Gallbladder Neuroendocrine Neoplasms in Dogs and Humans

Simple Summary Cancers of the gallbladder arise in dogs and humans. Neuroendocrine neoplasms are the rarest type of gallbladder cancer in both species. This review summarizes the characteristics of canine and human gallbladder neuroendocrine neoplasms with the objective of comparatively modeling clinical and diagnostic features of these spontaneously occurring neoplasms. Abstract Gallbladder neuroendocrine neoplasms (GB NENs) are among the rarest cancers reported in humans and dogs. This review provides a detailed review of the canine GB NEN literature and an interspecies comparison of demographics, clinical pathophysiology, pathobiology, and therapeutic response of GB NENs. The aim of this work is to explore the relevance of dogs as a spontaneous model for human GB NENs.


Introduction
Historically referred to as carcinoids (from karzinoide, meaning "carcinoma-like"), neuroendocrine neoplasms (NENs) comprise a heterogeneous group of tumors derived from cells of the neuroendocrine system.Neuroendocrine (NE) cells, which may be glandforming or diffusely distributed, characteristically express features of both endocrine cells and neurons [1].Functionally, NE cells synthesize bioactive amines, peptides, and hormones.Similar to serotonergic neurons, bioactive molecules are stored within dense core granules that are released in response to receptor-mediated stimuli [2].The specific hormonal products secreted by NE cells differ depending on their tissue niche, and NENs arising from anatomically distinct sites often exhibit diverse biologic behavior [1,[3][4][5].
NENs account for approximately 0.5% of all human malignancies.The majority of these neoplasms occur in the gastrointestinal and respiratory tracts, a pattern of distribution that reflects the high concentration of dispersed neuroendocrine cells within these organs.Less common sites of origin include the pancreas, skin, urogenital tract, and hepatobiliary system [6].Historically, NENs were classified according to their synthetic hormone profile, but this classification scheme was complicated by the complexity of NEN phenotypes.Nomenclature guidelines by the World Health Organization (WHO) have refined the terminology by dividing NENs into categories based on histopathologic features, including the well-differentiated neuroendocrine tumor (NET), poorly differentiated neuroendocrine carcinoma (NEC), and mixed neuroendocrine carcinomas which have features of NENs and non-neuroendocrine neoplasms (MiNEN) [7,8].NETs are further subdivided into grades 1-3, with grades 1 and 2 exhibiting indolent growth and a favorable prognosis, while grade 3 NETs have a high Ki67 index (>20%) and carry a poor prognosis.NECs, which are poorly differentiated, are considered high-grade.MiNENs are graded according to both their neuroendocrine and non-neuroendocrine components [7,8].
Reports of NENs in the veterinary literature are uncommon, although incidence has increased since the turn of the century as advances in diagnostic imaging have enabled earlier diagnosis of these neoplasms.In dogs, NENs of the pancreas, paraganglia, pituitary gland, and thyroid gland predominate, while less common locations include the gastrointestinal tract, the bronchopulmonary tree, the skin, and the hepatobiliary system [9][10][11][12][13][14][15][16][17][18].

Characteristics of Neuroendocrine Cells
The neuroendocrine system comprises a wide range of cells found throughout most mucosal organs in the body.NE cells are present in large aggregates comprising solid organs such as the adrenal medulla and pituitary gland, small focal aggregates such as those of the pancreatic islets or pulmonary neuroepithelial bodies (NEBs), and individualized cells scattered throughout the mucosa of numerous tissues.While the majority of the diffuse neuroendocrine cells of the body localize to the gastrointestinal tract, pancreas, and respiratory system, lower numbers of these cells can be found throughout many other tissues, including the urogenital system, the biliary tract, the skin, and the thymus.While NE cells do exhibit phenotypic differences depending on their specific tissue localization, they generally share a number of common characteristics, including the production and secretion of hormones and neurotransmitters.In response to environmental stimuli or neural input, neuroendocrine cells are stimulated to secrete granules containing bioactive molecules that may act on distant tissue sites (endocrine), neighboring cells (paracrine), or the cell of origin (autocrine) [19].

