinogenesis and progression of CG. N-myc downstream-regulated gene 1 (NDRG1) , a member of the N-myc downstream-regulated gene family, is associated with inhibition of tumor metastasis and tumor suppression. In CG, NDRG1 was expressed in 63.8 % of cases, specifically at the invasive front of the tumors, but not in normal gallbladder epithelium. This expression was significantly associated with high histologic grade, advanced T stage, positive nodal metastasis, venous/lymphatic invasion, and poor survival (Zhang et al. 2012). In CG, overexpression of HDAC 2 in tumor cell nuclei is correlated with higher histologic grade, and predicts unfavorable prognosis (Du et al. 2013a). There is evidence that factors involved in chromatin replication and chromatin remodeling are altered in CG and affect biology of disease. Rsf-1/HBXAP, a nuclear protein with histone chaperon function and member of the remodeling and spacing factor complex which mediates ATPase-dependent chromatin remodeling, is expressed in CG, and its overexpression confers aggressiveness and is associated with disease-specific survival (Chen et al. 2011a). Decreased expression of the chromodomain helicase DNA-binding protein 5 in CG cells is correlated with a higher lymph node metastasis rate and shorter disease-free survival and overall survival (Du et al. 2013b). Dicer and Drosha, two enzymes critically involved in the processing of microRNA, are significantly less expressed in CG than in dysplastic gallbladder epithelia and were less expressed in poorly differentiated carcinomas with lymph node metastases in comparison with well-differentiated adenocarcinomas. Loss of expression of Dicer and Drosha was associated with decreased overall survival (Shu et al. 2012).

Transcription Factors and Associated Protein Signaling Networks

SOX4, a member of the SRY-related HMG-box (SOX) transcription factor family, plays a significant role in carcinogenic pathways of various malignancies. SOX4 was expressed in 75 % of CG, but in normal gallbladder epithelium. SOX4 expression in CG was significantly associated with low histologic grade, low pathological T stage, early clinical stage, and better disease-free and overall survival (Wang et al. 2012a).

Sonic hedgehog, its receptor, patched, and its downstream transcription factor, Gli1 protein, are overexpressed in CG. The expression of these factors was significantly correlated with stage, lymph node metastasis, venous and lymphatic invasion, liver invasion, and lower survival rates (Li et al. 2012). Expression of the N-myc downstream-regulated gene 2 (NDRG2), a protein that may reduce the metastatic potential of cancers, was detected in 37.6 % of CG. Tumors with downregulation of NDRG2 more often had lymph node metastases and lymphovascular invasion and tended to have higher TNM stage; the patients also had poor prognosis (Song et al. 2012). Caudal-related homeobox protein (CDX2) , a homeodomain protein which plays an important role in the regulation of cell proliferation and differentiation of glandular epithelia and which is a regulator of intestinal metaplasia, is absent from the normal gallbladder, but is expressed in part of CG, mostly in well-differentiated tubular adenocarcinomas of the intestinal type (Wu et al. 2005).

