Neuroendocrine tumors of the gallbladder.

A gallbladder neuroendocrine neoplasm (GB-NEN) is a unique heterogeneous neoplasm, originating from neuroendocrine cells and able to secrete peptides as its neurotransmitter. Among all the neuroendocrine tumors (NETs), the GB-NEN prevalence is 0.5%, which accounts for ~2.1% of all gallbladder tumors. The current study aimed to present an up-to-date review of the clinical features, diagnosis and treatment of NETs of the gallbladder. A systematic literature search was performed in order to identify all relevant studies published. Thus far, the etiology and pathogenesis of GB-NEN remains unclear. Moreover, an ambiguous clinical manifestation exists as observed during laboratory examinations. As GB-NEN is a rare gallbladder lesion and is seldom seen in clinical practice, there is a limited availability of review reports, and it is therefore often considered only as a case study. It is difficult to distinguish GB-NEN from other gallbladder diseases using imaging diagnostic techniques. Although chemotherapy treatment provides prolonged progression-free survival, surgery is considered the best option. Thus, to overcome the inherent risks or shortfalls of traditional surgery; early detection, diagnosis and treatment of GB-NEN are required to improve patient longevity. Copyright: © Niu et al.

Introduction

A neuroendocrine neoplasm (NEN) is a type of heterogeneous tumor, which was first reported and identified as carcinoid by Oberndorfer in 1907. A NEN originates and spreads from neuroendocrine cells and peptidergic neural crest Kulchitsky cells (silver-addicted cells) (1). These cells perform essential biochemical functions such as the uptake of amine precursors, decarboxylation, and the release of bioactive peptide hormones and nerve mediators (2). As the occurrence of gallbladder NEN (GB-NEN) is low, it is difficult to obtain clear and large amounts of data on the etiology, pathogenesis, standard treatment plan and prognosis from large-scale studies conducted at multi-center research centers. As there is no standard procedure to identify and treat GB-NENs, the present study reviews the current clinical characteristics, diagnosis and treatment regimens available for patients with GB-NEN in order to better our understanding of the condition.

Epidemiology

The incidence of NEN is rare in clinical practice, it has been observed in only 115/100,000 cases worldwide in the last decade, which accounts for 1.25% of all malignancies (3). The average age of onset is 60 years and the majority of NENs are found in the gastrointestinal tract (66%), followed by the lungs (31%) (4). A hierarchical pattern of prevalence of NENs is found within the gastrointestinal tract, with increasing numbers found from the appendix, to the small intestine, to the rectum and finally to the colon. Although the occurrence of NENs is rare in the stomach and other parts of the gastrointestinal system, the ovaries, pancreas and testicles can be affected; however, occurrence in the liver and gallbladder is rare (5). Among all gastrointestinal carcinoids, extrahepatic biliary carcinoids account for 0.5–2.0% of cases and liver metastases remain common (6,7). Surveillance Epidemiology and End Result (SEER) research data indicate that GB-NENs account for 0.5% of all NETs and 2.1% of all gallbladder tumors (8). There are only a few reports available in the literature about GB-NENs and even fewer on cholecystic neuroendocrine carcinoma. From previous studies, there were three groups of large data reported, including two groups from Korea with 6 and 12 cases (9), respectively. The third group was from China with 10 cases (10). In addition to the above 10 cases, most of the literature in available in China about GB-NENs are individual case reports.

Causes

To identify the root cause of the GB-NEN, a thorough examination and understanding of the cellular nature of the disease is important. In the process of examination, minimal neuroendocrine cells can be found on the gallbladder mucosa, if any exist in this space (11–13). Further GB-NEN etiological identifiers may be as follows: i) The undifferentiated gallbladder stem cells separate into neuroendocrine cells. ii) The presence of gallbladder stones results in chronic inflammation of the gallbladder mucosa causing intestinal epithelium or gastric metaplasia, which are considered to be pathological metaplasia. In an advanced stage, this inflammation produces neuroendocrine cells at the lesion site eventually leading to the development of a GB-NEN (12,14,15). iii) In certain situations, the gallbladder adenocarcinoma function switches to a neuroendocrine one, resulting in chronic inflammation of gallbladder tissues and formation of stones, further exacerbating the other risk factors for gallbladder adenocarcinoma.