Neuroendocrine Neoplasms: Nomenclature and Grading
In 1963, the first formal classification scheme for NENs was established using the embryologic origin of the primary organ-specifically the foregut (respiratory tract, stomach, proximal duodenum, pancreas, hepatobiliary system), midgut (duodenum, ileum, appendix, ascending colon), or hindgut (transverse and descending colon, rectum).However, the wide variation in the behavior of NENs even within these embryonic segments limited the clinical utility of this classification system.Additionally, the notion that carcinoids of each embryonic segment had a distinct histologic appearance was later discredited, and it is now known that considerable histopathologic overlap exists between all NEN subtypes [8].
In 1999, the World Health Organization (WHO) attempted to clarify the nomenclature of NENs by establishing a classification scheme for pulmonary NENs consisting of four categories.Typical (low-grade) carcinoids were well-differentiated NENs consistent with the prototypic carcinoid first described by Oberndorfer, while atypical (intermediategrade) carcinoids had a slightly more aggressive clinical course while still appearing overall well differentiated.High-grade NENs were separated into the categories of largecell neuroendocrine carcinoma (LCNEN) and small-cell lung carcinoma (SCLC), both of which appeared poorly differentiated and exhibited malignant characteristics [2].The following year, the term "carcinoid" was dropped from gastrointestinal and pancreatic NENs altogether in favor of the broad category of "neuroendocrine neoplasm".Typical and atypical carcinoids were reclassified as grade 1 (G1) or grade 2 (G2) neuroendocrine tumors (NETs), respectively, while the LCNEC and SCLC were now considered subtypes of neuroendocrine carcinomas (NECs).In 2010, the NET/NEC classification system was expanded to encompass all neoplasms of neuroendocrine origin.Criteria for grading were also established for gastrointestinal and pancreatic NENs (GEP-NENs) using mitotic and Ki67 indices [20].
Over the following decade, evidence emerged for another category of NEN that did not fit within the 2010 WHO guidelines.Morphologically well-differentiated NENs with a Ki67 index exceeding 20% presented a diagnostic conundrum; while the histopathologic features would qualify these tumors as NETs, their proliferation index and clinical course were consistent with an NEC.The pancreas was the most common site for these "high-grade NETs", and the 2017 WHO guidelines for endocrine neoplasms expanded the grading system for pancreatic NENs to include a NET G3 category.In 2019, the use of the G3 category was extended to all GEP-NETs [21].
Vet. Sci.2024, 11, 371 3 of 13 Specific guidelines exist for grading NETs in each anatomic niche, but grading is generally based on mitotic index, Ki67 index, and/or the presence of necrosis.NECs are inherently considered to be high-grade.It was once accepted that NETs arose from differentiated neuroendocrine cells and that NECs were the result of NET progression to a malignant phenotype (sometimes referred to as the "NET-NEC sequence"); however, cell lineage tracing and genetic analyses have discredited the NET-NEC theory.The current understanding is that NENs are the result of neoplastic transformation of a stem cell precursor, and the resultant type of NEN depends on the stage of differentiation of that precursor (i.e., NECs arise from pluripotent stem cells, while NETs arise from committed neuroendocrine stem cells).No strong evidence exists for the progression of a NET to a NEC, and they likely represent separate and distinct entities [22,23].