Cell Cycle Regulation

High proliferative activity is generally associated with an aggressive phenotype of CG (Hui et al. 2002). CG expresses various factors involved in cell cycle regulation, including cyclin E (Mishra et al. 2009), cyclin D1 , and p16 (Xuan et al. 2005; Srivastava et al. 2013). The expression rate of cyclin D1 (68.3 %) in CG was significantly higher than in nonneoplastic disorders of the gallbladder, whereas the expression rates of p16 and retinoblastoma/Rb protein were significantly lower in CG compared to those in cholecystitis and adenoma (Ma et al. 2005). Cyclin D1 overexpression is a critical event in CG carcinogenesis and independently predicts decreased patient survival (Hui et al. 2000; Itoi et al. 2000). Loss of p16 in CG and bile duct cancers is associated with reduced survival of patients (Karamitopoulou et al. 2008), whereby loss of p16INK4 protein is correlated with overexpression of the retinoblastoma/Rb protein (Shi et al. 2000). In contrast, overexpression of cyclin E was not associated with depth of tumor invasion, tumor stage, or patient prognosis (Eguchi et al. 1999). p27 is a tumor suppressor protein which inhibits both Cdk2 /cyclin E and Cdk2/cyclin A complexes and prevents transition to the S phase of the cell division cycle. Expression of p27 is expressed in CG and was correlated with clinical stage of CG (Xuan et al. 2005; Alsheyab et al. 2007), and low p27Kip1 expression in CG is an independent prognostic factor associated with poor prognosis (Filipits et al. 2003). The expression pattern of p27 is critically modulated by two SCF(Skp2) ubiquitin ligase-related proteins, Skp2 and cyclin-dependent kinase subunit 1/Cks1, which are involved in the posttranscriptional degradation of the p27 tumor suppressor. Reduced p21(WAF1/CIP1) expression is an early event in CG carcinogenesis and is of prognostic significance (Li et al. 2001; Puhalla et al. 2007). Expression of Skp2 was found to be an important and independent adverse prognosticator in CG (Li et al. 2007). Cyclooxygenase-2 (COX-2) plays a role in promoting cell proliferation, growth, and metastasis of carcinoma cells. Intense immunostaining of COX-2 was found in hyperplastic gallbladder lesions (65 %), pT2 CG (76 %), and advanced stage CG (64 %), including the associated stroma. In pT2 CG, expression of COX-2 in the stroma adjacent to the carcinoma cells in the subserosal layer correlated with aggressiveness of disease, including the tendency for distant metastasis (Kawamoto et al. 2002). The mammalian target of rapamycin (mTOR) , a serine/threonine kinase, plays an essential role in the regulation of cell growth and is frequently deregulated in cancers. In CG, phospho-mTOR is an overexpression in early phases of cancer evolution, associated with poor prognosis (Leal et al. 2013). The sphingosine-1-phosphate receptor 1 (S1P1) is overexpressed in CG, and higher levels of S1P1 were significantly correlated with tumor differentiation, tumor mass, lymph node metastasis, invasion, and decreased survival time (Yuan et al. 2013). S1Ps are a class of G protein-coupled receptors that are a signaling target of sphingosine-1-phosphate and that are involved in the regulation of cell proliferation, cytoskeletal organization, and cell migration.

Other Factors Involved in Proliferation and Differentiation of Cells

Astrocyte elevated gene-1 is overexpressed in CG and is strongly correlated with the differentiation degree, stage, Ki-67 proliferation index, and liver invasion. Expression of this protein in CG is an independent prognostic marker for CG progression (Sun et al. 2011). Expression of connective tissue growth factor/CTGF in CG was correlated with better survival in two studies (Alvarez et al. 2008; Garcia et al. 2013), while in another investigation a role of CTGF expression in CG progression was found (Garcia et al. 2013). Part of CGs produce several classes of mucins (see below). Univariate analysis showed that MUC4 expression was significantly associated with poor survival, while expression of MUC1 and MUC2 was not correlated to survival. Backward stepwise multivariate analysis exhibited that MUC4 expression was a significant risk factor for poor outcome (Lee et al. 2012a). Expression of the multifunctional redox protein, human thioredoxin-1 (TRX-1), and its reducing enzyme, thioredoxin reductase (TRX-R), in CG affects tumor biology. Specifically, nuclear TRX-1 expression in cancer cells at the invasion front of CG is a significant prognostic marker (Nagano et al. 2012). Estrogen receptors (ERs) are expressed in part of CG cells (Gupta et al. 2012). ERbeta isoform was expressed in most specimens, but preferentially in central tumor parts. Absent ERbeta expression at the invasive front was significantly associated with lymph node metastasis, advanced stage, lymphatic invasion, and poor prognosis (Sumi et al. 2004).

Chemokines, Cytokines, and Other Factors Involved in Immune Responses

Chemokine (C-X-C motif) ligand 12 (CXCL12 ), important for the progression of various malignancies, was differentially expressed in CG tissue, together with its receptor CXCR4, and the magnitude of expression was associated with high histologic grade and nodal metastasis, and expression was a significant risk factor for survival. CXCL12 increased anchorage-dependent and anchorage-independent growth, adhesiveness, and migration and invasive properties of CG cells (Lee et al. 2012b). Toll-like receptors (TLRs), which play a crucial role in inflammatory reactions, including chronic cholecystitis, are also operational in various pathways involved in carcinogenesis. TLR4 is mainly expressed in the glandular and luminal epithelia of the gallbladder. TLR4 expression was lower in CG in comparison with chronic cholecystitis and normal gallbladder (Huan et al. 2012).