Molecular mechanisms

Despite inadequate understanding of the etiology and pathogenesis of GB-NENs, significant progress has been made in elucidating the molecular and biological pathways involved. These results encourage the creation of follow-up studies for the identification of novel diagnostic and treatment procedures for the disease. It has been confirmed in two previous studies (15,16) that activation of the epidermal growth factor receptor (EGFR) can upregulate downstream effector protein kinase B and extracellular signal regulate kinase expression (Fig. 1). Concurrently, the target protein of rapamycin in human cells regulates the growth, proliferation and motor activity of the cells. The expression levels of this protein and the proliferation index of the cells were determined to be positively correlated (13). The high expression of the aforementioned three molecules (the target protein of rapamycin, protein kinase B and extracellular signal regulate kinase) resulted in a poor prognosis in patients with GB-NENs (13). Scoazec described that angiogenesis serves an important role in NEN etiology and pathogenesis (17). It is also possible to treat NEN and prolong the survival of patients with the disease by blocking the high expression of vascular endothelial growth factor (VEGF) and its receptor. However, no specific mechanism backed by concrete evidence has been discovered to date for GB-NENs.

Figure 1.

Inhibitor (Cetuximab) of EGFR signaling in GB-NEN. Cetuximab blocks the interaction between the EGFR and EGF, thereby inhibiting the activation of downstream RAS-ERK and thereby blocking the occurrence of GB-NEN. EGF is a polypeptide molecule and EGFR is a typical receptor protein PTK. The signal transduction process of EGFR is: i) EGFR forms a dimer to change the conformation, PTK activity is enhanced, through self-phosphorylation of the receptor intracellular tyrosine residues phosphorylation. ii) EGFR phosphorylated by tyrosine binds to GRB2. iii) GRB2 activates RAS by collecting SOS, a positive regulator of RAS, promoting the release of GDP and binding to GTP. iv) Activated RAS (RAS-GTP) acts on RAF to activate it. Activated RAF acts on MEK, which acts on ERK to activate it. v) The activated ERK is translocated to the nucleus, which affects the expression of target genes and promotes the generation of GB-NEN. EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GRB2, growth factor receptor-bound protein 2; SOS, son of sevenless; GDP, guanine diphosphate; GTP, guanine triphosphate; MEK, mitogen-activated protein kinase; GB-NEN, gallbladder neuroendocrine neoplasm.

Pathological classification

There are three pathological classifications of GB-NEN: Carcinoid or typical carcinoid (low malignancy), atypical carcinoid (moderate malignancy) and small cell carcinoma (high malignancy). These classifications are based on GB-NEN histopathological structure, cell morphology and degree of differentiation, mitotic combination, and necrotic and biological behavior (18). Morphologically, the majority of neuroendocrine tumor (NET) cells are small, cone-shaped and polygonal, with no clear cell boundaries (18). GB-NEN cells have small nucleoli, granular chromatin, and relatively consistent tumor cell morphology, and are rich in interstitial blood vessels (19). According to the classification criteria of digestive system tumors by the World Health Organization in 2010, the well-differentiated NETs include grades G1 and G2, and the poorly differentiated NET (G3) is defined as a neuroendocrine carcinoma (NEC). The NEC is further classified based on the tumor cell size into large or small. G1 contains both the NEC and adenocarcinoma tumors and is therefore identified as a mixed gland neuroendocrine carcinoma (18).

The extrahepatic bile ducts and the ampullary region are more commonly observed in a GB-NEC of high malignancy, poor prognosis and little to no pathological differentiation (20). The occurrence of early metastasis is mainly identified through local infiltration and lymph node metastasis (21). The lymph nodes, liver, lungs and peritoneum are the most common metastasis sites of small cell GB-NEC (22). The American Cancer Institute Research reported 41 cases of gallbladder NEC of varying pathological grades between 1973 and 2005. The data revealed that high, medium and low/no differentiation pathological grade accounted for 2.4, 7.3 and 89.7% of the cases, respectively (8).