Epidemiology and Anatomic Distribution of Neuroendocrine Neoplasms
Though once considered to be rare, the reported incidence of NENs in humans has been steadily increasing.The rising number of cases is likely multifactorial, owing to improved awareness of these tumors and more accurate classification with modern immunohistochemical techniques.According to data from the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) Program, the incidence rate of NENs in the United States increased 6.4-fold from 1973 (1.09 per 100,000) to 2012 (6.98 per 100,000), with increases distributed across all anatomic sites [24].The greatest increases were observed for gastric (15-fold) and rectal (9-fold) NENs, which have been attributed to the widespread modern use of endoscopic procedures [24][25][26].
The gastrointestinal tract is the most common site of NEN development, accounting for 67.5% of NENs in one retrospective analysis of 13,715 cases.Of gastrointestinal NENs within that study, the small intestine was the most common site (41.8%),followed by the rectum (27.4%) and stomach (8.7%).The respiratory tract was found to be the second most common organ system of NEN origin (25.3%), with the remainder of cases distributed among less common locations, including the pancreas, urogenital tract, thymus, and hepatobiliary system.A slight overall female predominance was found for NENs as a whole (55.1%), with the highest female-to-male ratios observed in the stomach, colon, appendix, bronchopulmonary system, and gallbladder.Male predominance was noted in esophageal and thymic NENs.Metastatic disease was present in 12.9% of patients at the time of diagnosis, and the overall 5-year survival rate was 67.2%, although the range in overall survival was broad.Five-year survival rates were best for rectal (88.3%), bronchopulmonary (73.5%), and appendiceal (71.0%)NENs, while the poorest prognosis was observed with pancreatic (37.5%) and hepatic (18.4%) tumors [27,28].

Gallbladder Neuroendocrine Neoplasms
The mechanisms and regulation of neuroendocrine differentiation in the hepatobiliary tract have not been extensively clarified.It is now accepted that these sparse NE cells are of endodermal origin, derived from local populations of multipotent stem cells, rather than arising from the neural crest as was originally postulated.However, the exact molecular pathways governing their differentiation and the role these cells play in pathologic states is an area of ongoing study.
The origin of gallbladder NENs has been an issue of debate, as there are no neuroendocrine cells within the normal gallbladder mucosa.The theories proposed are that GB NENs arise either from NE cells in the gallbladder neck or from gastric or intestinal metaplasia, which may occur in the gallbladder secondary to conditions such as cholelithiasis.The fact that GB NENs are frequently observed in human patients with a history of cholelithiasis has been viewed as supporting evidence for the latter theory; while cholelithiasis is uncommon in dogs, 17.1% of the dogs described herein had choleliths concurrent with gallbladder NEN [10,39].Yet given the more recent revelations regarding the multipotent stem cell origin of most NENs, GB NENs may be derived from local progenitor cells rather than differentiated cells [10,23,[39][40][41][42].

Diagnosis of Gallbladder NENs: Cytopathology
Neuroendocrine neoplasms across anatomic locations share similar cytologic features in humans and dogs (Figure 1).Samples are commonly composed of loosely cohesive clusters of round to polygonal cells with indistinct borders, often forming palisades or rosettes.Characteristically, these samples tend to contain large numbers of intact free nuclei dispersed throughout a diffuse background of pale basophilic cytoplasm.In the majority of cases, anisokaryosis and nuclear atypia are minimal.Nuclear-to-cytoplasmic ratios are variable, and the cytoplasm may be lightly discretely vacuolated.Less commonly, nuclear pleomorphism may be more pronounced; karyomegaly and nuclear pseudoinclusions are rarely observed [5,8,43].For most cases, specific diagnosis requires interpretation of cytopathologic findings in conjunction with anatomic location and clinical features.
molecular pathways governing their differentiation and the role these cells play in pathologic states is an area of ongoing study.
The origin of gallbladder NENs has been an issue of debate, as there are no neuroendocrine cells within the normal gallbladder mucosa.The theories proposed are that GB NENs arise either from NE cells in the gallbladder neck or from gastric or intestinal metaplasia, which may occur in the gallbladder secondary to conditions such as cholelithiasis.The fact that GB NENs are frequently observed in human patients with a history of cholelithiasis has been viewed as supporting evidence for the latter theory; while cholelithiasis is uncommon in dogs, 17.1% of the dogs described herein had choleliths concurrent with gallbladder NEN [10,39].Yet given the more recent revelations regarding the multipotent stem cell origin of most NENs, GB NENs may be derived from local progenitor cells rather than differentiated cells [10,23,[39][40][41][42].