Mechanisms of Apoptosis

There are intimate relationships between CG and apoptosis (Zhang et al. 2003). Positive rates of Fas were not significantly different among CG, dysplasia, and gallbladder adenoma, but positive rates of Fas ligand/FasL in carcinoma were significantly higher than that in chronic cholecystitis, suggesting that FasL expression in CG permits tumor cells to escape from immune surveillance by inducing apoptosis in lymphocytes infiltrating CG tissue (Xu et al. 2005). Higher expression of Bcl-2 in CG is considered to affect growth and progression of the tumors via inhibition of apoptosis (Mikami et al. 1999; Karamitoupoulou et al. 2008). Expression of the cellular Fas-associated death domain-like interleukin-1-converting enzyme inhibitory protein (c-FLIP), an antiapoptotic protein, is upregulated in CG, potentially conferring a growth advantage (Zong et al. 2009). Expression of p53-upregulated modulator of apoptosis (PUMA) was significantly higher in adenocarcinoma of the gallbladder than in adenoma and chronic cholecystitis, whereby PUMA expression was lower in small (less than 2-cm diameter) and well-differentiated CG with N0 than in poorly differentiated larger (>2 cm) CG with lymph node metastasis and invasion of surrounding tissues (Cai et al. 2013). Receptor-binding cancer antigen expressed on SiSo cells (RCAS1), a protein that induces apoptosis in immune effector cells expressing the RCAS1 receptor , was overexpressed in 70 % of CG, but not in precursor lesions. RCAS1 expression was associated with depth of tumor invasion, venous involvement, perineural invasion, tumor stage, and the presence of metastases (Oshikiri et al. 2001). Expression of heat shock protein 70-interacting protein (CHIP), a U-box-type E3 ubiquitin ligase, in CG is associated with poor prognosis (Liang et al. 2013).

Cell Adhesion and Motility

Deranged cell-to-cell or cell-to-matrix contact mediated by adhesion molecules plays a crucial role within the invasion cascade. Several factors involved in adhesion are altered in CG. Epithelial cell adhesion molecule (Ep-CAM) is overexpressed in almost two thirds of CG, and overexpression of this factor is related to decreased overall survival and is an independent prognostic factor (Varga et al. 2004; Prince et al. 2008). The L1 adhesion molecule, associated with prognosis in several malignancies, is not expressed in normal gallbladder epithelium, but was detectable in 63.8 % of CG, specifically in cells located to the invasive front of the neoplasm. Expression of L1 in CG cells was correlated with high histologic grade, advanced T stage, and venous and lymphatic invasion and was an independent negative prognosticator in multivariate analysis (Choi et al. 2011). Expression of nectin-2, an adhesion molecule involved in calcium-independent cell adhesion, in CG is correlated to high T stage and poor prognosis (Miao et al. 2013). Thrombospondin-1 (TSP1), an extracellular glycoprotein that affects cell adhesion, motility, and growth, is mainly expressed in CG stromal cells and less so in cancer cells. Stromal expression of TSP1 increased as a function of increasing stage and was associated with lymph node metastasis and venous involvement, suggesting that TSP1 plays a role in CG spread and metastasis (Ohtani et al. 1999). Fascin, a protein interacting with thrombospondin-1, shows a marked overexpression in advanced CG, associated with aggressive clinical features and poor overall survival. Fascin, thrombospondin-1, and syndecan-1 interact to mediate this aggressive phenotype of CG (Roh et al. 2009). Syndecan-1 expression itself was detected in 58.1 % of CG, and syndecan-1-positive neoplasms more frequently showed lymph node metastasis and tended to have a deeper invasion depth and significantly shorter survival (Roh et al. 2008). Significant differences of E-cadherin and beta-catenin expression were detected between normal, inflamed, and cancerous gallbladder tissues (Puhalla et al. 2005). Loss of E-cadherin expression is high in CG (Priya et al. 2010), an alteration that may contribute to failure of intercellular adhesion, individualization of cells, and promotion of an invasive phenotype. N-cadherin and P-cadherin are biomarkers for invasion and metastasis in CG (Yi et al. 2014). Alterations of beta-catenin signaling are frequent events in CG, but these alterations seem to be minor contributors to CG carcinogenesis, but may be related to progression via loss of E-cadherin function (Kimura et al. 2003). Expression of the integrin-linked kinase (ILK) was found to be an independent poor prognostic predictor in CG (Li et al. 2013). CD97, a member of the EGF-TM7 adhesin family binding to its cellular receptor, CD55, plays an important role in invasiveness and aggressiveness of malignancies. It is a complement regulatory protein expressed by cells to protect them from bystander complement attack. CD97 and CD55 are absent or weakly expressed in normal gallbladder epithelium, but are overexpression CG, this expression being associated with high histologic grade, advanced T stage, angioinvasion, and reduced overall survival (Wu et al. 2012). CD44, a molecule involved in the metastatic pathway of cancers, exists in several variants or isoforms, including CD44s (standard variant), variant 3 (CD44v3), and variant 6 (CD44v6). CD44s is present in normal gallbladder epithelium and exhibits membranous immunostaining. In CG, CD44s is stained as strongly as normal epithelium, but reactivity for CD44 v3 and CD44v6 was also found. CD44s is significantly less expressed in well-differentiated CG in the invasive component of the tumor than in the intramucosal part, and both CD44v3 and CD44v6 are more strongly expressed in poorly differentiated CG than in well-differentiated CG, but these expression patterns were not associated with outcome (Yanagisawa et al. 2001). Trefoil factor family protein 1/TFF1 (pS2), a factor that interacts with mucins to protect gastrointestinal epithelial cells and which promotes epithelial cell migration, is expressed in low-stage CG, and expression decreases as a function of increased grade and stage. Patients with TFF1-positive CG revealed a more favorable outcome compared with TFF1-negative neoplasms (Kornprat et al. 2005). As other carcinomas, CG displays alterations of the cytoskeleton and cytoskeletal proteins, changes that affect cell motility and cancer cell migration as a component of the invasion cascade. Cofilin-1, an actin-modulating protein that depolymerizes filamentous F actin and inhibits the polymerization of monomeric G actin, is expressed in CG, whereby expression was significantly associated with large tumor size, high T stage, lymph node metastasis, and decreased overall survival (Yang et al. 2013).