The most common histological finding reported in gallbladder malignancy is adenocarcinoma (80–90%) followed by undifferentiated carcinoma (10%) and then squamous cell carcinoma/squamous adenocarcinoma (5%). Overall, the occurrence of NEC is rare.

Clinical manifestations

According to case reports, the occurrence of GB-NEN is higher among women (~68%) of cases identified (22,23). The majority of these patients do not have any manifestations of carcinoid syndrome (24). The majority of NECs in the gallbladder and extrahepatic bile duct are non-functional APUD (amine precursor uptake and decarboxylation) tumors, with low or no functional endocrine granules in tumor cells (25). Early clinical manifestations of NEC are similar to those of other types of gallbladder cancer and include abdominal distension pain, nausea and other non-specific symptoms (26). In order to distinguish from GB-NEC, symptoms specific to carcinoid syndrome include spasmodic abdominal pain, flushes, edema, wheezing, diarrhea and right heart valve disease, yet these account for only 1.0% of those symptoms reported in patients with NEC (26).

Diagnosis

The diagnosis of GB-NEN prior to surgery remains difficult. Currently, tumor markers and imaging examinations, such as ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI), are used. Limitations of the current techniques hinder the proper diagnosis of patients with GB-NENs. For instance, tumor markers that include carbohydrate antigen (CA)19-9, carcinoembryonic antigen and CA125 are often negative (27). Ultrasound examination detects only the thickening of gallbladder walls, gallbladder swelling-type lesions and a low echoic nodule (Fig. 2). CT and MRI examination generate impressions indicating that GB-NEN typically presents a thickening of the cystic wall on one side, with the mass protruding into or out of the cavity (Fig. 3). These scans also indicate a visible necrotic shadow in larger lesions (28). In studies examining the results of CT and MRI scans in GB-NEN, it was noted that after the enhancement of GB-NEN, the lesion continued to strengthen and the enhancement was slightly more obvious compared with that of adenocarcinoma (29). Endoscopic retrograde cholangiography revealed the defect of the tumor in the gallbladder (Fig. 4). However, the presence of lymph node metastasis around the gallbladder and retroperitoneum is difficult to distinguish from the other gallbladder tumors (30). Imaging examinations provide significant results only when used at an early stage and then provide aid in establishing a treatment plan (31).

Figure 2.

Ultrasonography images of GB-NEN. (A) Ultrasonography image presenting a 0.8-cm low echoic nodule (arrowheads) in the neck of the gallbladder. (B) Ultrasonography image identifying a low echoic mass of 39×30 mm, with clear boundaries to the liver from the fundus of the gallbladder (arrowheads). GB-NEN, gallbladder neuroendocrine neoplasm.

Figure 3.

Magnetic resonance images of GB-NEN. A, B, C and D are presenting the same mass in the gallbladder, which was confirmed as GB-NEN by pathology. Arrows indicate neuroendocrine tumor of the gallbladder. GB-NEN, gallbladder neuroendocrine neoplasm.

Figure 4.

Endoscopic retrograde cholangiography demonstrating no abnormalities of the bile duct, but an observable tumor defect in the gallbladder (arrowheads).

Diagnostic comparisons indicate that immunohistochemical staining is the most effective tool for the diagnosis of GB-NENs. Immunohistochemistry is divided into two parts (32). First, neuroendocrine cell markers such as neuron-specific enolase (NSE), synaptophysin (Syn), pheochromin-A (CHG-A), protein gene product and Rankine are positively detected (Fig. 5). Second, amines and amine hormones such as adrenocorticotropic hormone, growth hormone, human chorionic gonadotropin, 5-hydroxytryptamine, vasoactive polypeptide, insulin, gastrin, somatostatin, pancreatic polypeptide and calcitonin can simultaneously trigger expression of other hormones (33). Among all of the previously mentioned hormones CHG-A, Syn and NSE have the highest specificity (34). A study has demonstrated that CHG-A is a substance released by secretory particles in neuroendocrine cells to confer their secretory characteristics (35). Evidence demonstrated that the serum CHG-A level in 60–80% of patients with NEC of the digestive system was higher compared with that of normal patients (36). Therefore, the serum CHG-A test has the highest significance in the diagnosis of GB-NEN. Monier et al (37) further suggested that the urine detection of 5-hydroxyindole-acetic acid (5-HIAA) may aid in the diagnosis of GB-NEN. However, in this study, the positive rate reads were low due to the insufficiency or non-secretion of 5-HIAA in some patients with GB-NEN.