Diagnosis of Gallbladder NENs: Cytopathology
Neuroendocrine neoplasms across anatomic locations share similar cytologic features in humans and dogs (Figure 1).Samples are commonly composed of loosely cohesive clusters of round to polygonal cells with indistinct borders, often forming palisades or rosettes.Characteristically, these samples tend to contain large numbers of intact free nuclei dispersed throughout a diffuse background of pale basophilic cytoplasm.In the majority of cases, anisokaryosis and nuclear atypia are minimal.Nuclear-to-cytoplasmic ratios are variable, and the cytoplasm may be lightly discretely vacuolated.Less commonly, nuclear pleomorphism may be more pronounced; karyomegaly and nuclear pseudoinclusions are rarely observed [5,8,43].For most cases, specific diagnosis requires interpretation of cytopathologic findings in conjunction with anatomic location and clinical features.

Diagnosis of Gallbladder NENs: Histopathology
In general, the histopathologic characteristics of NENs in both humans and dogs are relatively consistent across anatomic niches.These neoplasms are composed of round to cuboidal cells arranged in one of several characteristic patterns, which include nests, rosettes, and solid cords interspersed by fibrovascular stroma.Well-differentiated NENs are composed of uniform polygonal cells, which can be arranged in solid nesting architecture, trabecular or ribbon-like morphology, or glandular patterns.Combinations of these patterns are sometimes observed, and palisading may be present near the periphery of nested arrangements.Abundant fibrous stroma is present in some cases, but more commonly, tumor cells are interspersed by a fine fibrovascular stroma [5,8,43].
Poorly differentiated NECs may be of large-or small-cell phenotype.Large-cell NECs (LCNECs) have low nuclear-to-cytoplasmic ratios, well-defined cell borders, and clear to

Diagnosis of Gallbladder NENs: Histopathology
In general, the histopathologic characteristics of NENs in both humans and dogs are relatively consistent across anatomic niches.These neoplasms are composed of round to cuboidal cells arranged in one of several characteristic patterns, which include nests, rosettes, and solid cords interspersed by fibrovascular stroma.Well-differentiated NENs are composed of uniform polygonal cells, which can be arranged in solid nesting architecture, trabecular or ribbon-like morphology, or glandular patterns.Combinations of these patterns are sometimes observed, and palisading may be present near the periphery of nested arrangements.Abundant fibrous stroma is present in some cases, but more commonly, tumor cells are interspersed by a fine fibrovascular stroma [5,8,43].
Poorly differentiated NECs may be of large-or small-cell phenotype.Large-cell NECs (LCNECs) have low nuclear-to-cytoplasmic ratios, well-defined cell borders, and clear to eosinophilic cytoplasm that may be granular.In contrast, small-cell NECs (SCNECs) have indistinct cell borders and small, hyperchromatic nuclei with scant cytoplasm.In both subtypes, cells are arranged predominantly in solid sheets or nodules, and necrosis is common [8].