Mechanisms of Invasion and Spread

CG cells express several members of matrix metalloproteinases (MMPs), enzymes that have a central role in the tumor invasion cascade, as they facilitate the egress of cancer cells from the primary tumor and the locomotion and migration of tumor cells in invaded tissues (Fan et al. 2002). MMP-9 and MMP-14 play a role in CG tumorigenesis (Karadag et al. 2008). MMP-1 and protease-activated receptor-1 are expressed in approximately 70 % of CG. CG expressing these two proteins more often showed lymph node metastasis, a deeper invasion depth, and more frequently lymphovascular invasion (Du et al. 2011). Expression of MMP-2 negatively affected the survival rate (Wu et al. 2009). In mucinous CG, which has a biology different from that of ordinary CG, MMP expression is lower (Karadag et al. 2008). The transcription and expression of MMPs in CG modulated by the neural precursor cell expressed developmentally downregulated 4-lik (Nedd4L) protein. Overexpression of Nedd4L in CG regulates the expression of MMP1 and MMP13 (Takeuchi et al. 2011). ADAM proteins, a multifunctional gene family of membrane proteins having a disintegrin and metalloprotease domain and involved in the metastatic cascade, are overexpressed in CG, this feature being associated with poor prognosis (Wu et al. 2011).

Expression of Differentiation Antigens and Factors Related to Cancer Cell Metabolism

Carcinoembryonic antigen (CEA) , which may function as a metastatic potentiator via modulation of intercellular adhesion and cell migration, is expressed in CG cells and CG stromal cells. Lymph node metastasis was frequently found in cytoplasmic and stromal cell CEA-positive CG (Dowaki et al. 2000). Stromal expression of the mucin MUC1 in CG is associated with tumor aggressiveness and a tendency to form distant organ metastases (Kashiwagi et al. 2001; Kawamoto et al. 2004), while expression of MUC2 was not significantly related to lymphatic invasion, lymph node metastasis, or prognosis (Kashiwagi et al. 2001). Tumor-associated glycoprotein 72 is expressed more frequently in CG of larger size, with lymph node metastasis, with higher stage, and with poor differentiation, suggesting that expression of this antigen is a marker of CG aggressiveness (Ouyang et al. 2010). CD24, a small surface protein, is a marker of malignancy and poor prognosis in CG (Liu et al. 2011). Part of CGs express the hepatocyte antigen (Hep). Expression of Hep was negatively correlated to the grade of differentiation, tumor size, and lymph node metastasis, and elevated Hep expression was associated with increased overall survival, Hep expression being an independent prognostic predictor (Li et al. 2011). Forced expression of LAPTM4B in a CG cell line increased the invasive potential (Zhou et al. 2007, 2010). CG expresses neurotrophin, nerve growth factor, and the receptor TrkA (Artico et al. 2010), but the biologic significance of this expression has not yet been elucidated. Glucose uptake is generally augmented in cancer cells. The glucose transporter, GLUT1, is expressed in CG, and this elevated expression is strongly associated with cancer progression (Kim et al. 2002).