Figure 5.

Histological examination of a gallbladder tumor. (A) Cellularity is very high with hyperchromatic nuclei and scant cytoplasm observed (H&E stain) (magnification, ×40). (B) Immunohistochemical staining of Ki-67. Tumor cells demonstrate a Ki-67 index <1.0% (magnification, ×40). (C) Tumor cells demonstrate positive expression of synaptophysin (magnification, ×40). (D) Positive expression of pheochromin-A is observed in tumor cells (magnification, ×40).

Differential diagnosis

Examination of patients presenting with GB-NEN symptoms and markers can identify differential disorders such as cholestasis, gallbladder polyps, gallbladder adenomyosis and gallbladder adenoma. Apart from the pathological immunohistochemical examinations, contrast-enhanced ultrasonography has differential clinical significance (38). Case reports using contrast-enhanced ultrasonography have revealed that biliary sludge is not enhanced (39). In these case reports, the gallbladder polyp was enhanced with grape-like fine pedicles and the three-layer structure of gallbladder wall was clear (40). For adenomyosis of the gallbladder, unenhanced vesiculoid echocardiography was observed and the inner and serous membranes of the gallbladder were intact (41). Gallbladder adenoma resulted in delayed enhancement that was identifiable by ‘fast in and slow out’ through the complete three-layer structure of the gallbladder wall (38). In conclusion, contrast-enhanced ultrasonography can improve the appearance of gallbladder carcinoma and facilitate an early differential diagnosis.

Treatment options

Surgical treatment

Advantages and disadvantages of the various treatment options discussed are listed in Table I: Due to its complexity, gallbladder cancer must be treated surgically by experienced biliary tract physicians and pathologists (42). GB-NEC is characterized by high malignancy, early lymphatic metastasis (the N2 lymph node metastasis rate is significantly higher compared with that of patients with adenocarcinoma in the same period) and a poor prognosis when compared with all other types of gallbladder cancer (20). Radical resection is considered to be the most effective and preferred method of surgical treatment for patients with GB-NEC (43). The purpose of a radical resection is to eliminate lesions, confirm a clear diagnosis, provide a basis for post-operative comprehensive treatment and improve the quality of life of affected patients (33). Surgical methods include simple, radical and expanded radical cholecystectomy, whereby the choice of surgical type is often discussed between the medical professional and patient (44). The progress made in recent years to expand the time period during which radical resection can be performed, including R0 resection for GB-NEC, has increased the overall long-term survival time of patients (31). With these promising results, expanded radical prostatectomy should also be attempted (45). Only under situations where the tumor invades the mucosa, submucosa or muscularis for GB-NET is simple cholecystectomy feasible (46). In the case of late-stage occurrence without distant metastasis, cholecystectomy combined with local liver resection and lymph node clearance is an option for obtaining a good surgical margin (47). The aforementioned study strongly recommends performing radical resection to the maximum possible extent even when liver metastases are limited. If radical resection is not feasible, then volume reduction surgery must be considered as an effective follow-up treatment to improve the quality of life of patients with GB-NENs (48).

Table I.

Comparison of advantages and disadvantages of different gallbladder neuroendocrine neoplasm treatment options.