Diagnosis of Gallbladder NENs: Immunohistochemistry
A key characteristic enabling the identification of neuroendocrine cells is the presence of a distinct profile of cell markers.Broad-spectrum markers that can be used to identify neuroendocrine differentiation include chromogranin A (humans and dogs), synaptophysin (humans and dogs), and insulinoma-associated protein 1 (only validated in humans) [8,10,44].
Granins are acidic hydrophilic glycoproteins found in large dense-core vesicles (LD-CVs) of neuroendocrine cells [45].They are initially synthesized at the rough endoplasmic reticulum (RER), before being transported to the Golgi complex, where they aggregate at the trans-Golgi network membrane and induce budding of LDCV.Granins also serve as precursors for numerous biologically active peptides, which induce a wide range of physiologic effects.The major granins include chromogranin A (CgA), chromogranin B, and secretogranin II.CgA is the most abundantly expressed granin, comprising 50% of the soluble protein content of adrenal medullary chromaffin secretory granules [1,46].CgA is very broadly expressed in neuroendocrine cells and is considered the most specific of the broad-spectrum neuroendocrine markers.However, since granins are found almost exclusively within secretory granules, poorly differentiated neuroendocrine cells with few mature granules may be negative for this marker.In some cases, a fine paranuclear dot-like pattern of positivity may be observed.
Synaptophysin (SYN), a transmembrane glycoprotein found on smaller synaptic-like microvesicles (SLMVs), is expressed by both normal and neoplastic NE cells [8,45].Its role in SLMV trafficking is still incompletely understood, but it appears to play a role in vesicle recycling following exocytosis.SYN is considered the most sensitive marker for both well-differentiated and poorly differentiated NENs but lacks complete specificity, as other neoplasms (e.g., adrenocortical carcinomas, neuroblastomas) may also be positive [45].
Insulinoma-associated protein 1 (INSM1), a zinc-finger transcription factor, is a relatively new broad-spectrum NE marker.Originally isolated from pancreatic insulinoma cells, INSM1 acts to repress transcriptional activity, regulate cell cycle entry, and control the expression of neuroendocrine phenotype [47].INSM1 plays a key role in the development of NE cells, and studies have demonstrated that it matches both the sensitivity of synaptophysin and the specificity of chromogranin A, making it a promising marker for the diagnosis of NENs.Additionally, the nuclear staining pattern of INSM1 is more readily identified in comparison to the cytoplasmic expression of CgA and SYN [48].To date, large-scale evaluation of the utility of INSM1 as an NE marker in dogs has not been reported.
Other broad-spectrum NE markers that have fallen out of common use include neuronspecific enolase (NSE), protein gene product 9.5 (PGP 9.5), and CD56 [3,43,49].NSE, an isozyme of the glycolytic enzyme enolase, is found in the cytoplasm of normal and neoplastic NE cells [50].NSE has classically been used as a marker of neuroendocrine differentiation, but its clinical utility is hampered by overall poor specificity, as anti-NSE antibodies have been shown to cross-react with enolase expressed in non-NE tissue such as striated muscle [51].Additionally, NSE positivity has been detected in some non-NE neoplasms of the pancreas.PGP 9.5 is a protease involved in the processing of ubiquitin precursors and is expressed by neurons and neuroendocrine cells; however, PGP 9.5 expression has also been documented in pancreatic adenocarcinomas and plasma cell neoplasms.CD56, also called neural cell adhesion molecule (N-CAM), is found in a wide range of cells including T cells, natural killer cells, thyrocytes, adrenocortical cells, and NE cells.It has a high sensitivity for neuroendocrine differentiation, but its specificity is poor, and positivity may be observed in numerous non-NE neoplasms [49].CD56 is still utilized sporadically in conjunction with CgA and SYN for the diagnosis of pulmonary NECs, but NSE and PGP 9.5 are not currently recommended for diagnostic use [8,52].

Gallbladder NENs in Humans
First described in 1929, NENs of the human gallbladder are rare, comprising only 0.5% of all NENs and approximately 2% of gallbladder neoplasms.They have a distinct female Vet.Sci.2024, 11, 371 6 of 13 predominance, with a female-to-male ratio of 4:1, and while the low number of documented cases and lack of consistent nomenclature in past studies limits the interpretation of their biologic behavior, they are generally considered to be aggressive neoplasms with a poor prognosis.Retrospective analyses have reported that NECs comprise 69.9-90.2% of GB NENs, with the majority being small cell carcinomas (SCCs).Abdominal pain is the most common presenting complaint, occurring in approximately half of patients, while less consistent clinical signs include vomiting, icterus, and abdominal distension [42,53].
Many human patients with GB NENs are initially asymptomatic or have non-specific clinical signs, making early identification of these neoplasms challenging; metastases are reported in 54-68% of cases at the time of diagnosis.Clinicopathologic features consistently correlated with prognosis include tumor grade, the presence of metastatic disease, and advanced age.Tumor size has also been reported as a prognostic feature, with more than 70% of tumors > 2 cm presenting with local or distant metastases [54].Prognostic information in human GB NENs is conflicting, complicated by lack of stratification by grade and the use of inconsistent terminology prior to the 2010 WHO reclassification of NENs; however, the prognosis in most cases is poor, with reported median survival times of 3-24 months.Grade 1 and G2 NETs have a more favorable prognosis if surgical excision is performed, but specific survival data for these grades are scarce [53].