Angiogenesis and Lymphangiogenesis

Angiogenesis of tumors, induced and controlled by complex signaling pathways involving VEGF, plays a significant role in tumor progression, including CG (Giatromanolaki et al. 2002; Harino et al. 2008). Increased tumor vascularization caused by angiogenic mechanisms plays a significant role in cancer progression. A high microvessel density in CG determined by Chalkley counting was associated with worse prognosis (da Rocha et al. 2009). In CG, microvessel density was found to increase as a function of depth of invasion, suggesting a role of angiogenesis in the invasive process (Kalekou and Miliaras 2011). Microvessel density correlated with tumor stage and liver metastasis (Chen et al. 2011b). However, microvessel density was not an independent prognosticator in multivariate analysis (Sugawara et al. 1999). Average microvessel counts were lower in cases of well-differentiated carcinoma, small tumor size (less than 2-cm diameter), and negative nodes, while higher microvessel counts and expression of CD146 in CG were associated with poor survival (Wang et al. 2012b). In a fraction of CG showing augmented angiogenesis, vascular endothelial growth factor/VEGF is expressed (Yamamoto et al. 1998; Giatromanolaki et al. 2003). The presence and expression level of VEGF were associated with tumor size, lymphatic invasion, and advanced disease stage (Okita et al. 1998; Letelier et al. 2014). Expression of platelet-derived endothelial growth factor/thymidine phosphorylase was detected in 63 % of CG, while hyperplastic epithelia or adenomas did not show significant expression. However, the magnitude of expression did not correlate with angiogenesis, but with depth of invasion, lymph node metastasis, and tumor stage (Yamamoto et al. 2000). Cyclooxygenase-2, which affects the expression of VEGF, induces angiogenesis in CG and is associated with poor prognosis (Zhi et al. 2005). A similar effect was found for the expression of inducible nitric oxide synthase (Niu et al. 2004). Tumor endothelial marker 8 (TEM8) protein is highly specific to angiogenesis in malignant neoplasms and is not required for normal adult angiogenesis. TEM8 is expressed in endothelial cells of CG, and its expression increased significantly with increasing CG stage (Maurya et al. 2011). TGF-beta, a protein with multiple functions, also promotes angiogenesis and is expressed in CG, where it augments angiogenesis and is associated with tumor progression (Kitamura et al. 2003).

CG shows vigorous lymphangiogenesis mediated by VEGF-C. TNF-alpha promotes formation of lymphatic vessels in CG through NF-kappaB-mediated upregulation of VEGF-C (Qiang et al. 2014). Expression of vascular endothelial growth factor-D in CG promotes growth, lymphangiogenesis, and lymphatic metastasis in CG and plays a role in CG progression (Lin et al. 2012).

Vasculogenic mimicry, a phenomenon associated with increased tumor-related mortality in several cancers, was observed in CG cell lines cultured in a 3D matrix, and vasculogenic mimicry in these cells was stimulated by HIF-1alpha. In CG analyzed in vivo, vasculogenic mimicry was also observed, and HIF-1alpha expression and vasculogenic mimicry were associated with poorer overall survival (Sun et al. 2012). Vasculogenic mimicry in CG is mediated by signaling pathways that involve PI3K/MMPs/Ln-5gamma2 and EphA2/FAK/paxillin networks (Lu et al. 2013).