Treatment options	Advantages	Disadvantages
Surgical treatment	1. Is the most effective treatment	1. Needs experienced biliary physicians and pathologists
	2. Eliminates lesions completely	2. Cholecystectomy alone is only feasible if the tumor invades the mucosa, submucosa or muscularis
	3. Makes the diagnosis clear
	4. Provides the basis for postoperative comprehensive treatment
	5. Improves the quality of life and the overall long-term survival rate
Chemotherapy	1. An important alternative therapy (for patients who are not suitable for surgery)	1. Has limited effect for tumors with high differentiation and slow growth
	2. Prolongs the survival of patients significantly	2. No unified standard chemotherapy treatment available currently
Molecular targeted therapy	Prolongs progression-free survival and overall survival times	No molecular targeted drug therapy has been widely recognized and used
Treatment with somatostatin analogs	1. Inhibits tumor progression	Only effective with positive expression of the somatostatin receptor
	2. Improves symptoms and the overall prognosis of patients
Radiotherapy and interventional therapy	1. Inhibits tumor growth	The clinical efficacy and safety have not been determined yet and need further study
	2. Interventional embolization of the hepatic artery is an effective method to treat hepatic metastasis

Chemotherapy

Patients with GB-NENs who are medically unfit for surgery must be given chemotherapy, a significant alternate treatment method (49). GB-NEC is highly invasive and develops early lymph node metastasis, hence surgery followed by radiotherapy and chemotherapy is recommended to help prolong the survival period of such patients (50). In the case of NEC with high differentiation and slow growth, the effect of chemotherapy has been observed to be limited (51). For rapidly growing tumors, chemotherapy response rates range from 20–60% worldwide (52). Based on the differing degrees of tumor differentiation, the most commonly used chemotherapy drugs include streptozotocin, 5-fluorouracil, adriamycin, cisplatin and etoposide (11). Due to the clinical incidence of GB-NEN being low, there are few associated studies available, which results in the lack of a unified standard chemotherapy program. Available studies have demonstrated that oxaliplatin plus gemcitabine is the most effective chemotherapy regimen for gallbladder cancer at present; however, cholecystic NEC has a poor response to this treatment. Therefore, chemotherapy drugs advocated for gastrointestinal and cholecystic NEC include etoposidem, cisplatin and Adriamycin (11). Associated case reports demonstrated that the post-operative use of gemcitabine, docetaxel, or cisplatin combined with cisplatin, sunitinib and docetaxel, respectively, resulted in a longer survival time in patients with GB-NENs (53,54). Inoue et al (55) reported that following combination treatment with cisplatin and irinotecan, the tumor size in a patient with GB-NEC was significantly reduced and the tumor-free survival period was significantly prolonged compared with cisplatin alone. In addition, in another study, it was reported that the response rate to etoposide and cisplatin increased up to 50–56% in poorly differentiated and rapidly growing NEC (56). In the cases investigated, a medium dose of 3–6 million units of α-interferon was used 3–7 times a week as adjuvant therapy for NEC (56).

Molecular targeted therapy

To date, there is no molecular targeted drug therapy of note for the treatment of patients with GB-NENs. However, there are limited drug therapies available under review. A study has reported progression of the disease coupled with an increased level of VEGF in the blood of patients with GB-NEN (57). This indicates that VEGF-mediated neovascularization plays an important role in the occurrence, progression, metastasis and recurrence of GB-NEN. Raymond et al (58) and Yao et al (59) confirmed that the targeted drug sunitinib extends the progression-free survival (P<0.001) and the overall survival (P=0.02) rate of pancreatic NEN patients by resisting the binding of the vascular endothelial growth factor and platelet-derived growth factor receptors to the corresponding ligand. To develop this as an effective treatment option, the mechanism by which sunitinib acts in patients with GB-NEN requires detailed investigation.

Treatment with somatostatin analogs

Caplin et al (60) conducted a randomized control trial where the progression-free survival rate of patients with NEN treated with a somatostatin analog (octreotide) was significantly higher compared with that of the placebo group (P<0.05). Other studies by Igaz (61) and Oberg et al (62) confirmed the significance of somatostatin analogs (octreotide) in inhibiting tumor progression, and improving symptoms and overall patient prognosis. Meanwhile, Oberg et al (62) also described that with the emergence of long-acting drugs, patients are able treat themselves using monthly injections. This treatment method is effective for patients with positive expression of somatostatin receptor GB-NEN.