Gallbladder NENs in Dogs
Primary gallbladder NENs in dogs were first described in 1988, and sporadic case reports or case series of gallbladder NENs have appeared in the literature since that time [10,11,18,39,44,[55][56][57][58][59][60][61].Grossly, these masses are firm, solitary, and variable in size.Necrosis and hemorrhage are common histopathologic features, and concurrent gallbladder pathology is frequently present, including cholecystitis, cystic mucosal hyperplasia, and cholelithiasis.Notably, the findings of these studies indicate that primary gallbladder NENs may carry a more favorable prognosis compared to gallbladder NENs in humans; however, the biologic behavior of canine NENs is poorly understood due to the lack of histologic grading [10].

Canine Survival Data
Censored survival data were available for 26 dogs (Table 1).Fifteen patients (57.69%) were still alive at the termination of data collection.Of the 11 patients that died, 5 deaths were directly due to the GB-NEN, whereas 6 patients were euthanized due to various causes (1 intermittent vomiting and diarrhea post-cholecystectomy, 1 lymphoma, 1 neurologic disorder, 1 pulmonary masses, and 1 cardiac mass, and one dog whose cause of death was not reported).Overall median survival time (MST) was 730 days (range: 240-2191).Metastasis was reported in 6 of the 35 dogs (17.14%), with sites including the liver (n = 4), lungs (n = 2), and mesentery (n = 1).

Summary Canine GB NENs
While the rarity of this neoplasm limits understanding of its behavior, the prognosis for canine patients with GB NENs appears to be good.In cases where mitotic index or Ki-67 has been reported, the majority of canine NENs can be classified as NETs.Mean survival times exceeding three years have been reported following cholecystectomy, which is consistent with reports of survival in human patients with completely excised G1 NETs.
It has been suggested that the production of gastrin by a portion of these neoplasms in dogs and the resultant clinical signs of vomiting and hematemesis may lead to an earlier diagnosis and contribute to a favorable prognosis; however, gastrointestinal signs have also been documented in a dog with a GB NEN that was negative for gastrin, indicating that the clinical presentation associated with this neoplasm is likely multifactorial [10,44].

Conclusions
NENs are a diverse group of neoplasms exhibiting significant behavioral heterogeneity, and the prognosis of primary gallbladder NENs in canine patients is better than that reported in humans.In contrast to NENs of the human gallbladder, which consist predominantly of NECs, the described histopathologic features and slower clinical course suggest that the majority of NENs in dogs are NETs.Improved histologic characterization of NENs in dogs, including more consistent reporting of proliferative indices, is needed to appropriately classify these tumors in the future.Further analysis of the molecular signatures of canine and human gallbladder NENs will contribute to improved understanding of their pathophysiology and prognosis.Furthermore, given the rarity of hepatobiliary NENs, a better understanding of these neoplasms will inform the development of therapeutic targets that may benefit both species.

Figure 1 .
Figure 1.Gallbladder neuroendocrine neoplasm cytology.Photomicrograph of a fine needle aspirate smear from a gallbladder NEN in a dog.Polygonal epithelial cells demonstrate overall mild anisokaryosis and variably distinct cell borders.Wright-Giemsa stain; Scale bar = 20 µm.

Figure 1 .
Figure 1.Gallbladder neuroendocrine neoplasm cytology.Photomicrograph of a fine needle aspirate smear from a gallbladder NEN in a dog.Polygonal epithelial cells demonstrate overall mild anisokaryosis and variably distinct cell borders.Wright-Giemsa stain; Scale bar = 20 µm.

Table 1 .
Summary of patient demographics, grading and mitotic index, and survival data.

Table 2 .
Immunohistochemical profiles of canine GB NEN.