Tumor Ploidy

Abnormal DNA contents were observed in 51 % of CG, whereby 44.4 % of tumors were aneuploid. In comparison, aneuploidy was detected in 81.3 % of metastatic lesions (Roa et al. 1993), suggesting effects of progressive genomic instability. Aneuploidy of CG was significantly associated with poorly differentiated adenocarcinoma, higher T stage, and a high mitotic index. A significant advantage in terms of 5-year survival was found in patients with diploid neoplasms in comparison with those having aneuploid tumors (Sato et al. 1993). In contrast, cytometric determination of DNA ploidy provided no prognostic information in CG as compared to conventional tumor staging in other studies (Yamamoto et al. 1990; Baretton et al. 1994).

MicroRNAs

MicroRNA-34 is associated with poor prognosis in CG through the regulation of telomere length in stem cells (Jin et al. 2014). An increased expression of microRNA-335 in CG predicts a favorable prognosis, but this miRNA is downregulated in the majority of CG, being one factor that may determine the aggressive biology of this cancer (Peng et al. 2013). Upregulation of the prometastatic microRNA-20a was closely associated with local invasion, distant metastasis, and poor prognosis (Chang et al. 2013). MicroRNA-155 was overexpressed in CG in comparison with nonneoplastic gallbladder, associated with the presence of nodal metastases and poor prognosis (Kono et al. 2013).

Staging

Determination of tumor stage is a decisive factor in prognostication of CG. However, up to 40 % of CG cases are diagnosed at an advance stage of disease (Henson et al. 1992), limiting the differential estimation of outcome as a function of stage. The current staging system formulated by the AJCC is shown in Table 2. It corresponds to the UICC system (Fong et al. 2006; Gore and Shelhamer 2007; Edge et al. 2010).

Table 2 AJCC staging of gallbladder carcinoma (7th edition; Edge et al. 2010)
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Table 3 Gallbladder cancer extension classified according to the 2001 version of the classification of biliary tract carcinoma of JSBS (2001)
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The advantages of pitfalls of the AJCC/UICC have recently been discussed (Adsay et al. 2012). The gallbladder does not have the distinct layering as other gastrointestinal organs do. Therefore, it was stated that the definitions of Tis/T1a/T1b may lack practicability, and therefore, the “early gallbladder carcinoma” category proposed in high regions may have to be recognized instead. Furthermore, it was proposed that documentation of hepatic versus serosal involvement should be performed in cases of advanced tumors (Adsay et al. 2012).

In addition to T stage, which describes depth of invasion and extension into adjacent organs and/or structures, other modes of invasion have been proposed as prognostically relevant parameters. T2 carcinomas with subserosal invasion form a distinct group of gallbladder cancers that may profit from radical surgery including resection of the gallbladder bed (Chijiiwa et al. 2001). In T2 tumors, free resection margins and the absence of perineural invasion and of lymph node metastasis are related to good prognosis. The depth of subserosal invasion in pT2 tumors has an impact on outcome in patients with pT2 neoplasms (Wakai et al. 2003). Depth of invasion of the subserosal layer was divided into three categories, i.e., ss1, ss2, and ss3, representing invasion of the upper, middle, or lower thirds of the subserosal layer, respectively (Sasaki et al. 2005). Expression of N-acetylglucosaminyl-transferase V, an enzyme that catalyzes the beta1-6 branching of N-acetyl-glucosamine on asparagine-like oligosaccharides of cellular proteins and enhances the malignant features of cancer cells, is expressed in CG located to the subserosal layer and correlates with postsurgical survival in pT2 tumors (Onuki et al. 2014). Expression of MUC1 mucins in the subserosal layer correlated with postsurgical prognosis (Kawamoto et al. 2001). A further prognostic feature related to stage is the mural invasion pattern of CG. Two intramural invasion patterns were defined as the infiltrative growth type (IG, infiltrative growth in the muscle layer without destruction) and a destructive growth type (DG, massive growth with destruction of the muscle layer; Okada et al. 2009a). Scirrhous growth was found more often in DG lesions, and the overall survival rate of patients with DG tumors was significantly lower than that of patients with IG tumors (Okada et al. 2009a). A negative effect of the DG pattern was also found in pT2 CG with a subserosa-invasive growth pattern (Okada et al. 2012). Tumors with a DG growth pattern show a higher proliferative activity, a poorer differentiation, a stromal laminin-5gamma2 chain expression, and a distant lymph node metastasis (Okada et al. 2009b).