Radiotherapy and interventional therapy

The molecular mechanisms that underpin the clinical effect of radioactive isotopes of peptide receptors and the somatostatin analogs are similar (63). Local radiotherapy inhibits tumor growth through use of the radioactive isotopes Y90 or Lu177, with good clinical tolerance in patients with GB-NEN (64). In addition, adjuvant treatment of GB-NEN also includes interventional therapy, namely radiofrequency ablation, seed implantation, arterial embolization and laser hyperthermia. Among them, interventional embolization of the hepatic artery is found to be effective for hepatic metastasis (65). Embolization agents such as anhydrous ethanol or chemotherapy drugs have also been used for patients with GB-NEN; however, the clinical effects and safety of the aforementioned treatment methods require further investigation in detailed studies (66). In clinical practice, it is suggested that targeted treatment is performed in accordance with relevant guidelines and by keeping the specific condition of the patient in mind (67).

Prevention

NEC is a slow-growing malignant tumor without any specific clinical manifestations (68). Preoperative diagnosis often depends on the typical oncoid syndrome. Since GB-NEN and oncoid syndrome are not associated, patients typically visit the doctor only during middle- or late-stage presentation with metastasis (44). Therefore, to increase the diagnosis rate and prevent progression of GB-NEN, an appropriate treatment is advised following complete routine examination for all patients with chronic cholecystitis and cholecystolithiasis. The presentation of GB-NEN is further complicated by the presence of gallbladder stones (29). Stimulation of the gallbladder wall by stones (69) and the occurrence of GB-NEN are related. The complexity of the disease is further increased as GB-NEN is also associated with small intestinal carcinoid. Thus, regular examination is strongly recommended for the correct diagnosis (5). Both gastrointestinal and pulmonary carcinoid cancers result in calcification via dystrophic calcification and endocrine hormone stimulation (70). However, to the best of our knowledge, there are no national or international cases of patients with GB-NEN in which calcification has been reported, providing some level of distinction.

Prognosis

The prognosis of GB-NEN depends on the pathological type under investigation. Atypical carcinoid, low-differentiated adenocarcinoma and small-cell carcinoma. The prognosis, in general, is acceptable for patients with GB-NET, especially G1 disease, as there is a low degree of clinical malignancy with no obvious early metastasis. However, due to the high malignancy and rapid progress of GB-NEC, lymph node and liver metastasis occur simultaneously at diagnosis and lead to a poor prognosis in practice (71). A study has indicated that a lower Ki-67 index with smaller tumor volume leads to a better prognosis for GB-NEN (72). Conversely, the prognosis is worse when the tumor sizes vary and the symptoms are more complex (73). A study by the Memorial Sloan Kettering Cancer Center demonstrated that the median survival time of 13 patients with GB-NEC was only slightly shorter compared with that of 435 patients with gallbladder cancer (9.8 months vs. 10.3 months, respectively) (14). Chiorean et al (11) demonstrated that GB-NEC 1-, 2- and 3-year survival rates were lower compared with those of other types of gallbladder cancer (20 vs. 38%; 10 vs. 31%; 0 vs. 30.1%, respectively) during the same period.

Conclusion

The current review demonstrates that GB-NEN is a relatively rare gallbladder lesion, unique in presentation and often relayed as a case study. Therefore, early detection, correct diagnosis and reasonable treatment of such tumors will help in extending the quality of life of affected patients. At present, the origin of GB-NEN is unclear, the clinical manifestations are atypical and the majority of laboratory and imaging examinations provide no specificity. The diagnosis of GB-NEN depends on pathological and immunohistochemical examinations utilizing markers such as Syn, NSE and CHG-A. In terms of treatment options, surgical treatment is the best choice and active multi-mode comprehensive treatment such as chemoradiotherapy, targeted therapy and somatostatin analogs also significantly prolong survival times in patients with GB-NEN. This review has certain limitations. Due to the low incidence and availability of studies, there is no uniform standard treatment identified for treating GB-NEN. Therefore, we suggest that treatment is provided on a case by case basis to comprehensively integrate the advantages of various treatment methods for providing targeted treatment and to maximize the benefits for patients with GB-NEN in clinical practice.